Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of SH2-domain inositol 5' phosphatase in the inhibition of macrophage activation Ming-Lum, Andrew N. 2012

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


24-ubc_2012_spring_minglum_andrew.pdf [ 17MB ]
JSON: 24-1.0072703.json
JSON-LD: 24-1.0072703-ld.json
RDF/XML (Pretty): 24-1.0072703-rdf.xml
RDF/JSON: 24-1.0072703-rdf.json
Turtle: 24-1.0072703-turtle.txt
N-Triples: 24-1.0072703-rdf-ntriples.txt
Original Record: 24-1.0072703-source.json
Full Text

Full Text

 THE ROLE OF SH2-DOMAIN INOSITOL 5ʹ′ PHOSPHATASE IN THE INHIBITION OF MACROPHAGE ACTIVATION by Andrew N. Ming-Lum B. Sc. (Hons), Queen’s University, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   April 2012   ©  Andrew N. Ming-Lum 2012  ii ABSTRACT  Interleukin-10 (IL-10) is an anti-inflammatory cytokine essential for maintaining immune homeostasis.  One of its major targets is the macrophage where it inhibits production of pro-inflammatory cytokines, chemokines and other soluble mediators. However, the intracellular signaling mechanisms by which IL-10 achieves macrophage deactivation remain under intense investigation.  Our studies suggest that in addition to canonical STAT3 signaling, IL-10 mediates its early phase anti-inflammatory reponse through SHIP1 in a STAT3-independent manner.  Upon macrophage activation by bacterial lipopolysaccharide, the phosphoinositide 3ʹ′ kinase (PI-3 kinase) pathway is activated to produce cytokines such as tumor necrosis factor α (TNFα).  SHIP1 is a negative regulator of the PI-3 kinase pathway and its activation downstream of the IL-10 receptor suppresses PI-3 kinase-initiated signals that trigger transcriptional elongation of TNFα and other pro-inflammatory related genes.  We next investigated whether SHIP1 activation could mimic the anti-inflammatory actions of IL-10.  We screened for small- molecule activators of SHIP1 and isolated the meroterpenoid compound Pelorol.  Pelorol and its derivatives specifically enhanced SHIP1’s phosphatase activity and thus suppressed inflammation in macrophage cultures, and in murine models of endotoxic shock, acute anaphylaxis, and inflammatory bowel disease.  Closer examination of SHIP1’s enzyme kinetics indicated that SHIP1 is subject to allosteric activation by its product phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2).  We subsequently identified a previously unrecognized C2 domain residing C-terminal of SHIP1’s phosphatase domain which is required for its allosteric activation and is the binding site for both PI-3,4-P2 and the small-molecule SHIP1 agonists.  Bioinformatic and structural analyses also revealed  iii another previously unappreciated domain located N-terminal of SHIP1’s catalytic domain.  Using NMR spectroscopy, we characterized this domain as having pleckstrin homology (PH) domain-like topology.  We demonstrate that SHIP1’s PH-related (PH-R) domain participates in recruiting SHIP1 to the plasma membrane upon cell stimulation via direct interactions with phosphatidylinositol-3,4,5-trisphosphate.   The PH-R domain is essential for SHIP1 inhibition of FcR-dependent phagocytosis and represents another target to which to develop modulators of SHIP1 function.  Together, this work suggests that IL-10 activation of SHIP1 is important in its inhibition of macrophage activation, and that mimicking IL-10 with small-molecule SHIP1 agonists could be an effective and viable approach to treating various inflammatory and autoimmune conditions.               iv PREFACE  Contributions of Collaborators: Design of all research, data analysis and manuscript preparation were completed with the assistance of Dr. Alice Mui. All experiments were performed solely by the author with the following exceptions: Chapter 2: Real-time quantitative PCR analysis of primary response genes were performed with the assistance of Erin McCarrell. Chapter 3: Structural identification of Pelorol and the synthesis of its structural analogues, AQX- 016A and AQX-MN100 (Figure 3.1A), were performed by our collaborators Drs. Raymond J. Andersen and Matthew Nodwell. Myeloperoxidase assays were performed with the assistance of Loutfig Demirjian (Figure 3.2B) Dosing and tissue harvest of IL-10-/- mice was performed with the assistance of Michael Kennah. Scoring of H&E stained tissue sections was performed with the assistance of Eva So (Figure 3.1 C).  v Expression of recombinant C2 domain and Protein-Lipid Overlay assays were performed with the assistance of Joseph Kim Chapter 4: NMR-spectroscopy structural determinations of the SHIP1 PH-R domain were performed by our collaborators Drs. Lawrence McIntosh and Shaheen Shojania (Figures 4.2, 4.3, and 4.7).  The corresponding materials and methods were prepared with their assistance. Confocal microscopy image capture and scoring were performed with the assistance of Eileen Shaw, Eva So, Erin McCarell and Tina Chang (Figures 4.5B and C, 4.9B, and 4.10) Bacterial expression of recombinant PH-R mutant domains and Protein-Lipid Overlay assays were performed with the assistance of Ida Wang and Eileen Shaw. Appendices: Figure A.1 were provided courtesy of Gary Golds. Data in Figures B.1 and E.1 were provided courtesy of Dr. Ali Ghanipour. List of Publications: The data presented in Chapter 2 are contained in the following manuscript that is currently under review for publication: Ming-Lum, A, Ghanipour, A, McCarrell, E, Qasimi, P, Rauh, M, Antignano, F, Sly, L, Chung, SW, Ong, CJ, Krystal, G, Mui, AL.  Interleukin-10 inhibits early phase macrophage activation via the Lipid Phosphatase SHIP1.  vi The majority of studies and figures described in Chapter 3 was originally published in Blood.  Ong, CJ, Ming-Lum, A, Nodwell, M, Ghanipour, A, Yang, L, Williams, DE, Demirjian, L, Qasimi, P, Ruschmann, J, Cao, L, Ma, K, Chung, SW, Duronio, V, Andersen, RJ, Krystal, G, Mui, AL. Activation of inositol phosphatases to inhibit the phosphoinositide 3-kinase pathway. Blood. 2007 Sep 15;110(6):1942-9. © the American Society of Hematology. The data presented in Chapter 4 are contained in the following manuscript that is currently under review for publication: Ming-Lum, A, Shojania, S, So, E, McCarrell, E, Shaw, E, Vu, D, Wang, I, Chang, T, Andersen, R.J., Ong, C.J., Krystal, G, McIntosh, L.P., Mui, A.L.  A pleckstrin homology- related domain in SHIP1 mediates membrane localization in Fcγ receptor induced phagocytosis.  Ethics Approval: All animal experiments were performed in accordance with the UBC Animal Care Committee guidelines under the following protocols: SHIP1-/- Mouse Colony:  A06-0336 IL-10-/- Mouse Colitis Model:  A08-0875-R001 Endotoxemia Model:    A11-0216 All biohazardous experiments were performed in accordance with the Health Canada, Laboratory Biosafety Guidelines under protocol B06-0140.     vii TABLE OF CONTENTS Abstract .............................................................................................................................. ii	
   Preface ............................................................................................................................... iv	
   List of Tables ..................................................................................................................... x	
   List of Figures ................................................................................................................... xi	
   List of Abbreviations ..................................................................................................... xiv	
   Acknowledgements ......................................................................................................... xx	
   Chapter 1:  Introduction .................................................................................................. 1	
   Inflammation .......................................................................................................... 2	
   Macrophages .......................................................................................................... 4	
   Monocyte/Macrophage heterogeneity ............................................... 5	
   Macrophage activation ....................................................................... 8	
   LPS  ..................................................................................................................... 11	
   LPS/Toll-like receptor 4 signaling ................................................... 13	
   Negative regulation of LPS/TLR4 signaling ................................... 18	
   Tumor necrosis factor α ....................................................................................... 20	
   TNFα in disease ............................................................................... 24	
   Transcriptional programs ..................................................................................... 24	
   Primary response genes ................................................................... 25	
   Interleukin-10 ....................................................................................................... 28	
   IL-10 signaling ................................................................................. 29	
   IL-10 biological activity .................................................................. 31   IL-10’s transcriptional and post-transcriptional regulation of TNFα ........................................................................................... 31 1.6.3	
   IL-10 in disease ................................................................................ 36	
  IL-10 in inflammatory bowel disease .......................................... 36	
   The PI-3 kinase pathway ...................................................................................... 37	
   The PI-3 kinase pathway in inflammation ....................................... 38	
   SHIP1 ................................................................................................................... 41	
   SHIP1’s biological activity .............................................................. 44	
  SHIP1’s role in myeloid cells ..................................................... 44	
  SHIP1’s role in other immune cells ............................................ 46	
   SHIP1 in disease .............................................................................. 49	
   Phagocytosis ......................................................................................................... 50	
   FcR-mediated phagocytosis ............................................................. 50	
   Objectives and aims ............................................................................................. 53	
   Chapter 2:	
  SHIP1 is required for mediating IL-10’s anti-inflammatory response . 55	
    viii 2.1  Introduction .......................................................................................................... 56	
   2.2  Materials and methods ......................................................................................... 58	
   Mice ................................................................................................. 58	
   Lentiviral constructs ........................................................................ 58	
   Cells ................................................................................................. 59	
   Immunoblot analysis of proteins ...................................................... 60	
   Analysis of AKT phosphorylation by flow cytometry .................... 61	
   Measurement of TNFα protein production ...................................... 61	
   Measurement of mRNA expression ................................................. 62	
   Chromatin immunoprecitiation ........................................................ 63	
   Mouse endotoxemia model .............................................................. 64	
   2.3  Results .................................................................................................................. 65	
   IL-10 inhibits LPS activation of PI-3 kinase through SHIP1 .......... 65	
   Macrophage production of TNFα occurs in two phases .................. 70	
   IL-10 inhibits Ser2 phosphorylation of RNA polymerase II associated with the TNFα promoter ................................................. 76	
   IL-10 inhibition of TNFα and CCL2 expression in mice requires SHIP1 ............................................................................................... 81	
   Discussion ............................................................................................................ 83	
   Chapter 3:  Allosteric activation of SHIP1 inhibits inflammation ............................. 87	
   Introduction .......................................................................................................... 88	
   Materials and methods ......................................................................................... 90	
   Formulation of compounds .............................................................. 90	
   Production of recombinant SHIP1 enzyme and SHIP1 C2 domain 90	
   in vitro SHIP1 enzyme assay ........................................................... 91	
   Production of SHIP1+/+ and SHIP1-/- in bone marrow derived macrophages .................................................................................... 91	
   LPS stimulation of macrophages ..................................................... 91	
   Mouse endotoxemia model .............................................................. 92	
   Mouse acute cutaneous anaphylaxis model ..................................... 93	
   Mouse colitis model ......................................................................... 93	
   Construction of the SHIP1 ΔC2 mutant and isolated C2 domain .... 94	
   Protein lipid overlay assays ............................................................. 94	
   Scintillation proximity assays .......................................................... 95	
   Results .................................................................................................................. 96	
   AQX-MN100 is as biologically active as AQX-016A .................... 96	
   AQX-MN100 is protective in in vivo models of inflammation ....... 98	
   SHIP1 is an allosterically activated enzyme .................................. 103	
   Discussion .......................................................................................................... 108	
    ix Chapter 4:	
  A pleckstrin homology-related domain in SHIP1 mediates membrane localization in FcγR-mediated phagocytosis ................................................... 112	
   Introduction ........................................................................................................ 113	
   Materials and methods ....................................................................................... 116	
   SHIP1 sequence domain identification .......................................... 116	
   Expression and purification of SHIP1 PH-R domain and K397A/K370A (KAKA) mutant domain ...................................... 116	
   Sequence of 6×His affinity tagged SHIP1 PH-R domain .............. 117	
   Expression and purification of the isotopically labeled PH-R domain   ......................................................................................................  117	
   NMR sample preparation ............................................................... 119	
   NMR spectral assignments ............................................................ 120	
   NMR-monitored titrations ............................................................. 120	
   Phosphoinositol binding (PLO assay) ............................................ 120	
   Cells and reagents .......................................................................... 121	
   GFP-tagged SHIP1 PH-R domains ................................................ 122	
   Scintillation proximity assays (SPA) ............................................. 122	
   Phagocytosis assays ....................................................................... 122	
   Production of recombinant SHIP1 WT and SHIP1 KAKA ........... 124	
   in vitro phosphatase assays ............................................................ 124	
   Results ................................................................................................................ 125	
   Identification of a PH related (PH-R) domain in SHIP1 ............... 125	
   Functional characterization of the PH-R domain .......................... 132	
   Identification of amino acid residues involved in PIP3 binding .... 138	
   The KAKA PH-R protein has impaired localization to the phagocytic cup ............................................................................... 139	
   SHIP1 with the KAKA substitution is still subject to allosteric regulation ....................................................................................... 143	
   SHIP1 with the KAKA substitution is not able to inhibit Fcγ-R mediated phagocytosis ................................................................... 146	
   Discussion .......................................................................................................... 151	
   Chapter 5: 	
  Conclusion ................................................................................................ 157	
   Conclusion .......................................................................................................... 158	
   Bibliography .................................................................................................................. 169	
   Appendices ..................................................................................................................... 206	
      x LIST OF TABLES Table 1.1:   Negative regulators of TLR4 signaling ....................................................... 19	
   Table 1.2:	
   The roles of SHIP1 in immune cells ............................................................ 48	
   Table A.1:	
   Primer sequences for real-time quantitative PCR using SYBR green detection. .................................................................................................... 208	
                       xi LIST OF FIGURES Figure 1.1	
   The development of macrophages in mice. ................................................. 7	
   Figure 1.2	
   Characteristics of M1 and M2 polarized macrophages. ............................ 10	
   Figure 1.3	
   The structure of LPS. ................................................................................ 12	
   Figure 1.4	
   The TLR4 signaling pathway. ................................................................... 16	
   Figure 1.5	
   The TNFα signaling pathway. ................................................................... 23	
   Figure 1.6	
   PRG transcriptional elongation control. .................................................... 27	
   Figure 1.7     IL-10 inhibition of the TLR4 signaling pathway and TNFα production. . 34	
   Figure 1.8	
   The domain structure of SHIP1 and its enzymatic reaction. ..................... 43	
   Figure 1.9	
   FcγR-mediated phagocytosis. .................................................................... 52	
   Figure 2.1     IL-10 inhibits PI-3 kinase pathway activation via SHIP1. ........................ 69	
   Figure 2.2     SHIP1 is required for IL-10’s early phase inhibition of TNFα production.  ................................................................................................................... 73	
   Figure 2.3    SHIP1 is required for IL-10 inhibition of the first peak of TNFα production but not the second. .................................................................. 75	
   Figure 2.4   IL-10 inhibits initiation of transctipional elongation ................................ 78	
   Figure 2.5   IL-10 suppression of primary response genes switches to enhancement in the absence of SHIP1 ................................................................................ 79	
   Figure 2.6	
   Absence of SHIP1 results in IL-10 enhancement of TNFα production during a specific phase of the first peak of production. ............................ 80	
   Figure 2.7	
   IL-10 requires the presence of SHIP1 in order to inhibit inflammation in an in vivo model of endotoxemia. ............................................................. 82	
   Figure 3.1	
   AQX-MN100 specifically targets SHIP1 to inhibit TNFα production. .... 97	
   Figure 3.2	
   AQX-MN100 inhibits inflammation in in vivo mouse models of inflammation. .......................................................................................... 102	
   Figure 3.3	
   The C2 domain is required for end-product allosteric activation of SHIP1 and binding of AQX-MN100 .................................................................. 107	
    xii Figure 4.1	
   Sequence alignment and phylogram of the PH-R domain. ..................... 127	
   Figure 4.2	
   SHIP1 contains an independently folded Pleckstrin Homolgy-Related (PH- R) domain. ............................................................................................... 129	
   Figure 4.3	
   SHIP1 contains an independently folded PH-R domain. ........................ 131	
   Figure 4.4	
   SHIP1’s PH-R domain preferentially binds PIP3 but does not bind the allosteric activator AQX-MN100. ........................................................... 135	
   Figure 4.5	
   The SHIP1 PH-R domain localizes to the phagocytic cup. ..................... 137	
   Figure 4.6	
   Mutation of SHIP1 PH-R domain residues K370 and K397 to alanines impairs its ability to interact with PIP3. ................................................... 141	
   Figure 4.7	
   Overlaid 1H–15N HSQC spectra of the His6-tagged wild type (blue) and K370A/K397A double mutant (red) PH-R domains at pH 5.8 and 25oC in 20 mM MES, 5 mM TCEP, and 200 mM Gdn-HCl. .............................. 142	
   Figure 4.8	
   Mutation of residues K370 and K397 to alanines in full-length SHIP1 abrogates in vitro PIP3 binding ability but does not affect its phosphatase activity. .................................................................................................... 145	
   Figure 4.9	
   SHIP1 with residues K370 and K397 mutated to alanines has impaired recruitment to the phagocytic cup. .......................................................... 149	
   Figure 4.10	
   SHIP1 with residues K370 and K397 mutated to alanines cannot restore normal regulation of FcγR-mediated phagocytosis. ................................ 150	
   Figure A.1	
   Cloning strategy for generation of inducible siRNA lentiviral vectors. . 207	
   Figure B.1	
   IL-10 activates SHIP1 ............................................................................. 209	
   Figure C.1	
   Continuous Flow Apparatus. ................................................................... 210	
   Figure D.1	
   Replicate experiments demonstrating IL-10 enhancement of TNFα production during a specific phase of the first peak of production in the absence of SHIP1. ................................................................................... 211	
   Figure E.1	
   Pelorol and AQX-016A enhance SHIP1 phosphatase activity. .............. 212	
   Figure F.1	
   AQX-MN100 specifically enhances SHIP1 phosphatase activity and has minimal off-target effects ........................................................................ 213	
   Figure G.1	
   Phosphatidyl inositol lipid binding ability of single mutant SHIP1 PH-R domains. .................................................................................................. 214	
    xiii Figure H.1	
   Double reciprocal plots of phosphatidyl inositol lipid binding ability of mutant SHIP1 PH-R domains ................................................................. 215	
                           xiv LIST OF ABBREVIATIONS  4-HT   4-hydroxytamoxifen 72-5ptase  72-kDa-5ʹ′ phosphatase AML   acute myeloid leukemia AP-1   activator protein 1 ARE   adenylate-uridylate rich element AREBP  ARE-binding protein ATF-2   activating transcription factor 2 BCL-3   B-cell lymphoma 3-encoded protein BCR-ABL  breakpoint cluster region – Abelson virus oncogene BMDM  bone marrow derived macrophages Brd4   bromodomain-containing protein 4 BSA   bovine serum albumin BTK   Bruton’s tyrosine kinase C/EBP   CCAAT-enhancer-binding protein CCR   chemokine (C-C motif) receptor (e.g. CCR2, CCR5) CD   cluster of differentiation (e.g. CD11b, CD45) CDK   cyclin-dependent kinase ChIP   chromatin immunoprecipitation CML   chronic myeloid leukemia CR3   complement receptor 3 CX3CR  CX3C chemokine receptor DAMP   danger-associated molecular pattern DAP10/DAP12 DNAX-activating protein of molecular mass 10/12 DC   dendritic cell DMEM  Dulbecco’s modified eagle medium  xv DNFB   2,4-dinitro-1-fluorobenzene Dox   doxycycline DSS   dextran sodium sulphate EC50   effective concentration (half maximal) EGR-1   early growth response protein 1 ER   estrogen receptor Erk   extracellular signal-regulated kinase ETV3   Ets (E26)-variant 3 FADD   Fas-associated death domain FcεR   Fcε-receptor FcγR   Fcγ-receptor FCS   fetal calf serum Fizz   fizzled 1 G-CSF   granulocyte-colony stimulating factor Gab1   Grb2-associated binding protein 1 GFP   green fluorescent protein GM-CFU  granulocyte/macrophage-colony forming unit GM-CSF  granulocyte/macrophage-colony stimulating factor Grb2   growth factor receptor-bound protein 2 H&E   hematoxylin and eosin HO-1   heme oxygenase 1 HPLC   high-performance liquid chromatography HSC   hemopoietic stem cell HTS   high throughput screen HuR   Hu-antigen R IBD   inflammatory bowel disease ICAM   inter-cellular adhesion molecule 1  xvi IFNγ   interferon γ IKK   inhibitor of κB  (IκB) kinase IL   interleukin (e.g. IL-10, IL-23) IP4   inositol-1,3,4,5-tetrakisphosphate Irgm1   immunity-related GTPase family M member 1 ITAM   immunoreceptor tyrosine-based activation motif ITIM   immunoreceptor tyrosine-based inhibitory motif Jak1   janus kinase 1 Jnk   c-Jun N-terminal kinase KC   keritinocyte chemoattractant KO   knockout LFA1   leukocyte function-associated antigen 1 LIF   leukemia inhibitory factor LPS   lipopolysaccharide Ly6C   lymphocyte antigen 6C LysM   lysozyme M M-CFU  macrophage-colony forming unit M-CSF  macropahge-colony stimulating factor M1   classically activated type 1 macrophage M2   alternatively activated type 2 macrophage Mac1   macrophage antigen 1 MAPK   mitogen-activated protein kinase MCP-1  monocyte chemotactic protein 1 MHC   major histocompatibility complex MIP1   macrophage inflammatory protein 1 MIR   myeloid immunoregulatory cells miRNA  micro-RNA  xvii MMP   matrix metalloproteinase MSC   myeloid suppressor cell MPO   myeloperoxidase NFAT   nuclear factor of activated T-cells NFkB   nuclear factor κ B NK   natural killer T-cell NMR   nuclear magnetic resonance NO   nitric oxide p-TEFb  positive transcription elongation factor b PAF   platelet activating factor PAMP   pathogen-associated molecular pattern PBS   phosphate buffered saline Pecam1  platelet/endothelial cell adhesion molecule 1 PH   pleckstrin homology PI   phosphatidyl inositol PI-3 kinase  phosphoinositide 3ʹ′-kinase PI-3-P   phosphatidylinositol-3-phosphate PI-3,4-P2  phosphatidylinositol-3,4-bisphosphate PI-4,5-P2  phosphatidylinositol-4,5-bisphosphate PIP3   phosphatidylinositol-3,4,5-trisphosphate PKC   protein kinase C PMΦ   peritoneal macrophage PRG   primary response gene PRR   pattern recognition receptor PRR   proline-rich region PTEN   phosphatase and tensin homolog PTPN1  protein tyrosine phosphatase, non-receptor type 1  xviii PX   phox homology RANTES  regulated on activation normal T-cell expressed and secreted RNAPolII  RNA polymerase II RT   room temperature RT-qPCR  reverse transcription – quantitative polymerase chain reaction s-SHIP   stem-cell (short) - SHIP SBNO2  strawberry notch homolog 2 Scrmb   scrambled siRNA SDS   sodium dodecyl sulphate SH2   Src-homology 2 Shc   Src-homology 2 containing protein SHIP1   SH2 domain-containing inositol 5ʹ′-phosphatase SHP-1   SH2 containing phosphatase (e.g. SHP-1, SHP-2) siRNA   small interfering RNA SMAD   Sma and Mad related protein SOCS   suppressor of cytokine signaling (e.g. SOCS1, SOCS3) Sp1   specificity protein 1 SRG   secondary response gene STAT   signal transducer and activator of transcription (e.g. STAT3) TACE   TNFα converting enzyme TCR   T-cell receptor TGFb   transforming growth factor β TH   T-cell helper (e.g. TH1, TH2, TH17) TIA-1   T-cell intracytoplasmic antigen 1 TLR   Toll-like receptor TNFα   tumor necrosis factor α TNFR   TNFα receptor  xix TRADD  TNFR associated death domain TRAF   TNFR associated factor TRIF   TIR-domain-containing adaptor-inducing interferon β TTP   tristetrapolin Tyk2   tyrosine kinase 2 UTR   untranslated region vIL-10   viral IL-10 VLA4   very-late antigen 4                    xx ACKNOWLEDGEMENTS  Foremost, I would like to thank my supervisor, Dr. Alice Mui for her mentorship, encouragement, guidance, and unwavering support.  This thesis would not have been possible without her steadfast belief in my abilities as a student and scientist for which I am eternally grateful.  My sincere thanks also to members of the Mui Lab, past and present, for all their help, support and friendship.  Many thanks to the members of my supervisory committee, Drs. Michael Gold and Ken Harder for their input, recommendations, and guidance throughout my Ph.D. studies.  I would also like to express my utmost gratitude to my program director, Dr. Vincent Duronio, for his kind help and support.  To my friends at the Jack Bell Research Centre and neighbouring research institutes, you have made the years fly by.  Thanks for the memories.  Lastly, I dedicate this thesis to my parents who have, as always, provided their unconditional love and support.   1            CHAPTER 1: INTRODUCTION               2 1.1 Inflammation Rubor et tumor cum calore et dolore – “redness and swelling with heat and pain” - were the four cardinal signs of inflammation first recorded by the ancient Roman encyclopedist, Aulus Cornelius Celsus (ca 25 – ca 50) 1.  Many years later, Rudolph Virchow (1821-1902) added a fifth sign, functio laesa (disturbance of function), which highlighted the cellular nature of the inflammatory process and is the only sign common to all presentations of inflammation 1,2.  Today, we recognize inflammation as a complex physiological process elicited in response to microbial pathogens, damaged cells, foreign cells, or chemical irritants.  The inflammatory response aims to combat and/or remove the insulting stimulus and initiate the reparative pathways leading to restoration of normal tissue structure and function.  However, despite having over two thousand years passed since the 4 cardinal symptoms were documented in Celsus’ De medicina, we are still unraveling the cellular and molecular mechanisms governing the inflammatory process. Inflammation begins with the activation of differentiated mast cells or macrophages residing in the tissues.  When these cells encounter allergens, pathogen associated molecular patterns (PAMPs) expressed on the surface of microbe 3-6, or damage associated molecular patterns (DAMPs) (intracellular molecules released into the extracellular environment by disrupted or dying cells) 7-9, they release a variety of soluble mediators.  These mediators include: cytokines such as tumor necrosis factor α (TNFα), interluekin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-12 (IL-12); chemokines such as monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP1); and chemical factors such as histamine, prostaglandins, and reactive oxygen and nitrogen species (reviewed in 10).  Collectively, these molecules  3 promote the activation of surrounding cells, the recruitment of other immune cells from circulating blood to the sites of infection or injury, and the direct killing of microbial pathogens. These molecules can also have local effects causing vasodilation and increased permeability of the local blood vessels 11-15.  This increased blood flow to the site of inflammation or infection is concurrent with the up-regulated expression of adhesion molecules on the luminal surface of the endothelial layer.  This, in turn, promotes the cell-to-cell contact between circulating leukocytes (primarily neutrophils) and endothelial cells– slowing their movement and causing them to “roll” along the endothelial wall. Once this occurs, leukocytes can then be sufficiently exposed to local inflammatory mediators – initiating cell activation, leukocyte spreading and firm adhesion to the endothelial cell lining.  Following a chemotactic gradient, leukocytes then transmigrate across the endothelial layer to the site of infection.  The recruited neutrophils, in concert with differentiated macrophages, mast cells, and plasma components such as antibodies and complement, then kill invading microorganisms or clear damaged cells and debris 11- 15. While inflammatory responses are beneficial to clear the body of invading microbes and pathogens, they must be quickly terminated to prevent inadvertent damage to normal, healthy cells and tissues.  Regulation of inflammation is achieved by several mechanisms.  One means of inhibiting inflammation is through the release of anti- inflammatory molecules such as interleukin-10 (IL-10), transforming growth factor-β (TGF- β), and glucocorticoids (reviewed in 10,16).  These molecules can function at multiple levels including: inhibiting the production of pro-inflammatory mediators,  4 inducing changes in the target cells that the pro-inflammatory mediators act upon (e.g. down-regulating the expression of a surface receptor), or interfering with the intracellular signaling pathways that pro-inflammatory mediators activate.  Altering signaling pathways, for example, can switch the conversion of arachadonic acid from pro- inflammatory prostaglandins to lipoxins.  Lipoxins can reduce vascular permeability, inhibit extravasation of neutrophils, and promote the recruitment of “healing” macrophages 16.  If these mechanisms fail to properly resolve an inflammatory response, various pathologies can arise including acute systemic inflammation in the form of sepsis 17, or chronic inflammatory diseases such as inflammatory bowel disease 18,19. 1.2 Macrophages Macrophages are the sentinels of the innate immune system whose various roles include phagocytosis of microbes, pathogens and cellular debris; activation of other cells to facilitate combat and clearance of the immune stimulus; and initiation of the pathways leading towards the development of an adaptive immune response (reviewed in 20-23). Macrophages are a component of the mononuclear-phagocyte system, which additionally includes the circulating blood monocytes from which they are differentiated and the lineage-committed, common myeloid progenitors residing in the bone marrow.  It is well recognized that macrophages are an incredibly heterogeneous population whose phenotype and function are profoundly influenced by the tissues within which they reside and the immunological microenvironment.    5 1.2.1 Monocyte/Macrophage heterogeneity Monocytes comprise approximately 5-10% of peripheral leukocytes in human blood and exhibit a high degree of variability with respect to size and granularity 24,25. Further, the identification of monocyte subsets with differential expression of cell surface markers has suggested that there may be monocyte subsets with specific physiologic functions.  One of the earliest monocyte classification schemes separated cells based on their expression of CD14, a component of the lipopolysaccharide (LPS) receptor complex, and CD16 (also known as FcγRIII) 26,27.  The CD14highCD16- and CD14+CD16+ monocytes defined in this classification were further identified to have differential expression of other surface molecules 28-32.  CD14highCD16- cells expressed CCR2 while CD14+CD16+ cells expressed higher levels of CD32 (also known as FcγRII) and CCR5 33,34.  CD14highCD16- monocytes are frequently referred to as classic “inflammatory” monocytes while CD14+CD16+ cells are considered “resident” monocytes and more closely resemble tissue-residing macrophages.  Over the years, additional subsets of monocytes have been defined including classification based on the expression of CD64 (also known as FcγRI) 35.  CD14+CD16+CD64+ monocytes produce larger quantities of TNFα and IL-6 and have greater phagocytic activity than CD14+CD16+CD64- cells and are able to stimulate lymphocytes to a greater degree than CD14highCD16- monocytes 35,36. Adding to the difficulty in characterizing distinct monocyte subsets, was the differing expression of markers between humans and mice.  It was not until 2003, when Geissmann et al. defined a murine classification analogous to the human CD14highCD16-/ CD14+CD16+ scheme 37.  Classic “inflammatory” monocytes were characterized as  6 CCR2+CD62L+CX3CR1lowLy6C+ while the equivalent of “resident” monocytes were CCR2-CD62L-CX3CR1highLy6C- (See Figure 1.1).  At the functional level, the “inflammatory” monocytes are believed to be released into the circulation from the bone marrow and are recruited by chemokines and other pro-inflammatory signals to inflamed tissues where they subsequently differentiate into mature macrophages or dendritic cells (DCs) and combat the inflammatory stimulus.  In contrast however, many questions still persist as to the origin of tissue-resident macrophages, which are believed to have multiple roles in the clearance of dead or damaged cells, repair and tissue remodeling after an inflammatory response.  Whether resident macrophages are differentiated from a specific subset of circulating monocyte, derived from the proliferation of pre-existing tissue macrophages, or if recruited “inflammatory” macrophages are capable of further differentiating into reparative “resident” macrophages, are all possibilities being investigated 38-41. Regardless of their origin,  “resident” macrophages adopt a phenotype unique and specialized to the tissue.  For example, alveolar macrophages and peritoneal macrophages express high levels of pattern recognition receptors (PRRs) and scavenger receptors 42-47, osteoclasts develop the ability to resorb bone 48, while macrophages residing in the lamina propria of the gut exhibit high phagocytic activity but low levels of secreted pro- inflammatory cytokines 49-51.      7                 Figure 1.1 The development of macrophages in mice. Monocytes are derived from a common myeloid progenitor cell that is shared with neutrophils.  Ly6C+ monocytes are released into the blood where they adopt a Ly6CMid phenotype and express CCR2, CCR7 and CCR8.  Under steady-state conditions, circulating monocytes lose their expression of Ly6C and upregulate their expression of CX3CR1.  These Ly6C- cells can migrate into the peripheral tissues to replenish resident macrophage populations.  Ly6C+ and Ly6CMid monocytes can respond to CCL2 and extravasate into the tissues towards sites of inflammation.  HSC, hemopoeitic stem cell, GM-CFU, granulocyte/macrophage colony forming unit, M-CFU, macrophage colony forming unit, CCR, CC-chemokine receptor, CCL, CC-chemokine ligand, CD, cluster of differentiation, CX3CR, CX3C-Chemokine Receptor, CX3CL, CX3C-chemokine ligand. Adapted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology] Gordon, S. & Taylor, P.R. Monocyte and macrophage heteropgeneity. Nat Rev Immunol 5, 953-964 (2005).  Copyright © (2005) 52.  8 1.2.2 Macrophage activation Circulating “inflammatory” monocytes, defined as being CCR2+ CD62L+Ly6C+, express LFA1, Mac1, Pecam1, and VLA4, which facilitate its endothelial adhesion and migration towards the site of inflammation 15.  The so-called “classical” activation of M1 macrophages is typically elicited in response to toll-like receptor (TLR) ligands (e.g. LPS) and interferon-γ (IFNγ) and yields an IL-12high, IL-23high, IL-10low phenotype53-58. Classically activated macrophages exhibit increased expression of pro-inflammatory cytokines (e.g. IL-1, IL-6 and TNFα) and reactive-oxygen species, enhanced microbicidal activity, and upregulation of major histocompatibility complex (MHC) class II and antigen presentation molecules 53-58.  However, in the presence of IL-4, IL-13, glucocorticoids, or TGFβ, macrophages become “alternatively” activated.  Unlike the M1 macrophages, these alternatively activated M2 macrophages are associated with humoral immunity and tissue repair 53-58.  They have an IL-12low, IL-23low, IL-10high, Fizz+, YM-1+ phenotype and have differing pro-inflammatory cytokine expression profiles depending on the instigating stimulus.  M2 macrophages also display enhanced endocytosis with increased expression of scavenger mannose and galactose-type receptors, and a switch in arginine metabolism from citrulline and nitric oxide (NO) production (as in M1 macrophages) to ornithine and polyamines.  The characteristic differences between M1 and M2 macrophages are described in Figure 1.2.  Much like the TH1 and TH2 paradigm in T-cells, the M1 and M2 classification of macrophage activation is an oversimplification and used more as a conceptual tool with the added appreciation that there is a far greater diversity in differentiation and activation states and that  9 macrophages may have the ability to move through these states through the course of an inflammatory event 59.                  10           Figure 1.2 Characteristics of M1 and M2 polarized macrophages. Classically activated “M1” macrophages are induced through TLR stimulation such as LPS.  They are characterized by the production of pro-inflammatory cytokines, chemokines, and reactive oxygen and nitrogen species.  M1 macrophages preferentially use transcription factors IRF5 and the p65 subunit of NFκB.  Alternatively activated “M2” macrophages are associated with immunosuppressive responses and tissue remodeling.  They produce high levels of the anti-inflammatory cytokine IL-10 and have high activity of Arg1.  M2 macrophages additionally have high expression of the chitinases Ym1 and Ym2, and the resistin-like protein, Fizz1.  M2 macrophages preferentially utilize transcription factors c-Maf, the p50 subunit of NFκB, STAT6, and C/EBPβ.  LPS, lipopolysaccharide, IFNγ, interferon-γ, iNOS, inducible nitric oxide synthase, ROI, reactive oxygen intermediates, RNI, reactive nitrogen intermediates, IL, interleukin, TNFα, tumor necrosis factor α, CXCL, chemokine (C-X-C motif) ligand, IRF, interferon regulatory factor, MHC, major histocompatibility complex, CD, cluster of differentiation, TLR, Toll-like receptor, MR, mannose receptor, SR, scavenger receptor52,53,55,58.  Adapted by permission from John Wiley & Sons Inc.: [European Journal or Immunology] Mantovani, A., et al. New Vistas on macrophage differentiation and activation. Eur J Immunol 37, 14-16 (2007).  Copyright © (2007) 52.  11 1.3 LPS Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria where it functions to structurally stabilize and protect the bacteria from certain forms of chemical attack 60,61.  LPS is a mammalian endotoxin and is a potent stimulator of the immune system by virtue of its ability to bind the CD14/TLR4/MD2 receptor complex to induce the production of pro-inflammatory cytokines and mediators 60-62. Structurally, as depicted in Figure 1.3, LPS is composed of three parts:  the O- antigen, the core oligosaccharide, and Lipid A.  The O-antigen forms the outermost domain of an LPS molecule.  It is an oligosaccharide chain whose structure and composition is specific for each bacterial strain and confers the immunogenicity of LPS. The O-antigen is covalently attached to the LPS core oligosaccharide with commonly contains multiple ketodeoxyoctocnic acids, heptose sugars, and non-carbohydrate components including phosphates and amino acids.  The core oligosaccharide, in turn, is covalently attached to Lipid A, which is typically a glucosamine disaccharide with varying numbers of fatty acid chains attached.  The fatty acid chains of Lipid A allow LPS to be inserted and anchored into the bacterial membrane.  It is the Lipid A component of LPS that interacts with TLR4 and stimulates the activation of immune cells 60-63.      12              Figure 1.3 The structure of LPS. LPS is composed of 3 regions: a hydrophilic polysaccharide O-Antigen, a core oligosaccharide, and a conserved hydrophobic Lipid A moiety.  KDO, 2-keto-3- deoxyoctonic acid, Hep, L-glycerol-D-manno-heptose, Gal, galactose, Glc, glucose, GlcNAc, N-acetyl-glucosamine, EtN, ethanolamine 61,63.  Adapted by permission from Elsevier Ltd.: [Pharmacology & Therapeutics] Fujihara, M., et al. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol Ther 100, 171-194 (2003).  Copyright © (2003) 61.      13 1.3.1 LPS/Toll-like receptor 4 signaling TLRs are one of the major families of PRRs, which recognize a variety of PAMPs including: lipopeptides (TLR1, TLR2, TLR6), double-stranded RNA (TLR3), LPS (TLR4), bacterial flagellin (TLR5), guanosine or uridine-rich single-stranded viral RNA (TLR7, TLR8), and unmethylated CpG DNA (TLR9) 64,65.  TLRs are type-1 membrane proteins with a leucine-rich extracellular domain and an intracellular signaling domain comprised of a conserved toll-IL-1 receptor (TIR) domain 65,66.  Upon receptor stimulation, the TIR domain facilitates protein-protein interactions with other TIR domain-containing adaptor proteins such as MyD88, Mal, TRIF, TRAM and SARM. Different TLRs employ various combinations of these adaptor proteins to mediate their pro-inflammatory signaling 64-68. TLR4 is the signaling receptor for LPS but additionally requires other proteins in order to properly bind its ligand.  LPS binding protein (LBP) is a serum lipid transferase that facilitates the transfer of LPS from the bacteria to the membrane glycoprotein CD14 on the surface of host cells.  CD14, in turn, presents LPS to TLR4.  MD2 is another membrane bound glycoprotein, which associates with TLR4 and also assists in LPS presentation 69-72. TLR4 is known to signal through two main pathways – the MyD88-dependent and independent pathways.  In the MyD88-dependent pathway, LPS binding promotes the interaction of the adaptor proteins Mal (also known as TIRAP) and MyD88 to TLR4. MyD88 contains a death domain, which mediates protein-protein interactions with the death domain containing IL-1 receptor associated kinase 4 (IRAK4).  At the membrane, IRAK4 becomes activated by phosphorylation and in turn, recruits IRAK1 and IRAK2 to  14 the receptor complex where they also become phosphorylated and activated.  The IRAKs then dissociate from the TLR4 receptor complex and interact with TNF receptor- associated factor 6 (TRAF6) in the cytoplasm.  TRAF6 is an E3 ubiquitin ligase that interacts with ubiquitin-conjugating enzyme (UBC13) and ubiquitin-conjugating enzyme E2 variant 1 (UEV1A), which together, promote the synthesis of lysine 63-linked polyubiquitin chains and activates TGFβ-activated protein kinase 1 (TAK1).  TAK1, a member of the MAP kinase kinase kinase (MAP3K) family, in addition with TAB1, TAB2, and TAB3 then activate kinases upstream of p38, JNK, and the IKK complex. The IKK complex is responsible for the phosphorylation of IκB, promoting its degradation and the subsequent liberation of NFκB, which translocates to the nucleus to upregulate the expression of pro-inflammatory genes.  Additional TRAF6-independent pathways have been described where, upon LPS stimulation, IRAK1 and IRAK4 activate the UBC-conjugating enzymes Pellino-1 and Pellino-2.  Pellino-1 and Pellino-2 then lysine 63-polyubiquitinate IRAK1, causing the recruitment and activation of the IKK complex 65,66,69-74. In the MyD88-independent pathway, LPS binding induces the association of TLR4 with TIR-domain-containing adapter-inducing interferon β (TRIF) via TRIF- related adaptor molecule (TRAM).  TRIF contains a C-terminal RIP homotypic interaction motif (RHIM) allowing it to bind and activate receptor-interacting protein 1 (RIP1).  RIP1, in turn, is able to activate the IKK complex leading to the expression of NFκB regulated gene products.  TRIF also contains an N-terminal region capable of binding TRAF3.  TRAF3 serves as an adaptor for the association of TRIF with TANK, TBK1 and IKKε.  These three kinases are then able to phorphorylate and activate  15 interferon regulatory factor 3 (IRF3), a transcription factor that induces the expression of type I interferon genes 65,66,69-74 (Figure 1.4).                  16                Figure 1.4 The TLR4 signaling pathway. LBP, CD14 and MD2 facilitate LPS interaction with TLR4.  Ligand binding activates the MyD88-dependent signaling pathway, which begins with recruitment of TIRAP and MyD88 to the receptor complex, which initiate signaling cascades leading to activation of the MAPKs p38 and Jnk as well as the NFκB transcrtiption factor.  LPS binding also activates MyD88-independent signaling, which starts with recruitment of TRIF and TRAM and ends with activation of IRF transcription factors.  TLR, Toll-like receptor, CD, cluster of differentiation, LBP, LPS binding protein, TRIF, TIR-domain-containing adaptor-inducing interferon-β, TRAM, TRIF-related adaptor molecule, RIP, receptor  17 interacting protein, TRAF, TNF receptor-associated factor, TBK, TANK-binding kinase, IKK, IκB kinase, IRF, interferon regulatory factor, TIRAP, TIR domain-containing adaptor protein, MyD88, myeloid differentiation primary response gene (88), IRAK, IL- 1R associated kinase, TAK, TGFβ activated kinase, TAB, Tak binding protein, MKK, Map kinase-kinase, Jnk, c-Jun N-terminal kinase, ATF, activating transcription factor, NFκB, nuclear factor κ B.  IκB, inhibitor of κB 61,73.  Adapted by permission from Elsevier Ltd.: [Pharmacology & Therapeutics] Fujihara, M., et al. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol Ther 100, 171-194 (2003).  Copyright © (2003) 61.                        18 1.3.2 Negative regulation of LPS/TLR4 signaling Although TLR4 signaling is essential for initiating protective inflammatory processes in response to an invading pathogen, its activity must be tightly controlled as exaggerated and/or sustained TLR4 signaling gives rise to various chronic and autoimmune pathologies.  There are numerous negative regulators of TLR4 signaling at every level of the signaling cascade.  This diversity in regulation allows for the fine- tuning of a cellular response so that it is tailored and appropriate for a given inflammatory stimulus.  Table 1.1 presents a summary of the reported negative regulators of the TLR4 signaling pathway.             19 Table 1.1:  Negative regulators of TLR4 signaling Cellular Compartment Protein Name Mechanism of Inhibition References Extracellular Soluble CD14 Soluble decoy.  Blocks interaction between LPS and membrane CD14. 75  Soluble MD2 Soluble decoy.  Blocks interaction between TLR4 and membrane MD2 . 76, 77  Soluble TLR4 Soluble decoy receptor.  Blocks interaction between TLR4 and CD14/MD2. 78-80 Transmembrane CD32A May inhibit TLR4 signaling by activating PI-3 kinases and preventing TIRAP recruitment by PI-4,5-P2. 81  DAP12/TREM1 Interacts with and activates PI-3 kinase and PLCγ.  Depletes PI-4,5-P2 and prevents TIRAP membrane localization. 82, 83  RP105 TLR4 homolog. Binds and sequester CD14/MD2. 84, 85  ST2 Sequesters MyD88 and TIRAP/Mal. Soluble ST2 binds to a putative receptor and suppresses NFκB binding to IL-6 promoter. 86-89  SIGIRR Inhibits MyD88 signaling.  Prevents TLR4 dimerization.  Complexes with IRAKs and TRAF6. 90-92  TRAILR Binds and stabilizes IκBα. 93 Intracellular A20 (TNFAIP3) Inhibits Ubc13 and UEV1A interactions with TRAF6 and subsequent recruitment of TAK1/Tab1 and Tab2. 94-96  ATF3 CREB family transcription factor.  Binds to acetylated histones and prevents NFκB binding. 97  β-Arrestin Binds to IκB and prevents its phosphorylation and ubiquitination.  Binds to TRAF6 and prevents its autophosphorylation and activation. 98-100  DUSP Negatively regulates MAPK activation. 101-104  FLIIH Binds TIR domains of TLR4 and MyD88 and prevents their mutual interaction. 105  FLN29 Binds TRAF6 and inhibits downstream signaling. 106  IRAK1c Splice variant.  Sequesters MyD88, IRAK2 and TRAF6 away from IRAK1. 107  IRAK-M Prevents dissociation of IRAK1/IRAK4 from the MyD88 complex and prevents the activation of TRAF6. 108-110  MyD88s MyD88 splice variant.  Forms MyD88/MyD88s dimmers that bind and sequester IRAK1. 111-113  PIN1 Binds phosphorylated IRF3 and promotes its ubiquitination and degradation. 114-116  RIP3 Inhibits RIP1 activity by sequestering it from TRIF. 117  Rab7b Small GTPase that promotes the lysosomal degradation of TLR4. 118  SARM Competitive inhibitor of TRIF. 119-121  SHP-2 Negatively regulates MyD88 independent signaling by binding to and inhibiting TBK-1. 122  SOCS1 Interacts with and potentially inhibits p65 subunit of NFκB.  Binds Mal and promotes its proteasomal degradation. 116, 123  TAG Splice variant of TRAM.  Disrupts TRAM-TRIF assocation. 124  TOLLIP Binds to TLR4 and IRAK and prevents IRAK phosphorylation. 125, 126  TRAF4 Interacts with p47Phox, TRAF6, TRIF and IRAK1 and inhibits NFκB activation. 127  TRIAD3A E3 ubiquitin ligase.  Targets RIP1, TRIF, TIRAP/Mal for proteasomal degradation. 128, 129  TRIM30α Targets TAB2/TAB3 for degradation via the lysosomal pathway. 130  20 1.4 Tumor necrosis factor α One of the hallmarks of inflammatory disease is heightened levels of pro- inflammatory cytokines such as TNFα.  TNFα was first described by Carswell et al. in 1975 as a factor in the serum of mice treated with endotoxin that was capable of inducing hemorrhagic necrosis of tumors 131.  Since that time, TNFα has become recognized as a potent activator of immune cells and as one of the earliest detected and most highly expressed cytokines in an inflammatory reaction 132-137. The TNFα gene is located on chromosome 6 (human) and 17 (mouse) and consists of 4 exons and 3 introns 138.  TNFα production is induced in a variety of cell types but most robustly in monocytes/macrophages.  In macrophages, the signaling pathways downstream of TLR4 activation that lead to increased transcription of TNFα have yet to be fully elucidated.  LPS stimulation has been reported to activate various kinases including IKK and MAPKs, which then go on to initiate the activity of multiple transcription factors including NFκB 139, AP-1 140, ATF-2 and Egr-1 141,142. Once activated, these transcription factors then bind to cis-acting DNA promoter elements upstream of the TNFα gene.  Both the signaling pathways and transcription factors employed appear to be macrophage type-specific 143.  TNFα is additionally subject to post-transcriptional mechanisms of regulation, which are dependent on the 5ʹ′ and 3ʹ′ untranslated regions (UTRs) of the TNFα mRNA 144,145.  Elements in these regions can interact with various RNA binding proteins to regulate both mRNA stability 146-153 and, in the case of 3ʹ′ UTR AU-rich elements (ARE), translation 154-160.  21 TNFα is first synthesized as a membrane-anchored, 27 kDa pre-protein which is then cleaved by TNFα-converting enzyme (TACE) to produce the mature 17 kDa protein 161-166.  Interestingly, it is believed that both membrane-bound and soluble TNFα are biologically active molecules with both shared and exclusive physiologic functions 167-174. TNFα mainly signals through two cysteine-rich receptors: TNF receptor 1 (TNFR1), which is constitutively expressed in almost all cell types; and TNF receptor 2 (TNFR2), which is expressed primarily on particular subsets of immune cells 175-178. Both receptors bind to TNFα with high affinity but with differing kinetics.  TNFR1 binds irreversibly to its ligand while TNFR2 binds with rapid association/dissociation kinetics 172,179,180.  The two receptors also differ in the structure of their cytoplasmic domains. While TNFR1 and TNFR2 both have cysteine rich extracellular domains, TNFR1 alone contains a cytoplasmic death domain capable of initiating apoptosis through the TNFR1- associated death domain (TRADD) and Fas-associated death domain (FADD) adaptor proteins 181,182.  TNFR2, alternatively, signals the transcription of cell survival genes via TNF receptor-associated factors (TRAFs) and activation of the NFκB pathway 183,184. Additionally, TNFR2 is most strongly activated by uncleaved, membrane-bound TNFα and to a lesser degree by the soluble form of TNFα 172,179,180.  The signaling pathways employed by TNFR1 and TNFR2 are represented in Figure 1.5. TNFα is produced by macrophages in response to TLR stimulation.  TNFα, in turn, is capable of activating macrophages with concomitant priming by interferon-γ (IFNγ).  IFNγ signaling promotes the nuclear sequestration of the signal transducer and activator of transcription 1α (STAT1α) and prevents it from interacting with TNFR1,  22 thereby potentiating TNFα stimulation of the NFκB pathway 185.  Similarly, it has been reported that TNFα can induce the sustained but low-level expression of Interferon-β, which synergizes with TNFα activation of the NFκB pathway and promotes the sustained expression of pro-inflammatory genes such as cxcl9, cxcl10, and cxcl11 and delayed expression of interferon response genes such as interferon response factor-1 (IRF-1) and STAT1 132.  Collectively, it is thought that these molecules establish the autocrine feedback loops that contribute to chronic inflammation such as those observed in various autoimmune diseases.             23            Figure 1.5 The TNFα signaling pathway. Ligand binding to TNFR1 recruits and activates TRADD, which then initiates a signaling cascade ending in the activation of caspases and induction of cell apoptosis.  Conversely, TNFα binding to TNFR2 recruits and activates TRAF2.  This in turn activates downstream MAPKs and NFκB leading towards the production of pro-inflammatory cytokines and cell survival.  TNFR, TNFα-receptor, TRADD, TNFR1-associated death domain, FADD, Fas-associated death domain, TRAF, TNFR-associated factor, RIP, receptor interacting protein, MKK, Map kinase-kinase, Jnk, c-Jun N-terminal kinase, AP1, activator protein 1, ATF, activating transcription factor, NFκB, nuclear factor κB, IκB, inhibitor of κB, c-FLIP, cellular FLICE-like inhibitory protein.  Adapted by permission from Macmillan Publishers Ltd: [Nature Reviews Drug Discovery] Faustman, D. & Davis, M. TNF receptor 2 pathway: drug target for autoimmune diseases. Nat Rev Drug Discov 9, 482-493 (2010).  Copyright © (2010) 178.  24 1.4.2 TNFα  in disease Despite TNFα’s integral role in defense against microbial infection, its overabundant production from macrophages has been attributed to various inflammatory diseases including IBD 186-188, sepsis 189-193, rheumatoid arthritis 194-197, and atherosclerosis 198-201.  Highlighting the central role that TNFα has in disease pathogenesis, anti-TNFα antibodies have shown clinical efficacy in the treatment of some of these conditions.  For example, in patients with Crohn’s disease or ulcerative colitis, administration of the murine anti-human TNFα monoclonal antibody, infliximab, resulted in reduced clinical inflammation and patients were able to discontinue use of standard corticosteroids 202-206.  Further, anti-TNFα antibody treatment has been reported to promote healing of the intestinal mucosa in patients who have become unresponsive to corticosteroid treatment 207,208.  Side-effects associated with anti-TNFα therapy, while rare, have been reported including an increased susceptibility to bacterial infection (particularly Mycobacterium tuberculosis), hematopoietic malignancies and disorders, congestive heart failure, and demyelinating diseases (reviewed in 209). 1.5 Transcriptional programs Stimulation with LPS causes the differential regulation of over 200 hundred genes within 1 or 2 hours 210-212.  These genes can be further grouped into clusters of genes that contribute towards a shared biological function such as phagocytosis, cell migration, and anti-microbial defense.  Genes grouped in these clusters are often subject to regulation by a defined and shared set of transcription factors.  Thus, modulated expression of groups of genes can be simultaneously coordinated based upon which transcription factors are activated.  Medzhitov and Horng 213 categorized transcription factors involved in the  25 inflammatory response into 3 categories.  Class I transcription factors are constitutively expressed as proteins and are regulated post-translationally by TLR stimulation, typically by phosphorylation events that regulate their nuclear translocation.  Class I transcription factors regulate the primary response genes (PRGs), which are rapidly induced upon stimulation.  Class II transcription factors are produced during the primary response by Class I transcription factors and regulate the expression of secondary response genes (SRGs).  Class III transcription factors are active during macrophage differentiation and are not directly regulated by inflammatory stimuli.  These transcription factors regulate the expression of genes involved in chromatin remodeling.  These 3 classes of transcription factors, in concert with covalent modifications in chromatin structure, and transcriptional co-activators and co-repressors, collectively form transcriptional programs capable of determining the type of stimulus-specific response elicited, duration of response and modes of regulation 213. 1.5.1 Primary response genes According to the transcriptional program classification of genes by Medzhitov and colleagues 213,214, TNFα is a PRG whose early increased expression following LPS stimulation is mediated by Class I transcription factors such as NFκB.  PRGs also have distinguishing characteristics with regard to their chromatin structure.  As opposed to SRGs, such as IL-6, PRGs have highly permissive chromatin in resting cells with high degrees of histone H3K4 trimethylation and H3K9 acetylation.  These covalent modifications correlate with high promoter GC content and facilitate the association of RNA polymerase II (RNAPolII) in the basal state of unstimulated cells.  When bound to PRG promoters in resting cells, RNAPolII is phosphorylated at Ser5 and is capable of  26 transcribing low levels of full-length, unspliced, unstable mRNA.  Upon LPS stimulation, bromodomain-containing protein 4 (Brd4) is recruited to the PRG promoters, which in turn recruits the P-TEFb complex composed of cyclin T1 and cyclin-dependent kinase 9 (CDK9).  The P-TEFb complex is responsible for the phosphorylation of RNAPolII at Ser2 in its C-terminal domain, which signals a switch of RNAPolII to a highly productive, elongation-competent producer of mature mRNA transcripts 213,214.  A graphical representation of PRG transcriptional elongation control is depicted in Figure 1.6. A report by Smallie et al. 215, further characterized a mechanism by which PRGs can be negatively regulated by the anti-inflammatory cytokine IL-10.  IL-10 was shown to inhibit the recruitment of the RelA (p65) component of the NFκB complex to κB sites in the TNFα promoter.  They additionally demonstrated that in a gene-specific manner, RelA recruits CDK9 to the promoters of PRGs.  Thus, by inhibiting RelA recruitment, IL-10 also inhibits the recruitment of CDK9 and the subsequent Ser2 phosphorylation of RNAPolII.  This prevents the signal switch to transcriptional elongation and inhibits the production of mature PRG mRNAs 215.       27           Figure 1.6 PRG transcriptional elongation control. In a basal state, primary response genes have GC-rich promoters that have high degrees of covalent histone modifications.  RNApolII is constitutively associated with the promoters of PRGs and is phosphorylated at Ser5, which activates low-level production of unspliced RNA.  Upon LPS stimulation, the P-TEFb complex composed of CyclinT1 and CDK9 is recruited, which phosphorylates RNAPolII at Ser2 switching it to produce high levels of mature, spliced RNA.  Secondary response genes have inaccessible chromatin and must undergo chromatin remodeling before transcription factors are capable of binding and subsequent recruitment of the transcriptional complex.  Met, methyl, Ac, acetyl, HAT, histone acetyltransferase, SP1, specificity protein 1, RNAPolII, RNA polymerase II, LPS, lipopolysaccharide, NFκB, nuclear factor κB, CDK9, cyclin dependent kinase 9, BRG1, BRM/SWI2-related gene 1, IRF, interferon-regulatory factor. Adapted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology] Medzhitov, R. & Horng, T. Transcriptional control of the inflammatory response. Nat Rev Immunol 9, 962-703 (2019).  Copyright © (2009) 213.    28 1.6 Interleukin-10 The discovery of interleukin-10 (IL-10) was first reported by researchers at the DNAX Research Institute in 1989 as a factor secreted by Th2 cells that inhibited Th1 cell cytokine production 216,217.  It was later shown that IL-10’s inhibition of T-cells and NK cells was via an indirect mechanism involving macrophages and that IL-10 was able to potently inhibit macrophage production of pro-inflammatory cytokines and chemokines, and surface expression of MHC Class II and co-stimulatory molecules 218. IL-10 is a glycosylated, 178 amino acid protein composed of an 18 amino acid signal sequence and the 160 amino acid mature polypeptide 219,220.  Structurally, IL-10 is composed of six α-helices.  Four amino-terminal helices form the core protein and two carboxy-terminal helices are necessary for protein-protein interactions with another IL-10 molecule, forming the fully-active, non-covalent, IL-10 homodimer (reviewed in 221). IL-10 is expressed by a wide variety of cells usually in response to a stimulus, such as LPS 222-225, and mediated by a number of transcription factors including Specificity protein-1 (Sp1) 226, Sp3 226,227, C/EBPs 228, and NFκB 229.  Evidence suggests that IL-10 is constitutively expressed in a number of cell types and that its expression is additionally regulated at the post-transcriptional level via mechanisms affecting IL-10 mRNA stability 230. The functional IL-10 receptor is a tetramer composed of two ligand-binding subunits, IL-10R1 (also referred to as IL-10Rα) 231,232, and two signaling subunits, IL- 10R2 (also referred to as IL-10R-β) 233,234.  IL-10R1 is expressed by almost all hemopoietic cells but most abundantly on monocytes/macrophages 222,235.  Further, while  29 IL-10R1 expression is downregulated by stimulation in T-cells, its expression is upregulated in macrophages upon activation by inflammatory stimuli consistent with IL- 10’s potent inhibitory activity on these cells 236-239.  IL-10R2 does not participate in interaction with IL-10 ligand, rather it initiates downstream signaling primarily through activation of the janus family kinases, Jak1 and tyrosine kinase-2 (Tyk2) 222,233,235,237. Unlike IL-10R1, IL-10R2 is constitutively expressed in almost all cells and its levels are unaffected by stimulation 222,232-234.  IL-10R2 is the shared signaling chain for at least 4 other cytokine receptors including IL-22R, IL-26R, IL-28R and IL-29R.  Interestingly, the pleiotropic activities of these cytokines are generally associated with pro- inflammatory responses as opposed to exerting anti-inflammatory activities, despite sharing the common IL-10R2 221,240. 1.6.1 IL-10 signaling In the current literature, it is believed that IL-10 signals solely through the canonical janus kinase (Jak)/STAT pathway 241,242.  Jak1 and Tyk2, which are constitutively associated with IL-10R1 and IL-10R2 respectively, become phosphorylated and activated upon IL-10/IL-10R binding 237,243.  In turn, these kinases then phosphorylate residues Y446 and Y496 on the intracellular domain of human IL- 10R1 (Y427 and Y477 on murine IL-10Rα) 237,244.  These phosphorylated tyrosines serve as docking sites for STAT3, which interact with IL-10R1 through its Src-homolgy 2 (SH2) domain 237,238.  STAT3 then becomes phosphorylated by the receptor-associated Jaks at Y705, which facilitates STAT3 dimerization, and S727, which enhances STAT3 transcriptional activity 237,238.  Upon dimerization and activation, STAT3 translocates to the nucleus where it interacts with STAT3 binding elements in the promoters of IL-10  30 responsive genes and thereby enhances the expression of genes associated with an anti- inflammatory response (e.g. SOCS3, HO-1, ETV3, SBNO2) and anti-apoptosis/cell-cycle progression (e.g. Bcl-3, c-Myc, Cyclins) 239,242,245-252. However, there is compelling evidence in the literature that suggests that IL-10 signals through pathways independent of the STAT3 transcription factor.  In a report published by Lang et al., STAT3 Wildtype bone marrow-derived macropahges (BMDMs) stimulated with LPS and treated with IL-10, exhibited a marginally reduced production of TNFα as compared to macrophages stimulated with LPS alone 247. BMDMs derived from STAT3flox/- LysMcre mice, however, produced significantly higher levels of TNFα when stimulated with LPS.  These higher levels of TNFα production additionally appeared to be subject to a greater fold inhibition by IL-10 than the STAT3 WT BMDMs, suggesting that IL-10 employs non-STAT3 dependent pathways to regulate TNFα expression.  Using a dominant negative STAT3, O’Farrell et al. demonstrated that IL-10 was still capable of inhibiting TNFα production in LPS- stimulated J774.1 cells 244.  In a similar study, using an adenovirally delivered STAT3 dominant negative, Williams et al. observed that STAT3 only contributed to a partial inhibition of TNFα production 242.  Further, when comparing macrophages stimulated with LPS for 1 hour and 2 hours, IL-10’s inhibition of TNFα production appears to have a greater dependence on STAT3 at longer timepoints whereas inhibition appears to be STAT3-independent at earlier, 1-hour timepoints.  In the same study, Williams et al. also demonstrated that at early timepoints, IL-10 does not require de novo protein synthesis in order to inhibit TNFα mRNA levels 242.  These data collectively suggest that there are  31 additional, STAT3-independent pathways utilized by IL-10, particularly at early timepoints. 1.6.2 IL-10 biological activity The biological activity of IL-10 is essential for regulating both the extent and duration of an inflammatory response.  Due to their high expression of IL-10R1, macrophages are generally considered to be the immune cell that is most sensitive to the effects of IL-10.  IL-10 is able to potently inhibit the production of cytokines (e.g. IL-1α, IL-1β, IL-6, IL-12, IL-18, GM-CSF, G-CSF, M-CSF, TNF, LIF, PAF) 224,253-256, chemokines (e.g. MCP1, MIP1α, RANTES, IL-8, KC) 257-260 and other soluble factors (e.g. prostaglandin E2, MMP2, MMP9) 261-267.  Additionally, IL-10 can alter the expression of molecules on the surface of macrophages. For example, it has been demonstrated that treatment of macrophages with IL-10 results in reduced expression of molecules involved in antigen presentation including MHCII and co-stimulatory molecules CD80 and CD86 268-271.  IL-10 has also been reported to inhibit the surface expression of the LPS receptor TLR4 272, as wells as CD54 (aka ICAM-1) 271 which is necessary for establishing cell-cell contacts between monocytes and endothelial cells during immune cell transmigration.  In contrast, IL-10 enhances the expression of CD16 and CD64 Fcγ-receptors (FcγR) 255,273,274, which promotes the phagocytic uptake of immunoglobulin (Ig)-opsonized microbes. IL-10’s transcriptional and post-transcriptional regulation of TNFα IL-10’s regulation of TNFα production is an area of intense research and at the molecular level, still not entirely clear.  IL-10 has been reported to regulate TNFα at both  32 transcriptional 275,276 and post-transcriptional levels 157,277,278.  With the current belief that STAT3 is required for mediating all activities of IL-10, much research has been invested in characterizing the gene products upregulated by IL-10 treatment.  IL-10 is known to induce expression of Suppressor of Cytokine Signaling 1 and 3 (SOCS1 and SOCS3) in a STAT3-depedent manner 235,248,269.  Both these proteins have been suggested to negatively regulate LPS-induced TNFα production directly by targeting and inhibiting components of the TLR4 signaling pathway 249,279 thereby preventing activation of pro- inflammatory transcription factors, as well as indirectly, by suppressing pathway components of the potentiating IFNα and IFNγ signaling pathways 280-282.  Tyrosine- protein phosphatase non-receptor Type 1 (PTPN1) is another gene upregulated by IL-10 treatment, which has been proposed to inhibit LPS-induced TNFα production due to its broad-spectrum phosphatase activity 283,284.  IL-10 has also been reported to induce the expression of transcriptional repressors such as Bcl-3 246,247, ETV3, and SBNO2 245.  Bcl- 3’s ability to associate with the p50 subunit of NFκB has been proposed as the mechanism by which it mediates IL-10’s inhibition of TNFα production.  By recruiting transcriptionally inactive p50 homodimers to the promoter of TNFα, Bcl-3 may prevent the recruitment of transcriptionally functional NFκB p50/p65 heterodimers 246,247,285,286. ETV3, which associates with the helicase co-repressor DDX20, and SBNO2, which contains a DExD/H helicase domain, are suggested to modify the chromatin structure of the TNFα promoter, thus inhibiting its transcription 245. There are also indications that IL-10 is able to regulate TNFα production via post- transcriptional mechanisms.  The TNFα mRNA contains AU-rich elements (AREs) in its  33 3ʹ′- untranslated region (UTR).  AREs confer a propensity for degradation unless the mRNA is stabilized by signals such as LPS stimulation 287.  IL-10 has been reported to inhibit translation 157 and stability 278 of TNFα mRNA by targeting the AREs.  By inhibiting the p38 MAPK/MK2 signaling axis, IL-10 is proposed to inhibit:  the stabilizing effect of ARE binding proteins (AREBPs), such as TIA-1 and HuR, and the AREBP-mediated recruitment of TNFα mRNA to actively translating polysome complexes 157,277,288.  IL-10 has also been shown to induce the expression of the mRNA destabilizing factor, tristetraprolin (TTP) 278 in a STAT3 dependent manner.  The multiple points of IL-10 regulation are depicted in Figure 1.7.            34            Figure 1.7  IL-10 inhibition of the TLR4 signaling pathway and TNFα production.  IL-10 has been reported to inhibit LPS-induced pathways at a number of points (indicated by red stars).  IL-10 and LPS both induce the expression of SOCS1 and SOCS3, which are capable of targeting proteins downstream of TLR4 (e.g. IRAK4) and IFNγR (e.g. STAT1/2) for proteasomal degradation.  IL-10 has also been reported to inhibit activation of p38 MAPK thereby preventing translation of LPS-induced pro- inflammatory proteins.  Additionally, IL-10-induced activation of phosphatases such as Protein tyrosine phosphatase nonreceptor Type I (PTPN1) and SHIP1 are capable of antagonizing the PI-3 kinase pathway, preventing activation of NFκB and transcription of pro-inflammatory mediators.  IL-10 has also been proposed to regulate LPS-induced pro- inflammatory mediators at the post-transcriptional level by influencing the proteins that regulate mRNA stability and decay, such as TIA-1, TTP and other AREBPs.  IL-10 can additionally upregulate the expression of various transcription factors such as Bcl-3, c- Maf and B-ATF, which can compete with NFκB for promoter binding.  IFN-R, interferon-receptor, STAT, signal transducer and activator of transcription, SOCS, suppressor of cytokine signaling, TLR, Toll-like receptor, CD, cluster of differentiation,  35 LBP, LPS binding protein, TRIF, TIR-domain-containing adaptor-inducing interferon-β, TRAM, TRIF-related adaptor molecule, TRAF, TNF receptor-associated factor, TBK, TANK-binding kinase, IKK, IκB kinase, IRF, interferon regulatory factor, TIRAP, TIR domain-containing adaptor protein, MyD88, myeloid differentiation primary response gene (88), IRAK, IL-1R associated kinase, TAK, TGFβ activated kinase, TAB, Tak binding protein, MKK, Map kinase-kinase, Jnk, c-Jun N-terminal kinase, ATF, activating transcription factor, NFκB, nuclear factor κ B.  IκB, inhibitor of κB, PI-3 K, Phosphoinositide-3ʹ′ kinase, Btk, Bruton’s tyrosine kinase, GSK3b, glycogen synthase kinase 3-β, TTP, tristetraprolin, TIA, T-cell intracellular antigen, MK2, MAPK-activated protein kinase-2, AREBP, AU-rich element binding protein, IP-10, interferon γ-inducible protein-10, iNOS, inducible nitric oxide synthase, RANTES, regulated on activation, normal T expressed and secreted, Bcl3, B-cell lymphoma 3, c-Maf, c- musculoaponeurotic fibrosarcoma oncogene homolog 236,283.  Adapted by permission from John Wiley & Sons Inc.: [Immunology] Williams, L.M., et al. Interleukin-10 suppression of myeloid cell activation - a continuing puzzle. Immunology 113, 281-292 (2004).  Copyright © (2004) 236.             36 1.6.3 IL-10 in disease Due to its potent anti-inflammatory actions, defects giving rise to either increased or decreased activity of IL-10 are associated with disease.  Through evolution, many pathogens have developed mechanisms of exploiting IL-10’s activity of inhibiting the immune system in order to establish a state of chronic infection.  M. tuberculosis 289,290, Candida albicans 291, Shistosoma mansoni 292, Toxoplasma gondii 293,294, Leishmania major 295 and lymphocytic choriomeningitis virus 296 are all infectious pathogens that have been demonstrated to manipulate the increased production of IL-10 from macrophages and DCs to prevent immune clearance of the infectious pathogen.  Of note, several viruses including Epstein-Barr virus and human cytomegalovirus are capable of producing viral IL-10 (vIL-10), which mimics the biological activity of mammalian IL- 10 and can inhibit immune activation 220,297. IL-10 in inflammatory bowel disease The vital importance of IL-10 in maintaining proper homeostasis is underscored by genome-wide association studies that have identified polymorphisms in both the IL-10 and IL-10R loci 298,299, which confer an increased susceptibility to the development of IBD.  These observations are consistent with the phenotype of the IL10-/- and IL10rb-/- mice which spontaneously develop colitis 234,300.  IL-10 is essential for the negative regulation of mucosal inflammation since it inhibits the infiltration and activation of leukocytes, suppresses the production of pro-inflammatory cytokines from the gut- resident immune cells, and prevents epithelial cell damage.  Further, characterization of the phenotype of IL10-/- mice revealed that the lack of IL-10 fails to suppress the production of the key cytokine, IL-23, from macrophages and DCs in the gut in response  37 to intestinal flora 301.  IL-23 is then thought to mediate many of the pathogenic responses leading to colitis.  IL-23 induces the expression of IL-17 and IL-6, which promote the development of pathogenic TH17 cells. While recombinant IL-10 has shown some efficacy in treating IBD in animal models 302,303, treating human IBD using this strategy has not been as successful 304. Several reasons for this difference have been postulated.  It is possible that systemic delivery of recombinant protein is insufficient for achieving high enough concentrations in the gut in order for IL-10 to perform its anti-inflammatory action.  To address these issues, researchers have investigated the use of lactobacilli modified to express human IL-10 as a novel means of protein delivery with indications that biologically functional levels of IL-10 are achieved to reduce Crohn’s disease activity index scores 305.   There has also been a report to suggest that systemic administration of recombinant IL-10 stimulates a pro-inflammatory response marked by an increase in IFNγ levels 306. Furthermore, patients who harbor specific single nucleotide polymorphisms in the IL- 10R may be unresponsive to IL-10 treatment 299. 1.7 The PI-3 kinase pathway The phosphoinositide 3ʹ′-kinases (PI-3 kinases) are a group of enzymes, which phosphorylate membrane phosphatidyl inositol lipids (PIs) at the 3ʹ′-hydroxyl position. The resulting 3ʹ′-PI products then act as second messengers to recruit proteins containing lipid interacting domains such as pleckstrin homology (PH) and phox homology (PX) domains.  Once localized at the membrane, these effector proteins become activated and initiate signaling cascades that mediate a broad range of cellular functions including  38 cellular activation, proliferation, growth, and motility.  Defects in PI-3 kinase pathway signaling have been implicated in a variety of diseases including cancer, inflammatory diseases, cardiovascular diseases and metabolic disorders (reviewed in 307,308). There are 8 PI-3 kinase isoforms that are categorized into 3 classes based upon their PI substrate specificities and their domain structure.  The class I PI-3 kinases are further sub-categorized into Class IA, which includes p110α, p110β and p110δ, and Class IB, comprised of p110γ.  Each of these PI-3 kinase catalytic subunits are associated with a regulatory subunit, p85 and p101 for Class IA and Class IB respectively.  While all Class I isoforms are expressed in mammalian cells, p110δ and p110γ exhibit particularly enriched expression in immune cells (reviewed in 307-312).  Class II and Class III PI-3 kinases are less well characterized and their biological roles yet to be clearly defined. Class II PI-3 kinases have been reported to be activated downstream of integrins and certain growth factor and chemokine receptors.  Class III PI-3 kinase exclusively generates PI-3-P as its product and has been reported to have roles in vesicular trafficking (reviewed in 307-312). 1.7.1  The PI-3 kinase pathway in inflammation The PI-3 kinase pathway is known to have important roles in the immune system, however, delineating its contribution to various cellular responses has been difficult for a number of reasons.  Firstly, the PI-3 kinase pathway appears to be activated by nearly all receptors on immune cells, which poses the challenging question of how such a commonly used signaling pathway can give rise to such a diverse array of biological activities in response to different stimuli.  Secondly, different PI-3 kinase isoforms are activated depending on the cell type and stimulus and may signal in isolation or in  39 parallel with other isoforms in a redundant or non-redundant fashion.  Thirdly, the tools that we have used to study the PI-3 kinase pathway have confounded our understanding due to issues with specificity, in the case of small molecule PI-3 kinase inhibitors 313-315, and compensatory actions of other PI-3 kinase isoforms, in the case of isoform-specific knock-out models 308,316. These difficulties have contributed towards the debate regarding the pro- or anti- inflammatory effects of LPS-triggered PI-3 kinase.  Several groups have demonstrated with the use of inhibitors or PI-3 kinase isoform-specific knockout cells that the PI-3 kinase pathway positively contributes to cellular activation 317-325.  In contrast, other groups, primarily through the use of pan-PI-3 kinase inhibitors, have reported that the PI- 3 kinase pathway attenuates TLR4-induced activation 326-337.  One potential source for these discrepancies is the contributing effects of autocrine IL-10 production; these would vary according to which cell types are used and the timepoint at which indices of inflammation and activation are being assessed.  Studies in our lab and others have observed that in vitro cultured BMDMs are high producers of IL-10 in comparison to more mature macrophages such as peritoneal elicited macrophages or the RAW264.7 or J774.1 cell lines 247.  It is possible that the autocrine effects of these high IL-10 levels could alter both the basal activation and responsiveness of the PI-3 kinase pathway downstream of TLR4 stimulation.  The timepoint at which measurements are made – the time post-LPS stimulation that supernatant TNFα-levels are measured, for example – is also a critical parameter to consider when interpreting the role of the PI-3 kinase pathway in TLR4 signaling.  LPS stimulation, in addition to inducing the production of pro- inflammatory cytokines like TNFα, IL-1β, and IL-12, also induces the expression of IL-  40 10 within 2 hours 247.  Thus, it is possible that the effects of autocrine IL-10 could confound measurements of pro-inflammatory cytokine production or macrophage activation taken past 2 hours of LPS stimulation and mask the positive role of the PI-3 kinase pathway in LPS signaling.  The concentration of LPS used in experiments is another possible source for the controversy.  Data from our lab suggests that macrophages respond quite differently in response to low amounts versus supra- physiological amounts of LPS, which may induce additional signaling pathways that are not biologically relevant in vivo or render cells less capable of activating the PI-3 kinase pathway in response to TLR4 agonist.  The specificity of the inhibitors used to evaluate the role of the PI-3 kinase pathway must also be considered as a source for the differing results.  Wortmannin and LY-294002 have predominantly been used to investigate PI-3 kinase contributions to TLR signaling.  It has been shown that both inhibitors have off target effects 313,314,338-340 and the advent of Class I isoform-specific PI-3 kinase inhibitors, knockout animals, and siRNA targeting strategies has demonstrated that broad-spectrum inhibitors can mask the biological role of the individual isoforms.  Of note, a report from Kevan Shokat’s group has highlighted the anti-inflammatory actions of PI-3 kinase γ, δ, and dual γ/δ inhibitors and has suggested that additional inhibition of other isoforms may decrease the inhibitory capacity of these compounds 341.  However, despite the advent of these improved isoform-specific tools, controversy still exists in the literature with conflicting reports attributing both positive and negative roles of particular PI-3 kinase isoforms to immune cell activation 319,323,342.    41 1.8  SHIP1 The PI-3 kinase pathway is tightly controlled in cells by mechanisms regulating PI-3 kinase enzyme activation as well as the activity of the PIP3 metabolizing enzymes phosphatase and tensin homolog (PTEN), which degrades PIP3 into PI-4,5-P2, and SH2 domain-containing inositol 5ʹ′-phosphatases 1 and 2 (SHIP1 and SHIP2), which degrade PIP3 into PI-3,4-P2.  Of these inositol phosphatases, SHIP1 is of particular interest with regards to immune regulation as it has been suggested that its activity is elicited only in the context of immune cell stimulation, whereas PTEN is believed to have constitutive, low-level enzymatic activity (reviewed in 343).  Further, unlike PTEN and SHIP2, which exhibit ubiquitous expression in virtually all cell types, SHIP1’s expression is predominantly restricted to hemopoeitic cells making it an attractive target for the development of immune-cell specific anti-inflammatory therapeutics. 3 independent research groups first cloned SHIP1 in 1996 as a 145 kDa protein that interacted with the Shc adaptor protein following cytokine and growth factor stimulation of immune cells 344-346.  SHIP1 contains an amino-terminal SH2 domain, a 5ʹ′- phosphatase domain, and a carboxy-terminal proline rich region (PRR) containing 2 NPXY motifs.  The central phosphatase domain performs SHIP1’s enzymatic activity of degrading PIP3 to PI-3,4-P2.  SHIP1 has been demonstrated to specifically degrade inositol-1,3,4,5-tertrakisphosphatase (IP4) in vitro 347 and PIP3 in vitro  and in vivo 348. SHIP1’s SH2 domain facilitates interactions with various tyrosine-phosphorylated proteins including FcεRI, FcγRIIa, and CD3 via immune receptor tyrosine-based activating motifs (ITAMs) 349-351, FcγRIIb via immune receptor tyrosine-based inhibition motifs (ITIMs) 352-355, Shc and Grb2 associated proteins (GABs) 344,345,356-358.  Likewise,  42 SHIP1’s PRR mediates interactions with numerous proteins capable of binding to its phosphorylated NPXY motifs including Shc, Doks 1 and 2, Grb2, and the p85α regulatory subunit of PI-3 kinase 356,359-362.  The domain structure of SHIP1 and its enzymatic reaction are illustrated in Figure 1.8.  In addition to the 145 kDa SHIP1 protein, SHIP1 variants have been reported that are generated via alternative splicing mechanisms or truncations, which effectively remove either the SH2 domain or PRR 361- 365.  Of note, a 104 kDa s-SHIP is also generated by transcription from an internal promoter between exons 5 and 6 of the SHIP1 gene.  This isoform lacks an SH2 domain and has been suggested to be necessary for pluripotent stemcell growth and survival 366- 369. While SHIP1 expression is restricted to hemopoietic cell lineages, there is evidence that the degree of SHIP1 expression can differ depending on the cell type and that these levels can contribute greatly to the biological function of different immune cell subsets.  SHIP1 expression can be regulated at multiple levels.  The SMAD transcription factors have been implicated in the transcriptional regulation of SHIP1 expression 347,370. Sly et al. have further demonstrated that LPS can induce SHIP1 expression via the autocrine production of TGFβ 371.  Additionally, SHIP1 levels are subject to post- transcriptional regulation by microRNAs, particularly mIR-155 372-376, as well as post- translational control by proteasomal degradation 377.      43          Figure 1.8 The domain structure of SHIP1 and its enzymatic reaction. SHIP1 is structurally comprised of an N-terminal SH2 domain, a central phosphatase domain and a C-terminal proline rich region containing NPXY and PXXP motifs.  PI-3 kinase catalyzes the conversion of PI-4,5-P2 to PIP3.  The lipid phosphatases PTEN and SHIP1 antagonize PI-3 kinase activity by degrading PIP3 into PI-4,5-P2 and PI-3,4-P2 respectively.  PI-3 KINASE SHIP1 PTEN  44  1.8.1 SHIP1’s biological activity SHIP1 is a negative regulator of immune cell signaling.  By opposing the PI-3 kinase pathway, SHIP1 had been demonstrated to inhibit the membrane recruitment and activation of Tec kinases 378-383, Akt, and PLCγ.  It has also been shown to inhibit the Erk, Jnk, and p38 MAPK pathways and the activation of transcription factors such as NFκB and NFAT 382,384-389.  As well, SHIP1 negatively regulates immune cell activation by virtue of its ability to act as an adaptor protein 381,390,391.  For example, by acting as an adapter between the inhibitory FcγRIIb and SHP-2, SHIP1 is able to negatively regulate the formation of the PI-3 kinase signaling complex by dephosphorylating Gab1 390. SHIP1 can also function by blocking the recruitment of other signaling enzymes such as SHP-1 to the 2B4 receptor in NK-cells and PI-3 kinase recruitment to DAP10 and DAP12 in osteoclasts and BMDMs 82,392,393. SHIP1’s role in myeloid cells Much of our knowledge with regards to SHIP1’s role in different immune cell lineages has been derived from experiments using SHIP1-/- mice and conditional SHIP1 knock-outs.  The phenotype of SHIP1-/- mice includes a shortened life-span, a Paget’s- like osteoporosis, splenomegaly, and an asthma-like syndrome (reviewed in 394).  These pathologies can be attributed to the profound defects in the myeloid cell compartment in germline SHIP-/- mice.  Enhanced osteoclast proliferation and activity results in accelerated bone resorption, myeloid proliferation in the bone marrow causes extramedullary hemopoeisis in the spleen, and myeloid cells accumulate in the lungs of SHIP1-/- mice impairing respiratory function due to increased fibrosis and deposition of Ym1 crystals 395.  45 SHIP1 is also involved in regulating myeloid cell phagocytic activity where it inhibits FcγR and CR3-mediated phagocytosis in macrophages 396,397.  Interestingly, a report by Tiwari et al. 398 demonstrated that SHIP1-mediated generation of PI-3,4-P2 was necessary for phagosome maturation and uptake of M. tuberculosis via the recruitment of the GTPase, Irgm1.  These results, in addition to other reports suggest that while SHIP1’s overall net effect may be the negative regulation of phagocytosis, its presence at the phagocytic cup may be necessary for the proper formation and membrane sealing of the mature phagosome 399,400. SHIP1-/- mice are also hypersensitive to immune stimulation such as when they are challenged with bacterial LPS.  SHIP1-/- macrophages produce elevated levels of IFNβ, IL-1β, IL-6, TNFα and reactive oxygen species 325,397,401.  This phenotype led to the discovery that SHIP1 is necessary for establishing endotoxin tolerance by inhibiting the production of IFNβ and arresting the propagation of the pro-inflammatory response 371.  Rauh et al. also made the discovery that SHIP1-/- macrophages are skewed towards an alternatively activated (or M2) phenotype, suggesting that SHIP1 is necessary for suppressing the development of these so-called “healing” macrophages 395.  Reports from William Kerr’s lab have observed the expansion of a similar macrophage subset in SHIP- /- mice that they referred to as “myeloid immuno regulatory” cells or MIR, which were able to suppress TH1 responses in Graft-Versus-Host Disease 402,403.  Weisser et al. have also demonstrated that SHIP1-/- M2 macrophages confer protection against an experimental model of colitis induced by dextran sodium sulfate (DSS) 404. It is worthy of mention that two independent groups have generated LysMCre conditional SHIP1 KO mice and have reported differing phenotypes.  In a review, Kerr  46 reports unpublished findings that LysMCre SHIP1-/- mice do not exhibit a myeloproliferative disorder, which suggests that the expansion of myeloid cells observed in germline SHIP1 KO cells is due to disturbances in the stromal microenvironment and myeloid growth factor production 405.  Leung et al., however, observe that their LysMCre SHIP1-/- mice suffer from a milder myeloproliferative disease as compared to germline SHIP1 KO mice and that the T-cells are TH17 skewed due to the increased levels of IL-6 being produced by SHIP1-deficient macrophages 406.  Leung et al. also note that their LysMCre SHIP1-/- lack suppressor macrophage cells 406.  Further studies will need to be performed in order to identify the source of these apparently disparate phenotypes. SHIP1’s role in other immune cells In vitro studies in T-cell lines have shown that SHIP1 inhibits T-Cell receptor (TCR) signaling but unlike the behaviour of SHIP1-/- myeloid cells, enhanced T-cell proliferation is not observed in germline SHIP1-/- mice 382,407-412.  Kashiwada et al. reported elevated levels of CD4+CD25+FoxP3+ regulatory T-cells (Treg) in germline SHIP1-/- mice, however, studies using mice with T-cell restricted SHIP1 deletion have suggested that the increased Treg population is not attributable to inherent T-cell defects but via a myeloid cell intermediate 405,406,413 SHIP1 has been shown to be necessary for FcγRIIB inhibition of activating signals initiated by immune complex binding to the B-Cell Receptor (BCR) 387,414. Germline SHIP1-/- mice show reduced numbers of circulating B-Cells, spontaneous formation of germinal centres and increased isotype switching towards low-affinity receptors 410,415.  These phenotypes were also observed in CD19cre conditional KO mice but like the T-cell compartment, there does not appear to be any intrinsic defect in B-cell  47 development in the bone marrow 406.  Rather, reduced circulating B-cell numbers are attributed to alterations in CD22-mediated homing of IgM+IgD+ cells from the spleen to the bone marrow 416.  SHIP1 has been reported to directly interact with CD22 ITIMs 417,418. Work by Kerr et al. have demonstrated a significant role of SHIP1 in the signaling pathways downstream of NK cell inhibitory receptors such as 2B4/CD244 392,409.  In SHIP1-/- mice, there is increased expression of 2B4 that predisposes NK cells to a hypo-responsive state.  SHIP1-/- NK cells also exhibit impaired production of IFNγ 419. Together, these effects impair NK cell mediated rejection in MHC-I mismatched bone marrow reconstitution experiments leading the authors to suggest SHIP1 knockdown as a potential strategy for bone marrow transplant therapy. In neutrophils, Nishio et al. have demonstrated that SHIP1 is required for the coordination of polarization and motility during chemotaxis in response to fMLP 420. SHIP1 has been shown to accumulate at the leading edge of the migrating neutrophil where the accumulation of PIP3 and PI-3,4-P2 is coupled to cytoskeletal rearrangements and actin polymerization. By opposing the PI-3 kinase pathway, SHIP1 has also been demonstrated to inhibit mast cell proliferation in response to FcεRI and cytokine stimulation in vitro 380,421,422.  Reconstitution of mast cell-deficient mice with wildtype and KO SHIP1 mast cells has further demonstrated that loss of SHIP1 in mast cells results in an enhanced anaphylactic allergic response characterized by mast cell hyperplasia and elevated levels of IL-6, TNFα and IL-5 423.  48 Two studies by Kuroda et al. have elegantly described the role of SHIP1 in basophils 424,425.  By suppressing IL-3 activated PI-3 kinase, particularly the p110α isoform, SHIP1 inhibits the production of IL-4 from basophils, which is capable of skewing macrophages and T-cells towards M2 and TH2 phenotypes respectively. The multiple roles of SHIP1 in immune cells are described in Table 1.2.  Table 1.2: The roles of SHIP1 in immune cells Cell Type Function References Monocytes/Macrophages Negatively regulates M-CSF induced proliferation 405,426   Inhibits Fcγ and CR3-mediated phagocytosis 396,397   Required for phagosome maturation 398   Negatively regulates oxidative burst 397,400   Inhibits pro-inflammatory cytokine production 397   Required for endotoxin tolerance 371   Required for development of marginal zone macrophages 412   Required for development of myeloid suppressor cells 402,403,427 T-Cells Negatively regulates TCR stimulation 407,408,428   Negatively regulates Treg development 413,429   Promotes TH17 development 430   Suppresses TH1 skewing 406,431 B-Cells Inhibits FcγRIIb signaling 355,414,417   Contributes to the positive selection of high-affinity receptor B-Cells in germinal centres 406   Participates in CD22-mediated homing of IgM+IgD+ cells from spleen to bone marrow 406,416,432 NK Cells Necessary for CD16-mediated cytotoxicity 433,434   Required for NKG2D and Ly49H-mediated cytotoxicity 369,392,435 Neutrophils Involved in establishing polarization necessary for chemotaxis 420 Mast Cells Negatively regulates Igε-induced degranulation 379,380,415,421- 423 Basophils Inhibits IL-4 production 42 ,425      49 1.8.2 SHIP1 in disease Mutations or altered expression of SHIP1 have been implicated in a number of human disorders.  In acute myelogenous leukemia (AML) and acute lymphoblastic leukemia inactivating mutations have been identified in the phosphatase domain and proline-rich regions of SHIP1 suggesting that SHIP1 functions as a tumour suppressor in hemopoietic progenitor cells 436.  SHIP1 has also been reported to be constitutively phosphorylated and associated with Shc in CD34- chronic myelogenous leukemia (CML) progenitors 437.  Further, inverse relationships have been noted between oncogenic BCR- ABL and KITK641E with SHIP1 indicating that reduced SHIP1 levels may be a requirement for tumour progression in CML and AML 438-440.  Heterozygosity at the 2q36 locus, the chromosomal location of SHIP1, has also been reported in a subset of patients with familial Paget-like osteoporosis supporting the animal model observations that reduced levels of SHIP1 results in increased bone resorption due to enhanced activity and proliferation of osteoclasts 441.  Patients with chronic idiopathic urticaria (hives) exhibit reduced expression of SHIP1 in their basophils, which potentiatiates FcεRI mediated histamine release and allergic responses 442,443.  SHIP1 levels are elevated in the oral mucosa of patients with chronic periodontitis, which the authors of the study propose is a compensatory mechanism to induce tolerance towards the Gram negative bacteria in the dental plaques 444.  In knockout mouse models, deficiency of SHIP1 has been implicated in the development of inflammatory bowel disease 445,446.  However, Arijs et al. have reported a significantly increased level of SHIP1 mRNA expression and SHIP1 immunohistological staining in mucosal biopsies taken from IBD patients with active  50 disease 447.  Whether these increased levels contribute to disease pathogenesis or are a compensatory reaction to increased gut inflammation has yet to be determined. 1.9 Phagocytosis Phagocytosis is the cellular ingestion of particles greater than 0.5 µm in diameter. The phagocytic process functions in two major capacities: 1) microbial killing as microbes are digested through successive phagosome/phagolysosome stages and 2) antigen presentation to cells of the adaptive immune system.  Various cell surface receptors, signaling pathways, and cytoskeletal proteins are known to be involved in phagocytosis but how all these components are coordinated to achieve particle ingestion is still an area of active research.  Numerous cell surface receptors are capable of initiating phagocytosis including the complement receptor 3 (CR3), the mannose receptor, scavenger receptors AI and AII, and Dectin I but FcγR-mediated phagocytosis has been the most thoroughly characterized (reviewed in 448-450). 1.9.1 FcR-mediated phagocytosis Opsonization of particles with immunoglobulins (Ig) marks them for rapid uptake and clearance by professional phagocytes such as neutrophils and macrophages.  The conserved Fc domain of the immunoglobulin molecule binds to its cognate Fc-receptor, initiating signaling pathways that trigger the formation of actin-dependent pseudopodial extensions around the particle being ingested as well as the coupled production of reactive oxygen species and inflammatory cytokines 451,452.  While the less abundant IgA and IgE immunoglobulins are capable of initiating phagocytosis 453,454, IgG is the primary opsonin, which is capable of binding the five isoforms of the FcγR: FcγRI (CD64), FcγRIIa (CD32), FcγRIIb1, FcγRIIb2, and FcγRIII (CD16) on the surface of phagocytes  51 450,455.  All the FcγRs can support phagocytosis with the exception of FcγRIIb, which negatively regulates phagocytosis and phagocyte activation 450,455-457.  Upon ligand binding, FcγRs cluster within cholesterol-rich microdomains and are rapidly phosphorylated within their ITAMs by Src family kinasases 458.  Phosphorylated ITAMs then serve as docking sites for Syk.  Syk, in turn, facilitates the recruitment of the Grb2 and Gab2 adaptor proteins 459, PLCγ1 and the Class I PI-3 kinases 396,460.  Generation of PIP3 by Class I PI-3 kinases, recruits a number of downstream signaling proteins involved in actin cytoskeleton rearrangement including the Rho family GTPases Cdc42 and Rac1 461-463.  Cdc42 localizes to the tips of advancing pseudopodia and stimulates localized actin polymerization through interactions with WASP 464,465.  When in its GTP- bound form, Rac activates a number of downstream proteins including Pak1, p67phox, WAVE and PI-4P-kinase, which collectively contribute towards phagosome closure 461,466-470.  Coupled to the activation of the cytoskeletal machinery are dynamic changes in the lipid composition at the phagocytic cup.  PI-4P-kinase, PI-3 kinase, PLA2, PLC, PLD, and SHIP1 are lipid modifying enzymes whose products have been demonstrated to regulate the phagocytic process either by coordinating actin polymerization or the rate at which phagocytosis occurs 349,396,397,399,400,432,450,471-477 (Figure 1.9).      52             Figure 1.9 FcγR-mediated phagocytosis. Binding of opsonized particles causes clustering of FcγRs and phosphorylation of ITAM motifs within their cytoplasmic domains.  Src family kinases and Syk are recruited and activated, which then initiate a number of downstream signaling pathways including activation of PLCγ, PLD, PKC and PI-3 kinase.  These pathways promote actin reorganization and polymerization, which is required for particle uptake and/or contributes to regulating the rate at which phagoyctosis occurs.  Ig, immunoglobulin, ITAM, immunoreceptor tyrosine-based activation motif, FcγR, Fcγ-receptor, PL, phospholipase, IP3, inositol-trisphosphate, DAG, diacylglycerol, PA, phosphatidic acid, PAP1, PA-phosphatase 1, AA, arachidonic acid, PI3K, Phosphoinositide 3ʹ′-kinase, CDC42, cell division control protein 42, ERK, extracellular signal-regulated kinase, MLCK, myosin light chain kinase.  Adapted by permission from The Journal of Leukocyte Biology: Garcia-Garcia, E. & Rosales, C. Signal transduction during Fc receptor-mediated phagocytosis. J Leukoc Biol 72, 1092-1108 (2002).  Copyright © (2002) 454.  53 1.10 Objectives and aims  The overall objective of this thesis was to further characterize the molecular mechanisms regulating SHIP1 activity and its contributions to the inhibition of macrophage activation and function.  Previous preliminary studies from our lab identified that treatment of macrophages with IL-10 resulted in a rapid increase in SHIP1 tyrosine phosphorylation.  This increase in phosphorylation correlated to enhanced SHIP1 phosphatase activity.  We hypothesized that in addition to the canonical STAT3-mediated anti-inflammatory response, IL-10 is also capable of signaling through SHIP1 in a STAT3-independent manner to inhibit the production of pro-inflammatory cytokines such as TNFα.  We also hypothesized that unlike STAT3-dependent signaling, which requires de novo gene transcription, signaling though SHIP1 would be more immediate and serve to inhibit the early phases of pro-inflammatory cytokine production.  Work from our lab also identified small molecule SHIP1 agonists that were able to enhance SHIP1 activity in in vitro phosphatase assays.  We hypothesized that these small molecule activators could be mimicking IL-10 activation of SHIP1 and sought to determine the mechanism by which these compounds regulated SHIP1 phosphatase activity.  The first aim of my thesis was to characterize IL-10’s SHIP1-dependent regulation of TNFα production.  Work performed by past students in our lab suggested that IL-10 was able to regulate TNFα mRNA post-transcriptional association with polysomes in a SHIP1-dependent manner.  However, we specifically asked whether SHIP1 was able to mediate IL-10’s negative regulation of TNFα production at the transcriptional level.  The second aim of my thesis was to characterize the mechanism by  54 which small molecule activators of SHIP1 enhanced its phosphatase activity and the resulting impact on macrophage activation.  Further, we wanted to determine the efficacy of these drugs in treating a number of inflammatory disorders using in vivo murine models.  Through bioinformatic and structural analyses of how the SHIP1 activators stimulated enzyme activity, we identified two previously unrecognized structural domains flanking SHIP1’s phosphatase domain.  The final chapter of my thesis aims to characterize the structure and function of the newly identified domain amino-terminal of the catalytic phosphatase domain.  In particular, we investigated its potential role in regulating SHIP1 activity by facilitating SHIP1’s membrane recruitment upon macrophage activation.  Together, the findings from this thesis lend deeper insight into the function of SHIP1 in macrophages and highlights that SHIP1 is subject to more mechanisms of regulation than has been previously appreciated.   In addition, we have demonstrated that small molecule activators of SHIP1 can be used to mimic the activity of IL-10 and inhibit inflammation in vivo.   55            CHAPTER 2: SHIP1 IS REQUIRED FOR MEDIATING IL- 10’S ANTI-INFLAMMATORY RESPONSE               56 2.1 Introduction IL-10 limits the magnitude and duration of the inflammatory response and loss of normal levels of IL-10 production or defects in its signaling results in immune dysfunction and inflammatory diseases such as colitis.  These defects are clearly evident as was first observed in IL-10 deficient mice, which develop spontaneous colon inflammation in response to normal gut flora 300.  In humans, polymorphisms in the IL-10 gene are associated with ulcerative colitis 298, while homozygous loss-of-function mutations in the IL-10R subunits results in early onset colitis 299.  Thus, understanding the mechanism by which IL-10 exerts its action on target cells may provide insight for the development of therapeutics to treat inflammatory disease. One of the core in vivo functions of IL-10 is inhibition of macrophage activation and it is the loss of IL-10 inhibition of myeloid cells, such as macrophages, which contributes most to the development of colitis in IL-10-/- mice 222,301.   The IL-10R in macrophages consists of a ligand-specific IL-10R1 subunit and a second subunit, IL- 10R2, which is also found in other cytokine receptor complexes 222.  Analysis of intracellular signaling pathways downstream of the receptor has lead to the suggestion that activation of the STAT3 transcription factor and expression of STAT3-regulated gene products is sufficient to mediate the anti-inflammatory actions of IL-10 276. However, as detailed in this chapter, we now show that LPS-stimulated activation of PI-3 kinase pathway signaling in macrophages is additionally countered by IL-10 activation of the SHIP1 inositol phosphatase.  SHIP1 is expressed predominantly in hemopoietic cells and negatively regulates PI-3 kinase signaling by dephosphorylating the PI-3 kinase product PIP3 365.  Robust production of pro-inflammatory cytokines, such as TNFα, is one  57 of the hallmarks of TLR stimulation of macrophages.  Using continuous flow cultures we found that LPS-induced TNFα is produced in two distinct phases, and that IL-10 differentially inhibits the first and second phases by signaling through SHIP1 and STAT3 respectively. TNFα is a PRG, as defined and classified by Hargreaves et al 214.  PRGs are a group of genes whose expression are quickly induced upon exposure to LPS.  Rapid expression of PRGs is facilitated by virtue of the GC content and epigenetic modifications at their promoters, which maintain their permissivity and allow RNApolII to be constitutively associated with them, even when cells are in a resting state 213,214,478. In un-activated cells, RNAPolII at PRG promoters is phosphorylated on Ser5 but not Ser2.  Phosphorylation of Ser2 by the P-TEFb kinase complex is necessary for successful transcription elongation and proper processing of the primary transcript 214,479,480.  We now show that IL-10 treatment reduces the amount of Ser2 phosphorylated RNAPolII bound to the TNFα promoter within minutes of IL-10 addition and that this is dependent on SHIP1.   Furthermore, we found that IL-10 inhibited the expression of 17 out of the approximate 50 LPS-induced PRGs defined by Medzhitov’s group 213,214.  These 17 include TNFα, other pro-inflammatory cytokines, chemokines, and transcription factors associated with macrophage activation.  Remarkably, in SHIP1 deficient cells, IL-10 enhanced rather than inhibited the expression of the majority of these genes.     58 2.2 Materials and methods 2.2.1 Mice Balb/c SHIP1+/+ and SHIP1-/- mice (kindy provided by Dr. Gerald Krystal, BC Cancer Research Centre, Vancouver, B.C.) used for the experiments described in this chapter were housed and maintained in accordance with ethics protocols approved by the University of British Columbia Animal Care Committee. 2.2.2 Lentiviral constructs Small interfering RNA (siRNA) constructs specifically targeting STAT3 and SHIP1, and a scrambled control siRNA sequence (Scrmb) were designed and generated using the BLOCK-iT RNAi Expression system (Invitrogen, Mississauga, ON) with a protocol modification whereby, using Invitrogen’s Gateway technology, the shRNA sequences were recombined into a modified form of the tetracycline-inducible pTRIPZ lentiviral vector (Thermo Fisher Scientific, Nepean, ON).  Please refer to Figure A.1 in Appendices for a more detailed description of the lentiviral constucts and cloning strategy.   A plasmid construct containing a modified form of human AKT was kindly provided by Dr. Megan Levings (Univeristy of British Columbia, Vancouver, B.C.).  In this construct, the PH domain is removed from AKT, a src myristoylation signal sequence is added to the amino terminal end and the steroid binding domain of the estrogen receptor (ER) and a hemagglutinin tag are added to the carboxy terminal end 481.  This AKT-ER sequence was sub-cloned into the pENTR-1A vector (Invitrogen, Mississauga, ON) and recombined into a modified lentiviral vector.  VSV-­‐pseudotyped	
  ultracentrifugation.  59 2.2.3 Cells RAW264.7 cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 9% fetal calf serum (FCS, Thermo Fisher Scientific, Nepean, ON).  To generate stably transduced cell lines expressing SCRMB, STAT3, and SHIP1 siRNA constructs and AKT-ER, RAW264.7 cells were transduced with concentrated lentivirus in the presence of 8 µg/ml of protamine sulfate (Sigma, Burlington, ON).  Transduced cells were selected according to the antibiotic resistances conferred by their respective lentiviral vectors or further purified by fluorescence activated cell sorting 24-hour doxycycline-treated cells for green-fluorescent protein (GFP) positive events.  For experiments, siRNA lentivirus- transduced cells were treated with 2 µg/ml of doxycycline (Sigma, Burlington, ON) for 48 hours prior to stimulation.  AKT-ER lentivirus-transduced cells were treated with 150 nM 4-hydroxy tamoxifen (4-HT, Sigma, Burlington, ON) prior to stimulation.  For in vitro signaling and cytokine production experiments, primary cells from male and female Balb/c SHIP1+/+ or SHIP1-/- mice aged 6-12 weeks were isolated by peritoneal lavage with 3-5 ml of sterile phosphate buffered saline (PBS, Thermo Fisher Scientific, Nepean, ON).  Cells were transferred to Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific, Nepean, ON), supplemented with 10% FBS, 10 µM β-mercaptoethanol, 150 µM monothioglycolate, and 1 mM L-glutamine, and allowed to adhere to tissue-culture treated plates for 4 hours in a 37°C, 5% CO2, 95% humidty incubator, prior to stimulation.    60 2.2.4 Immunoblot analysis of proteins Cells plated at a density of 1.5 X 106 cells per well on a 6-well tissue culture plate were stimulated with 10 ng/ml of LPS (E. coli Serotype 0111:B4, Sigma, Burlington, ON) +/- 100 ng/ml IL-10 (Recombinant murine IL-10 protein was cloned, expressed, and purified in house).  Cells were then lysed with 500 µl of Nonidet P-40 (NP-40) lysis buffer (50 mM HEPES, 2 mM EDTA, 1 mM NaVO4, 100 mM NaF, 50 mM NaPPi, 1% NP40, supplemented with Complete Protease Inhibitor Cocktail, Roche Diagnostics, Laval, QC). Lysates were rocked at 4°C for 30 minutes and clarified by centrifugation for 20 minutes at 12,000 G.  Lysates were then made 1X in Laemmli buffer, boiled 3 minutes, loaded onto 7.5% SDS-polyacrylamide gels and run under constant voltage. Alternatively, proteins were extracted using TRIzol reagent (Invitrogen, Mississauga, ON) concurrently with total RNA preparation as per the manufacturer’s protocol.  Resolved proteins were immobilized onto PVDF membrane (Millipore, Etobicoke, ON) using a semi-dry blotting apparatus (Biorad, Mississauga, ON), blocked with 3% bovine serum albumin (BSA, Sigma, Burlington, ON) in Tris-Buffered Saline, probed with specific protein or phospho- protein antibodies, detected with fluorescence-conjugated secondary antibodies, and developed using a Li-Cor Odyssey Infrared Imager (Lincoln, NE).  Antibodies used in this chapter include: anti-Phospho AKT (Ser473), anti-AKT, anti-Phospho CDK9 (Thr186) purchased from Cell Signaling Technologies (Pickering, ON), anti-SHIP1 purchased from BD Biosciences (Mississauga, ON), anti-STAT3 purchased from Upstate Biotechnology (Lake Placid, NY), and anti-Actin purchased from Sigma (Burlington, ON).   61 2.2.5 Analysis of AKT phosphorylation by flow cytometry Peritoneal macrophages (PMΦs) were treated with 10 ng/ml LPS +/- 100 ng/ml IL-10 for times indicated ranging between 10 and 30 minutes.  Stimulation media was removed and cells were immediately washed in ice-cold PBS.  Cells were incubated in Fixation/Permeabilization solution (eBioscience, San Diego, CA) for 30 minutes at 4°C, washed 2 times in permeabilization buffer (eBioscience, San Diego, CA), then incubated with anti-CD16/32 antibody (Fc Block, BD Biosciences, Mississauga, ON) for 30 minutes at 4°C.  Cells were then incubated with anti-CD11b-PE and anti-Phospho AKT (Ser473)- FITC (BD Biosciences, Mississauga, ON) for 45 minutes at 4°C.  Cells were washed 3 times in permeabilization buffer then samples were read on a BD FACS Canto and analyzed with FlowJo Version 8.7 (Ashland, OR). 2.2.6 Measurement of TNFα protein production For standard stimulations, RAW264.7 cells were plated in a 24-well plate (2.5X105 cells per well in 500 µl volume) and incubated overnight at 37°C, 5% CO2, 95% humidity in complete growth medium.  Following overnight incubation, media was replaced with fresh growth medium and stimulated with 10 ng/ml of LPS +/- 10 ng/ml of IL-10.  Cell supernatants were collected after 1, 2 and 24 hours post-stimulation and analyzed using a BD OptEIA Mouse TNFα Enzyme-Linked Immunosorbent Assay (ELISA) kit (BD Biosciences, Mississauga, ON).  AKT-ER transduced cells were similarly plated and stimulated with the added modification that cells were treated with or without 150 nM of 4-hydroxytamoxifen (4-HT, Sigma, Burlington, ON) 20 minutes prior to LPS stimulation. SCRMB, STAT3, and SHIP siRNA transduced cells were similarly plated and stimulated as RAW264.7 parental cells with the exception that these cells were treated for 48 hours  62 with 2 µg/ml doxycycline (Sigma, Burlington, ON) prior to LPS stimulation.  For PMΦ stimulation, cells were plated in a 24-well plate (2.5X105 macrophages/well) in supplemented IMDM for 4 hours prior to LPS +/- IL-10 treatment.  In continuous flow experiments, RAW264.7 parental, siRNA and AKT-ER transduced, and PMΦs were plated as per standard stimulation conditions. Cells were removed from culture media and equilibrated in Leibovitz’s L-15 media (Invitrogen, Mississauga, ON) supplemented with 9% FCS for 1 hour at 37°C with some wells being treated with 25 µM LY294002 (Sigma, Burlington, ON) for the last 30 minutes of the hour equilibration and AKT-ER cells being treated with 150nM 4-HT for the last 20 minutes of equilibration.  Following equilibration, cells were placed in a continuous flow apparatus where 37°C stimulation media was passed through a modified well inlet over cell monolayers by a syringe pump (New Era Syringe Pumps Inc., Farmingdale, NY) set to a constant flow rate of 150 µl per min.  Concurrently, cell supernatants were removed at a constant flow rate of 150 µl per minute and fractions were collected at 5-minute intervals over the course of 4 hours. Fractions were analyzed for TNFα levels using a BD OptEIA ELISA kit (BD Biosciences, Mississauga, ON) 2.2.7 Measurement of mRNA expression Cells were stimulated with LPS +/- IL-10 for 0.5 or 1 hour, lysed in TRIzol reagent (Invitrogen, Mississauga, ON) and total RNA extracted as per the manufacturer’s recommended protocol.  Purified RNA was then treated with DNase (Roche Diagnostics, Laval, QC) reverse transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON) and the resulting cDNA analyzed by Sybrgreen-based real-time quantitative PCR (RT-qPCR) using a 7300 Real-Time PCR  63 apparatus (Applied Biosystems, Foster City, CA) and gene specific primers for PRGs, and a GAPDH normalization control (See	
  sequences). 2.2.8 Chromatin immunoprecitiation Cells plated at a density of 1 X 107 cells on a 100 mm tissue culture dish were treated with vehicle, IL-10 (100 ng/ml), or LY294002 (25 μM) for 30 minutes prior to LPS stimulation for 30 minutes.  Following LPS stimulation, proteins were cross-linked by adding freshly prepared formaldehyde to a final concentration of 1% for 10 minutes at room temperature (RT).  A final concentration of 125 mM glycine was added for 5 minutes at RT to quench the cross-linking reaction.  Cells were then washed with ice-cold PBS and lysed with SDS Lysis buffer (50 mM Tris-HCl, pH 8.1, 1% SDS, 10 mM EDTA, 150 mM NaCl, and Protease Inhibitor Cocktail, Roche Diagnostics, Laval, QC) for 10 minutes on ice.  Lysates were sonicated to shear cross-linked DNA into 200-1000 bp fragments.  Insoluble materials were then removed from lysates by centrifuging samples at 10,000X G for 10 minutes at 4°C.  Clarified lysates were diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris- HCl pH 8.1, 167 mM NaCl).  Samples were then pre-cleared with Protein G Agarose beads (Sigma, Burlington, ON) that had already been pre-adsorbed with sonicated salmon sperm DNA (Invitrogen, Mississauga, ON) for 1 hour at 4°C with rotation.  Beads were removed by centrifugation at 1500X G for 5 minutes.  Supernatants were transferred to fresh microfuge tubes and 1 µg of Phospho-Ser2 RNA PolII (Abcam, Cambridge, MA) or isotype control antibody (Sigma, Burlington, ON) was added and incubated for 18 hours with rotation at 4°C.  Following overnight incubation, Protein-G Agarose beads were  64 added to the samples and incubated for an additional hour at 4°C with rotation.  Protein G beads were pelleted and washed with low-salt wash buffer (20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 0.1% SDS, 1% NP-40, 2 mM EDTA, and Protease Inhibitor Cocktail), high- salt wash buffer (low-salt wash buffer with 500 mM NaCl), and LiCl wash buffer (10 mM Tris-HCl, pH 8.1, 0.25 mM LiCl, 1% NP-40, 1% Deoxycholate, 1 mM EDTA and Protease Inhibitor Cocktail) for 5 minutes each with rotation at 4°C.  Beads were then washed with 2X 5 minute TE washes with rotation at 4°C.  Protein/DNA complexes were eluted from the beads by incubating beads in elution buffer (25 mM Tris-HCl, pH 10, 1 mM EDTA, and 1 mM CaCl2) for 15 minutes at RT with rotation.  A final 200 mM concentration of NaCl was added to samples and incubated at 65°C for 18 hours to reverse the DNA-protein crosslinks.  RNase A (Sigma, Burlington, ON) was added and incubated for 30 minutes at 37°C followed by addition of 10 mM EDTA, 50 mM Tris- HCl, pH 10, and Proteinase K and further incubation for 1 hour at 45°C.  DNA was purified using standard phenol-chloroform extraction techniques then used as template for Sybrgreen-based RT-qPCR with murine TNFα promoter-specific primers (See Table A.1 in Appedices for sequence). 2.2.9 Mouse endotoxemia model Groups of 6-8 week old Male and Female Balb/C SHIP1+/+ and SHIP1-/- mice were intraperitoneally injected with either 1 or 5 mg/kg of LPS with or without co- administration of 1 mg/kg of IL-10.  Blood was drawn 1 hour later by cardiac puncture for determination of plasma cytokine levels by ELISA.  ELISA kits were purchased from BD Biosciences (Mississauga, ON) and eBioscience (San Diego, CA) for TNFα and CCL2 respectively.  65 2.3 Results 2.3.1 IL-10 inhibits LPS activation of PI-3 kinase through SHIP1 The PI-3 kinases are a family of enzymes which are activated in response to specific extracellular signals to phosphorylate the membrane lipid PI-4,5-P2 to generate the second messenger PIP3.  PIP3 interacts with and activates PH domain-containing proteins such as the protein kinase Akt 308,310,311.  As part of signal downregulation, the PIP3 is then degraded through the action of inositol phosphatases that remove either the 3' or 5' phosphate to produce PI-4,5-P2 or PI-3,4-P2 (reviewed in 343,482,483).  Previous studies in our lab investigating the contribution of IL-10 in regulating LPS-induced PI-3 kinase signaling, observed that treatment of 32P-orthophosphate labeled J2M macrophages with LPS resulted in an anticipated increase in membrane PIP3 within 15 minutes of stimulation, as determined by methanol/chloroform lipid extraction and subsequent HPLC analysis 484-486.  In cells stimulated with LPS and IL-10, PIP3 levels were dramatically reduced with a corresponding increase in membrane PI-3,4-P2.  These initial results suggested that LPS activates PI-3 kinase to produce PIP3 and that treatment with IL-10 promotes its dephosphorylation to PI-3,4-P2. (See Figure B.1 in Appendices). Since the LPS and IL-10 induced PIP3 and PI-3,4-P2 expression patterns indicated the action of a 5'-phosphatase, we examined whether IL-10 might activate SHIP1 to antagonize the PI-3 kinase pathway.  We found that IL-10 treatment of RAW264.7 macrophage cells results in rapid phosphorylation of SHIP1 on tyrosine 1020 and this phosphorylation event required tyrosines 446 and 496 on the IL10R2 cytoplasmic domain (See Figure B.1 in Appendices).  These residues were previously described to be required for the anti-inflammatory action of IL10 and involved in recruiting STAT3 220,222,244,487.  66 However, we found that the phosphorylated, but not unphosphorylated, form of these residues can also interact with SHIP1 (See Figure B.1 in Appendices).  These data suggest that IL10R signaling includes recruitment and activation of SHIP1. Continuing this line of investigation, we then wanted to confirm if activation of SHIP1 and alterations in membrane PIP3 levels correlated with modifications in downstream PI-3 kinase pathway signaling.  We measured the phosphorylation state of Akt at residue serine 473 (S473) as an indication of Akt activation.  Consistent with the previous data, LPS induction of Akt phosphorylation and the inhibition of this by IL-10 was observed in both peritoneal macrophages (PMΦs, Figure 2.1A) and in the RAW264.7 macrophage cell line (Figure 2.1B) We next compared the ability of IL-10 to inhibit LPS-induced Akt phosphorylation and TNFα production in PMΦs isolated from wild-type (SHIP1+/+) or SHIP1 deficient (SHIP1-/-) mice.  Cells were stimulated with LPS +/- IL-10 for 0-30 minutes and Akt activation status assessed by intracellular staining with phospho-AKT S473 antibodies and subsequent analysis by flow cytometry.  In SHIP1+/+ cells, LPS treatment induced phospho-AKT S473 staining, and the presence of IL-10 reduced the induction (Figure 2.1C Left).  In contrast, IL-10 was not able to inhibit LPS-induced phospho-Akt S473 induction in SHIP1-/- cells (Figure 2.1C Right). Next, SHIP1+/+ and SHIP1-/- PMΦs were treated with LPS and 0-100 ng/mL of IL-10 for 1 hour prior to collection of culture supernatant for TNFα protein determination.   As shown in Figure 2.1D, SHIP1+/+ cells were significantly more sensitive to inhibition by IL-10 than SHIP1-/- cells with the greatest difference between  67 SHIP1+/+ and SHIP1-/- cells observed at ~10 ng/mL of IL-10.  Treatment with IL-10 also resulted in a greater inhibition of LPS-induced TNFα mRNA levels in SHIP1+/+ than SHIP1-/- cells (Figure 2.1E).  Interestingly, in SHIP1-/- cells, low concentrations (0.01 and 0.1 ng/mL) of IL-10 enhanced rather than inhibited LPS-induced TNFα mRNA levels.                68     -         5        10      15      20       25       30     -         5        10      15      20       25       30 p-AKT [Ser473] AKT LPS + IL-10LPS Time (min) B      0     10   20   30    40   60  10    20   30  40   60 LPS LPS + IL-10A C 0 10 20 30 40 0.0 2.5 5.0 7.5 10.0 *** *** *** *** *** T ime p- AK T [S 47 3] (6 G eo m et ric  M ea n)  SHIP1+/+ 0 10 20 30 40 0.00 1.25 2.50 3.75 5.00 LPS LPS + IL-10 T ime SHIP1-/- p- AK T [S 47 3] (6 G eo m et ric  M ea n)  D 0 0 25 50 75 100 125 0.1 1 10 100 * ****** ****** [IL-10] (ng/ml) %  M ax  T NF _ P ro te in 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 SHIP1 WT SHIP1 KO 0.01 0.1 1 10 100 ** ** ** ** ** [IL-10] (ng/ml) Re lat ive  T NF _  m RN A ov er  G AP DH SHIP1 WT SHIP1 KO E p-AKT [Ser473] AKT Time (min)                    Figure 2.1  69 Figure 2.1 IL-10 inhibits PI-3 kinase pathway activation via SHIP1. Immunoblot analysis of Balb/C PMΦs (A) or RAW264.7 cells (B) stimulated with LPS (10 ng/ml) or LPS + IL-10 (100 ng/ml) for the indicated times, probed with anti- Phospho-AKT (Ser473) and protein AKT (Loading Control).  (C) Flow cytometric analysis of wildtype (Left) or SHIP1-/- PMΦs (Right) stimulated with LPS (10 ng/ml) (n) or LPS + IL-10 (100 ng/ml) (Δ) for the times indicated.  Data represent differences between geometric means of treated and untreated samples gated on CD11b/CD86 positive events ± s.d. (n=3).  Geometric Means of SHIP+/+ LPS treated, SHIP1+/+ LPS + IL-10 Treated, SHIP1-/- LPS treated and SHIP1-/- LPS + IL-10 treated samples at 30 minutes were 26.0 ± 0.2, 22.8 ± 0.2, 18.6 ± 0.4, 18.2 ± 0.3 respectively.  ***p<0.001 when comparing LPS treated to LPS + IL-10 treated cells (Two-way ANOVA).  (D) TNFα Enzyme-linked immunosorbent assay (ELISA) of cell supernatants collected from SHIP1+/+ or SHIP1-/- PMΦs stimulated with LPS (10 ng/ml) and IL-10 at the various concentrations indicated for 1 hour.  Data represent mean TNFα levels as a percentage of LPS alone treated samples ± s.d. (n=3).  TNFα levels of LPS alone treated samples were 49.16 ± 2.41 and 41.69 ± 1.54 pg/ml for SHIP1+/+ and SHIP1-/- cells respectively.  * p<0.05, ***p<0.001 when comparing SHIP1 wildtype to knockout (Two-way ANOVA). (E) Real-Time quantitative PCR of cDNA prepared from SHIP1+/+  (n) or SHIP1-/- (Δ) PMΦs stimulated for 1 hour with LPS (10 ng/ml) with or without IL-10 at the concentrations indicated.  Data represent mean TNFα expression levels relative to GAPDH ± s.d. (n=3). *p<0.05, **p<0.01 when comparing SHIP1 wildtype to knockout (Two-way ANOVA).              70 2.3.2 Macrophage production of TNFα occurs in two phases To probe the relative contribution of STAT3 and SHIP1 to IL-10’s inhibition of LPS-induced TNFα production, we generated RAW264.7 cell lines in which STAT3 and SHIP1 protein expression were reduced by RNA silencing.  siRNA sequences targeting STAT3, SHIP1 or a scrambled sequence were cloned into a lentiviral vector which contained microRNA (miRNA)-like processing elements to express these siRNA sequences in the context of a doxycycline (Dox) regulated promoter (See Figure A.1 in Appendices).  As shown in Figure 2.2A, the addition of Dox to the SHIP1 siRNA knock- down cells inhibited SHIP1 protein expression by 98% and reduced STAT3 expression by 57% in STAT3 siRNA knock-down cells.  Several targeting sequences for STAT3 were tested, however this was the greatest degree of knockdown that could be achieved, perhaps because STAT3 appears to be necessary for cell survival. These siRNA-transduced cells were treated for 48 hour with Dox, then stimulated with LPS + IL-10 for 1, 2 and 24 hours.   TNFα levels in the culture supernatant were determined and graphed in Figure 2.2B as a percent of the TNFα levels in parallel cultures stimulated with LPS alone. We found that IL-10 inhibited TNFα protein expression similarly at all timepoints in parental and control scrambled (Scrmb) siRNA expressing cells.  Consistent with the reported resistance of macrophages from STAT3 deficient mice to the inhibitory effect of IL-10 238, we observed IL-10 hypo- responsiveness in the STAT3 siRNA knockdown cells, but notably, this was only observed at the longer 2 and 24-hour timepoints.  At 1 hour, the STAT3 knockdown cells responded to IL-10 equally well as the parental or SCRMB siRNA expressing cells.  71 Conversely, SHIP1 knock-down cells were hypo-responsive to IL-10 at the 1 and 2 hr timepoints, but responded normally to IL-10 at 24 hours. We investigated the kinetics of IL-10 inhibition of TNFα protein more precisely using a continuous-flow culture system.  This apparatus (See Figure C.1 in Appendices) allows for cytokine production to be monitored over a continuous period of time.  Figure 2.3 shows that LPS induces two peaks of TNFα protein expression with the first peaking around 50 minutes and the second peaking around 110 minutes.  This bi-phasic TNFα production profile was observed in all 4 RAW264.7 cell types (parental, SCRMB siRNA, STAT3 siRNA and SHIP1 siRNA, Figure 2.3A), as well as in SHIP+/+ PMΦs (Figure 2.3B).  The 2nd peak of TNFα production may result, in part, from the action of autocrine factors produced during the first 90 min of stimulation since diluted culture supernatants from cells treated with low-dose LPS (0.25 ng/ml) collected at 45 or 90 minutes stimulated TNFα production when added to naïve cells (Figure 2.3C). IL-10 inhibited both peaks of TNFα production in:  parental and SCRMB siRNA expressing RAW264.7 cells, and SHIP1+/+ PMΦs.  However, only the 1st peak was inhibited by IL-10 in STAT3 knockdown cells.  Alternately, IL-10 did not inhibit the 1st peak of TNFα in either SHIP1 siRNA-expressing RAW264.7 cells or SHIP1-/- PMΦs. We also examined the effect of adding the PI-3 kinase inhibitor LY294002 and found that it profoundly inhibited both peaks of LPS-induced TNFα (Figure 2.3D).  Collectively, these data demonstrate that IL-10 inhibits the initial phase of LPS-stimulated TNFα production through a SHIP1-dependent mechanism.  Interestingly, inhibition of the 2nd peak was impaired in both SHIP1 and STAT3 knock-down cells, suggesting that although IL-10 can use SHIP1-independent (i.e. STAT3-dependent) mechanisms to  72 inhibit the 2nd phase of TNFα protein expression, the degree of inhibition is impaired in the absence of 1st phase inhibition. Since IL-10 inhibits Akt activation through SHIP1, we hypothesized that ectopic activation of Akt would overcome the inhibitory action of IL-10.   To test this, we expressed an Akt-estrogen receptor (ER) fusion protein in RAW264.7 cells.  The addition of 4-hydroxytamoxifen (4-HT) activates Akt-ER by displacing Hsp90 bound to the ER ligand-binding domain, relieving steric hindrance and allowing the constitutively active Akt access to its substrates 481,488.  We found that IL-10 could not inhibit TNFα protein (Figure 2.3E) expression in 4-HT treated Akt-ER cells.  4-HT did not alter the IL-10 responsiveness of parental RAW264.7 cells (data not shown).  Interestingly, the presence of IL-10 enhanced TNFα production in 4-HT treated Akt-ER cells.           73          Figure 2.2  SHIP1 is required for IL-10’s early phase inhibition of TNFα production. (A) Immunoblot analysis and densitometry quantification of RAW264.7 Parental cells and cells stably transduced with inducible lentiviral constructs containing a scrambled siRNA sequence (SCRMB), or siRNA sequences targeting STAT3  (STAT3) or SHIP1 (SHIP1), cultured in the presence or absence of doxycycline (2 µg/ml) for 48 hours.  Blot was probed with anti-SHIP1 and anti-STAT3.  Data are representative of 3 independent experiments.  (B) TNFα ELISA of cell supernatants from RAW264.7 Parental or SCRMB, STAT3 or SHIP1 siRNA transduced cells cultured in the presence of doxycycline (2 µg/ml) for 48 hours then treated with LPS (10 ng/ml) or LPS + IL-10 (100 ng/ml) for the times indicated.  Data represent mean TNFα levels as a percentage of LPS alone treated samples ± s.d. (n=3). TNFα levels of LPS alone stimulated samples were 1.59 ± 0.20, 5.09 ± 0.13, and 56.43 ± 0.91 ng/ml for RAW264.7 parental cells at 1, 2 and 24 hours respectively, 1.16 ± 0.015, 4.02 ± 0.10,and 55.55 ± 0.093 ng/ml for SCRMB siRNA cells at 1, 2 and 24 hours respectively, 0.94 ± 0.013, 2.84 ± 0.073, 47.2 ± 0.091 ng/ml for STAT3 siRNA cells at 1, 2 and 24 hours respectively, and 1.27 ± 0.018, 3.07 ± 0.054, and 51.4 ± 0.37 ng/ml for SHIP1 siRNA cells at 1, 2 and 24 hours respectively. ***p<0.001 when comparing siRNA transduced cell lines to RAW264.7 parental cells. (Two-Way ANOVA)  74 A B 0 50 100 150 200 250 0 50 100 150 200 LPS LPS + IL-10 [T NF _ ] ( pg /m l) 0 50 100 150 200 250 0 100 300 500 700 0 50 100 150 200 250 0 100 200 300 0 50 100 150 200 250 0 100 200 300 400 500 Time (min) Time (min) Time (min) Time (min) Parental SCRMB STAT3 SHIP1 0 50 100 150 200 0 500 1000 1500 2000 0 50 100 150 200 0 250 500 750 1000 Time (min) Time (min) [T NF _ ] ( pg /m l) SHIP1 WT SHIP1 KO LPS LPS + IL-10 Unstim . LPS LPS D il. 45 m in. Fraction D il. 90 m in. Fraction D il. 0 500 1000 1500 2000 2500 [T NF _ ] ( pg /m l) C [T NF _ ] ( pg /m l) 0 50 100 150 200 250 0 500 1000 1500 LPS LPS + LY294002 0 50 100 150 200 250 0 250 500 750 1000 [T NF _ ] ( pg /m l) [T NF _ ] ( pg /m l) Time (min) Time (min) LPS + 4-HT LPS + IL-10 + 4-HT D E * *** 7500                  Figure 2.3  75 Figure 2.3 SHIP1 is required for IL-10 inhibition of the first peak of TNFα production but not the second. (A) TNFα ELISA of fractions collected from RAW 264.7 Parental or SCRMB, STAT3 or SHIP1 siRNA transduced cells treated with doxycycline (2 µg/ml) for 48 hours prior to continuous-flow apparatus stimulation with LPS (10 ng/ml) (n) or LPS + IL-10 (10 ng/ml) (Δ).  Data represent TNFα concentrations of each 5 min fraction over the course of 4 hours stimulation.   Data are representative of 2 independent experiments.  (B) TNFα ELISA of fractions collected from SHIP1+/+ (Left) or SHIP1-/- (Right) PMΦs stimulated with LPS (10 ng/ml) (n) or LPS + IL-10 (10 ng/ml) (Δ).  Data represent TNFα concentrations of each 5 min fraction over the course of 4 hours stimulation.   Data are representative of 2 independent experiments.  (C) RAW264.7 macrophages were stimulated with low dose LPS (250 pg/ml), a 1:100 LPS dilution (2.5 pg/ml, LPS Dil.) or 1:100 dilutions of continuous-flow fractions collected at 45 and 90 minutes following stimulation with 250 pg/ml of LPS (45 min. Fraction Dil. and 90 min. Fraction Dil., respectively).  Cells were stimulated overnight and supernatants analyzed for TNFα production by ELISA. (D) TNFα ELISA of fractions collected from RAW 264.7 Parental cells treated with LY-294002 (25 µM) for 30 minutes prior to continuous flow apparatus stimulation with LPS (10 ng/ml) + Vehicle (n) or LPS + LY294002 (Δ).  Data represent TNFα concentrations of each 5 min fraction over the course of 4 hours stimulation in the continuous flow apparatus.  Data are representative of 2 independent experiments.  (E) TNFα ELISA of fractions collected from AKT-ER transduced RAW264.7 cells treated with 4-HT (150 nM) for 20 minutes prior to continuous-flow apparatus stimulation with LPS (10 ng/ml) (n) or LPS + IL-10 (10 ng/ml) (Δ).  Data represent TNFα concentrations of each 5 min fraction over the course of 4 hours stimulation in the continuous flow apparatus.  Data are representative of 2 independent experiments.         76 2.3.3 IL-10 inhibits Ser2 phosphorylation of RNA polymerase II associated with the TNFα promoter Hargreaves et al. classified TNFα as belonging to a subclass of PRGs, termed PRG-I, whose expression are controlled by signals initiating transcriptional elongation rather than through the classical mechanism of RNAPolII recruitment to gene promoters 214.  PRG-I genes have pre-assembled RNAPolII at their promoters and a high degree of H3K4 trimethylation and H3K9 acetylation in basal, resting cells.  RNAPolII associated with PRG-I promoters is phosphorylated at Ser 5 (but not Ser 2) and supports the production of low levels of full-length, unspliced transcripts which fail to make mature, protein-coding mRNAs.  Stimulus-induced phosphorylation at Ser 2 by the P-TEFb kinase then activates RNAPolII transcriptional elongation and processing of functional PRG-1 mRNAs.  This preassembly of RNAPolII at PRG-I promoters is thought to allow rapid mRNA expression of these genes.  Since IL-10 appears to regulate the rapid, initial phase of TNFα expression through SHIP1, we examined whether IL-10 controlled RNAPolII Ser 2 phosphorylation and whether this occurred in a SHIP1 dependent manner. Parental or SHIP1 siRNA-expressing cells were stimulated for 15 minutes with LPS +/- IL-10 prior to genomic DNA extraction for chromatin immunoprecipitation (ChIP) with an antibody against the Ser2 phosphorylated form of RNAPolII and qPCR primers specific for the TNFα promoter region.  As shown in Figure 2.4A, LPS stimulated an increased amount of phospho-Ser2 RNAPolII associated with the TNFα promoter in both parental and SHIP1 siRNA cells.  IL-10 reduced this to levels comparable to un-stimulated cells in parental cells but not SHIP1 knock-down cells.  In  77 contrast, treatment with LY294002 reduced the amount of phospho-Ser2 RNAPolII associated with the TNFα promoter in both parental and SHIP1 siRNA-expressing cells. We next determined whether IL-10 regulated RNAPolII Ser2 phosphorylation by altering the activity of the P-TEFb complex.  Using a phospho-specifc antibody for CDK9, the catalytic component of the P-TEFb complex, we observed that IL-10 treatment of LPS-stimulated macrophages was able to inhibit CDK9 phosphorylation at Thr186 in parental RAW264.7 cells but SHIP1 siRNA-expressing cells appeared resistant to this inhibition (Figure 2.4B).  However, CDK9 phosphorylation was inhibited in both parental and SHIP1 knock-down cells when treated with LY294002 (Figure 2.4C). Together, these data suggest that PI-3 kinase activation is upstream of CDK9 phosphorylation of RNAPolII and that IL-10 inhibition of Ser 2 phosphorylation during at least the first 15 minutes of LPS stimulation is mediated by SHIP1. We then examined whether other LPS-induced PRGs were inhibited by IL-10 through a SHIP1-dependent mechanism.  Of the PRG-Is defined by Hargreaves et al. in BMDMs 214, 17 (including TNFα) were induced by LPS in both PMΦs and RAW264.7 cells, and inhibited by IL-10 in SHIP1+/+ PMΦs, parental and Scrmb siRNA-expressing RAW264.7 cells (Figures 2.5A and 2.5B).   However, IL-10 increased rather than inhibited the expression of 16 of these PRGs (including TNFα) in SHIP1-/- PMΦs and 11 PRGs in SHIP1 knock-down RAW264.7 cells.   We investigated the IL-10 enhancement of TNFα expression in SHIP1-/- PMΦs further and found that the increase in mRNA was reflected in increases in protein during a specific phase of TNFα production around 100 minutes after LPS stimulation (Figures 2.6A, 2.6B and Figure D.1 in Appendices).  78             Figure 2.4  IL-10 inhibits initiation of transctipional elongation (A) Chromatin Immunopreciptation analysis of RAW264.7 parental or SHIP1 siRNA transduced cells treated with LY-294002 (25 µM) or vehicle for 30 minutes prior to stimulation with LPS (10 ng/ml) with or without IL-10 (10 ng/ml) using antibody that recognizes phosphorylated RNAPolII at Ser2.  Data represent mean TNFα promoter association with Phospho Ser2 RNA PolII relative to GAPDH promoter association ± s.d. (n=3) as determined by Real-time quantitative PCR using promoter specific primers. ***p<0.001 when comparing to LPS stimulated cells (One-way ANOVA).  (B) Immunoblot analysis of RAW264.7 parental or SHIP1 siRNA cells stimulated with LPS (10 ng/ml) with or without IL-10 (10 ng/ml) for the indicated times, probed with anti- Phospho CDK9 (Thr186) and actin (Loading Control).  (C) Immunoblot analysis of RAW264.7 parental or SHIP siRNA cells treated with LY-294002 (25 µM) or vehicle for 30 minutes prior to LPS (10 ng/ml) stimulation of 1 hour, probed with anti-Phospho CDK9 (Thr186) and actin (Loading Control).  79                Figure 2.5  IL-10 suppression of primary response genes switches to enhancement in the absence of SHIP1 SHIP1 WT or KO PMΦs (A) and RAW264.7 parental, SCRMB, and SHIP1 siRNA expressing cells (B) were treated with LPS (10 ng/ml) with or without IL-10 (100 pg/ml) for 30 minutes.  Data represent mean mRNA expression relative to GAPDH as determined by RT-qPCR and values expressed as a percentage of LPS alone treated samples ± s.d. (n=3).  80              Figure 2.6 Absence of SHIP1 results in IL-10 enhancement of TNFα production during a specific phase of the first peak of production. (A) TNFα ELISA of fractions collected from SHIP1+/+ and SHIP1-/- PMΦs stimulated with LPS (1 ng/ml) with or without IL-10 (100 pg/ml) under continuous-flow conditions. Mean TNFα levels in fractions collected at 100 minutes post-stimulation from 3 independent experiments are presented in (B) ***p<0.001 when comparing to LPS stimulated cells (Two-way ANOVA).    81 2.3.4 IL-10 inhibition of TNFα and CCL2 expression in mice requires SHIP1 To study the contribution of SHIP1 to IL-10 action in vivo, we compared the ability of IL-10 to inhibit LPS-induced increases in two PRG-Is, TNFα and CCL2, in SHIP1+/+ and SHIP1-/- mice.  Since SHIP1-/- mice are hypersensitive to LPS, we first determined the dose of LPS required in each of the two strains of mice to give similar levels of serum TNFα and CCL2.    As shown in Figures 2.7A and 2.7B, injection of 5 mg/kg and 1 mg/kg of LPS into SHIP1+/+ and SHIP1-/- mice respectively, induces similar levels of TNFα and CCL2 detectable in serum.  Co-administration of IL-10 at 1 mg/kg reduced TNFα and CCL2 in the SHIP1+/+ but not the SHIP1-/- mice.   These findings are consistent with our in vitro observations that TNFα and CCL2 are both PRGs whose transcription are regulated by IL-10 in a SHIP1-dependent manner.           82          Figure 2.7 IL-10 requires the presence of SHIP1 in order to inhibit inflammation in an in vivo model of endotoxemia. TNFα (A) and CCL2 (B) ELISA of serum samples prepared from wildtype or SHIP1-/- mice intra-peritoneally injected with LPS or co-administered LPS + IL-10 at the concentrations indicated for 1 hour.  Data represent means of n=6.  **p<0.01 when comparing to LPS alone stimulated mice (One-way ANOVA)        83 2.4 Discussion The anti-inflammatory actions of IL-10 are essential for maintaining proper immune homeostasis.  In its absence, stimulatory pathways are left unchecked and lead to various acute and chronic pathologies 221.  It is currently believed that the transcription factor, STAT3, mediates all of IL-10’s anti-inflammatory signaling where it upregulates the expression of various genes whose products then go on to inhibit pro-inflammatory pathways at the level of transcription 242,276.  This can be regarded as a relatively lengthy process, requiring translocation of STAT3 from the membrane to the nucleus, de novo gene transcription, and translation of the nascent polypeptide.  However, data from our lab and others have shown that IL-10 is able to inhibit LPS-induced production of pro- inflammatory cytokines, such as TNFα, within the first 30 minutes of IL-10 treatment (data not shown).  This time scale is not in agreement with STAT3-exclusive regulation. We thus investigated other signaling pathways that could potentially mediate IL-10’s early phase anti-inflammatory activity.  In this chapter, we characterize IL-10’s signaling through the lipid phosphatase SHIP1, a key negative regulator of the PI-3 kinase pathway. Using regulation of TNFα production as a model for similar pro-inflammatory PRGs as described by Hargreaves et al. 214, we made the observation that TNFα production occurs in two phases within the first 2 hours of LPS stimulation.  Of note, a bi-phasic profile for TNFα production has also been reported for macrophages stimulated by Staphylococcal enterotoxins B (SEB) with similar kinetics 489.  Whereas STAT3 was required for mediating IL-10 inhibition during the second phase of TNFα production, it was not necessary for IL-10 mediated inhibition of the first peak of TNFα.  In contrast,  84 the presence of SHIP1 was required for inhibition of the first peak of TNFα.  These results provide evidence for STAT3-independent signaling pathways utilized by IL-10, which predominate during IL-10’s early phase anti-inflammatory action. To further characterize the mechanism by which SHIP1 was mediating IL-10’s early phase regulation, we compared downstream signaling pathways between parental RAW264.7 macrophages and SHIP1 siRNA expressing macrophages.  Using ChIP and phospho-specific antibodies, we determined that in the presence of SHIP1, IL-10 inhibited CDK9 phosphorylation, which prevented Ser2 phosphorylation on RNAPolII. TNFα transcription is not regulated by RNAPolII recruitment to the tnfα promoter, rather, it is constitutively associated with the promoter in resting cells 213. Phosphorylation of RNAPolII at Ser2 is required for signaling a switch from basal, low- level production of full-length non-spliced transcripts to high levels of mature, full- length, spliced mRNAs.  Smallie et al. have reported a unique requirement for NFκB binding motifs within TNFα’s 3ʹ′ UTR that is necessary for both LPS induction of TNFα and IL-10’s inhibition of TNFα transcription 215.  These studies demonstrated that IL-10 acts as a general block of RelA (the p65 subunit of NFκB) recruitment to TNFα’s 3ʹ′ UTR and thus prevents NFκB-dependent CDK9 recruitment.  Whether SHIP1 is also necessary for mediating IL-10’s inhibition of NFκB recruitment is an intriguing hypothesis worth pursuing in future investigation. Medzhitov’s group has classified a number of genes induced upon LPS- stimulation based upon the transcription factors and chromatin modifications that regulate their expression 213,214.  For PRGs, TLR stimulation post-translationally activates  85 transcription factors such as NFκB and IRFs, which bind to promoter regions of genes that are enriched with histone H3K4 trimethylations and H3K9 acetylations.  These covalent modifications in chromatin structure maintain the promoter regions in a permissive state allowing constitutive association of RNAPolII and thus rapid transcription upon cell stimulation.  SRGs, on the other hand, are regulated by transcription factors that are de novo transcribed and translated upon TLR stimulation, such as ATF3 and C/EBPδ.  These transcription factors then bind to promoter elements and recruit chromatin modifying enzymes and the transcriptional machinery.  Due to these limitations, SRG expression typically occurs at later timepoints (2-8 hours post- stimulation). 17 of the genes classified by Hargreaves et al. 214 as PRGs were both upregulated by LPS and inhibited by IL-10 in SHIP1 WT PMφs, Parental RAW264.7 cells and RAW264.7 cells expressing a control scrambled siRNA sequence. In SHIP1 KO PMφs or SHIP1 specific siRNA expressing cells, however, treatment with 100 pg/mL IL-10 had the reverse effect on the majority of these IL-10 regulated genes in that their expression was enhanced.  These findings were surprising as IL-10 is the prototypical anti- inflammatory cytokine and the capacity for IL-10 to stimulate macrophage pro- inflammatory responses has never before been directly addressed in the literature.  It is worthy of note that IL-6, an archetypal pro-inflammatory cytokine, has conversely been reported to switch to an anti-inflammatory response when SOCS3 is deleted 490. Although STAT3 is activated downstream of both the IL-6 and IL-10 receptors, SOCS3 only binds to the IL-6R.  Consequently, IL-10R stimulation results in sustained STAT3 activation while IL-6R stimulation transiently activates STAT3 before being targeted for  86 proteasomal degradation by SOCS3.  Akin to SOCS3 being the molecular switch determining whether IL-6 exerts a pro or anti-inflammatory effect, SHIP1 may be a molecular determinant of whether IL-10 acts as an anti-inflammatory or pro- inflammatory agent. We also investigated the contribution of SHIP1 to IL-10 activity in vivo.  The presence of SHIP1 was necessary for mediating IL-10’s inhibition of two PRGs, TNFα and CCL2, in a mouse model of endotoxemia.  These results suggest that despite having intact STAT3, the lack of SHIP1 renders SHIP-/- mice incapable of suppressing the initial phase of TNFα production.  Furthermore, inhibition of this early phase appears to be necessary for IL-10’s attenuation of subsequent phases of pro-inflammatory cytokine production via STAT3. In summary, we have demonstrated the existence and biological relevance of STAT3-independent signaling pathways utilized by IL-10 through SHIP1.  This regulatory pathway predominates during the early phases of IL-10 signaling and acts to suppress the expression of a sub-set of PRGs by preventing their transcription by RNAPolII.  In so doing, IL-10 is able to temper and fine-tune the activity of a transcriptional program in a more expedient manner than would be achievable by STAT3-mediated transcriptional regulation alone.  Thus, targeted manipulation of SHIP1 activity may represent a potential strategy for treatment of IL-10 deficiency-related diseases.    87          CHAPTER 3: ALLOSTERIC ACTIVATION OF SHIP1 INHIBITS INFLAMMATION               88 3.1 Introduction As described in the previous chapter, in response to extracellular signals, PI-3 kinase becomes activated to phosphorylate PI-4,5-P2 within the plasma membrane to generate PIP3.  PIP3 then initiates a cascade of downstream signaling pathways by interacting with PH domain-containing proteins, such as Akt, that regulate cellular activation, proliferation or survival, depending on the cell type and stimulus 491.  Cellular levels of PIP3 are normally tightly regulated by modulation of: (i) PI-3 kinase activity, (ii) the 5′inositol phosphatases SHIP1, SHIP2, and 72-kDa 5′-phosphatase (72-5ptase) and (iii) the 3′inositol phosphatase PTEN 492-494.   Of these, SHIP1 is unique in that its expression is restricted mainly to immune and hemopoietic cells 344,492.  SHIP1’s role in immune cell homeostasis is shown both by the myeloproliferative syndrome observed in SHIP1-/- mice, as well as the hypersensitivity of SHIP1-/- mice and cells to immune stimulation 371,411.  SHIP1 mediates signaling from the inhibitory FcγRIIB receptor 495, and is important in terminating signal transduction from activating immune/hemopoietic cell receptor systems 365.  Diminished SHIP1 activity or expression has been observed in human inflammatory diseases 443 and hemopoietic malignancies 440,496-498. Since dysregulated activation of the PI-3 kinase pathway contributes to inflammatory/immune disorders and cancer, much effort has been invested into the development of inhibitors of PI-3 kinase itself, as well as downstream protein kinases 499- 504.  The precedent for discovery and biological efficacy of kinase inhibitors is well established and a number of promising new PI-3 kinase isoform specific inhibitors have recently been developed and used in mouse models of inflammatory disease 505-508 and glioma 509 with minimal toxicities.  However, because of the dynamic interplay between  89 phosphatases and kinases in regulating biological processes, inositol phosphatase activators may provide an alternate and complementary approach to inhibit PIP3 levels (discussed in Knight and Shokat 502).   Of the four phosphatidylinositol phosphatases that have been reported to degrade PIP3, SHIP1 is a particularly ideal target for development of potential therapeutics for treating immune and hemopoetic disorders because its hemopoietic-restricted expression would limit their action to only these cell types. Having observed in the experiments described in Chapter 2 that IL-10 signals through SHIP1 to mediate its early-phase anti-inflammatory action, we hypothesized that activating SHIP1 using small-molecule agonists could mimic the biological effects of IL- 10.  In search of small molecule modifiers of SHIP1, our lab previously developed a chromogenic enzyme assay to monitor SHIP1 phosphatase activity.  We identified the meroterpenoid Pelorol, isolated from a marine invertebrate extract library, as a potent SHIP1 activator (See Figure E.1 in Appendices).  Structural analogues of Pelorol, AQX- 016A and AQX-MN100, were synthesized by our collaborators, Drs. Raymond Andersen and Matthew Nodwell (University of British Columbia, Departments of Chemistry and Earth & Ocean Sciences, Vancouver, B.C.) 510, which exhibited greater SHIP1 activating activities than the parent compound.  The data presented in this chapter show that these small molecule agonists could selectively activate SHIP1 in intact cells and were protective when administered in mouse models of inflammatory disease.  We further applied these SHIP1 activating compounds as molecular tools to further characterize SHIP1 enzyme activity and revealed that SHIP1 is subject to a previously unrecognized allosteric regulation by its product, PI-3,4-P2.   90 3.2 Materials and methods 3.2.1 Formulation of compounds For in vitro testing in the SHIP1 enzyme assay, AQX-016A and AQX-MN100 were dissolved in EtOH and diluted into aqueous buffer (20 mM Tris-HCl, pH 7.5 and 10 mM MgCl2).  The actual concentration of drug in solution was determined by optical density measurement at 280 nM (λmax of both compounds) after high speed centrifugation at 14 000 X g for 30 min to remove precipitated drug.  For testing on cells, compounds were formulated in the carrier cyclodextrin (Cyclodex Technologies, High Springs, FL) at 6 mM (2 mg/mL).   For oral administration to animals, compounds were dissolved in 100% cremophore EL (Sigma-Aldrich Canada, Oakville, Ontario) at 150 mM (50 mg/mL) prior to dilution to 6 mM in phosphate buffer saline.  Compounds caged in cyclodextrin or formulated in cremophore EL micelles are very soluble in aqueous solution, however they could not be used in the SHIP1 enzyme assays because of interference from both cyclodextrin and cremophore EL. 3.2.2 Production of recombinant SHIP1 enzyme and SHIP1 C2 domain Recombinant, N-terminal His6 tagged SHIP1 enzyme was expressed in mammalian 293T cells by transient transfection with pME18S-His-SHIP1 plasmid and purified to >95% homogeneity by Ni-chelating bead chromatography (Qiagen, Mississauga, Ontario) as assessed by Coomassie Blue visualization of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separated material.  Recombinant SHIP1 C2 domain (amino acid residues 725 to 863) was expressed in E. coli transformed with a pET28C expression vector constructed as described below.  Recombinant protein purified from the  91 cell lysates by Ni-chelating bead chromatography was >95% pure by SDS-PAGE analysis. 3.2.3 in vitro SHIP1 enzyme assay The SHIP1 enzyme assay was performed in 96-well microtitre plates with 10 ng of enzyme/well in a total volume of 25 µL of 20 mM Tris-HCl, pH 7.5 and 10 mM MgCl2. SHIP1 enzyme was incubated with test extracts (provided in DMSO) or vehicle for 15 min at 23°C before the addition of 100 µM inositol-1,3,4,5-tetrakisphosphate (Echelon Biosciences Inc, Salt Lake City, Utah).  The reaction was allowed to proceed for 20 min at 37°C and the amount of inorganic phosphate released assessed by the addition of malachite green reagent followed by an absorbance measurement at 650 nm 511. SHIP2 enzyme was purchased from Echelon Biosciences (Salt Lake City, Utah) and an equivalent amount of inositol phosphatase activity was used in the in vitro enzyme assay. Enzyme data are expressed as the mean of triplicates +/- SEM.  Experiments were performed at least 3 times. 3.2.4 Production of SHIP1+/+ and SHIP1-/- in bone marrow derived macrophages Bone marrow derived macrophages from SHIP1+/+ and SHIP1-/-mice were obtained as described previously 371 and maintained in IMDM supplemented with 10% FCS, 150 µM MTG, 2% C127 cell conditioned medium as a source of macrophage colony stimulating factor (M-CSF) (macrophage medium). 3.2.5 LPS stimulation of macrophages For the analysis of LPS-stimulated TNFα production, 2 x105 cells were plated the night before in 24 well plates in macrophage medium.  The next day, the medium was changed  92 and AQX-016A or carrier was added to cells at the indicated concentrations for 30 min prior to the addition of 10 ng/mL LPS.  Supernatants were collected after 1 hr for TNFα determination by ELISA (BD Biosciences, Mississauga, ON, Canada).    For analysis of intracellular signaling, 2 x106 cells were plated the night before in 6 cm tissue culture plates.  The next day, the cells were cultured in macrophage medium without M-CSF for 1 hr at 37ºC and then pretreated with AQX-016A or carrier for 30 min prior to the addition of 10 ng/mL LPS for 15 min.  Cells were washed with 4ºC PBS and resuspended in lysis buffer (50 mM Hepes, 2 mM EDTA, 1mM NaVO4, 100 mM NaF, 50 mM NaPPi and 1%NP40) supplemented with Complete Protease Inhibitor Cocktail (Roche, Montreal, Canada).  Lysates were rocked at 4ºC for 30 min and clarified by centrifuging 20 min at 12000 x g.  Lysates were then made 1 x in Laemmli’s buffer, boiled 2 min and loaded onto 7.5% SDS polyacrylamide cells.  Immunoblot analysis for phospho PKB (Cell Signaling Technology, Pickering, Ont), SHIP1 and actin (Santa Cruz, Santa Cruz, CA) were carried out as described previously 371. 3.2.6 Mouse endotoxemia model 6-8 week old C57Bl/6 mice (VCHRI Mammalian Model of Human Disease Core Facility, Vancouver, BC) were orally administered the indicated dose of AQX-016A, AQX-MN100 or dexamethasone or carrier 30 min prior to an IP injection of 2 mg/kg of LPS (E. Coli serotype 0111:B4, Sigma, Oakville, Ont).  Blood was drawn 2 hrs later for determination of plasma TNFα by ELISA.  Results are representative of 3 independent experiments.   93 3.2.7 Mouse acute cutaneous anaphylaxis model 6-8 week old CD1 mice (VCHRI Mammalian Model of Human Disease Core Facility, Vancouver, BC) were sensitized to the hapten DNP by cutaneous application of 25 µL of 0.5% dinitroflourobenzene (DNFB) (Sigma, Oakville, Ont) in acetone to the shaved abdomen of mice for two consecutive days.  One week later, test substances (dissolved in 10 µL of 1:2 DMSO:MeOH) were painted on the right ear while the left ear received vehicle control.  30 min after drug application, DNFB was applied to both ears to induce mast cell degranulation.  A 6 mm punch was taken from the ear and immediately frozen on dry ice for subsequent determination of neutrophil myeloperoxidase (MPO) activity as described 512. 3.2.8 Mouse colitis model Colitis was induced in 6-8 week old Balb/c IL-10-/- mice (VCHRI Mammalian Model of Human Disease Core Facility, Vancouver, BC) by administering the colonic contents of conventional C57Bl/6 mice diluted 1:10 in PBS by oral gavage 513.  Mouse weights and fecal consistencies were monitored and colitis allowed to develop for 4 weeks.  Ethanol (Vehicle) and AQX-MN100 (3 mg/kg) was diluted in cage drinking water and dexamethasone (0.4 mg/kg) was administered every 2 days by oral gavage for 3 weeks. At the end of the dosing period, proximal, medial and distal colon sections were collected for paraffin embedding or stored in RNALater (Invitrogen, Mississauga, ON) for RNA extraction.  Slides were prepared, stained with hematoxylin and eosin, and mounted by the UBC Department of Pathology and Laboratory Medicine Histochemistry Facility. Specimens were assigned pathological scores by 3, independent, blinded investigators according to a method described by Madsen et al. 514.  In brief, colonic inflammation was  94 graded using a 4-point system assessing submucosal edema, immune cell infiltration, goblet cell ablation, and integrity of the epithelial layer.  For analysis of mRNA expression, colon sections were homogenized and total RNA extracted using TriZOL Reagent (Invitrogen, Mississauga, ON) as per the manufacturer’s protocol.  Purified RNA was then treated with DNase (Roche Diagnostics, Laval, QC) reverse transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON) and the resulting cDNA analyzed by Sybrgreen-based real-time quantitative PCR (RT- qPCR) using a 7300 Real-Time PCR apparatus (Applied Biosystems, Foster City, CA) and gene specific primers for IL-17, CCL2, and a GAPDH normalization control (See	
  sequences). 3.2.9 Construction of the SHIP1 ΔC2 mutant and isolated C2 domain A His6 tagged SHIP1 ΔC2 domain deletion mutant (deleted amino acid residues 725 to 863) in the mammalian expression vector pME18S was generated by a standard PCR- based methodology.    An N-terminal His6 C2 domain construct was also generated by PCR inserted into the pET28C bacterial expression vector using EcoRI and NdeI restriction sites. 3.2.10 Protein lipid overlay assays Protein lipid overlay (PLO) assays were performed essentially as described 515 with minor modifications. Lyophilized phosphatidylinositol-3,4-bisphosphate diC16 (PIP2, Echelon Biosciences, Salt Lake City, UT) was reconstituted in a 2:1.8 solution of methanol and water. PVDF membranes (Millipore, Missisauga, ON) were initially wetted in methanol for 1 minute, and washed 3 X 5 min with water, and gently agitated in TBST buffer (20 mM Tris-HCl pH 7.5, 0.15 M NaCl (TBS) with 0.05% Tween 20) at 23°C overnight. The  95 treated membranes were air-dried and dilutions of reconstituted lipids were spotted in 1 µl aliquots to give the indicated amount of PIP2 per membrane spot. The membranes were dried completely and blocked with blocking buffer (3% BSA in TBS with 0.05% NaN3) for 1 h at 23°C.   Purified, recombinant C2 domain was diluted into blocking buffer (5 µM final) and treated with 4 µM AQX-MN100 or EtOH control for 30 min at 23°C prior to overnight incubation with the PIP2 spotted membranes.  Membranes were washed 10 times over 50 min in TBST buffer at 23°C and incubated with anti-His6 mouse IgG (Qiagen, Missisauga, ON) for 1 h at 23°C. Membranes were washed as above and incubated with Alexa Fluor 660 anti-mouse goat anti-mouse IgG (Invitrogen, Mississauga, ON) for 1 h at 23°C. After washing, bound proteins were detected and quantified on a Li-Cor Odyssey scanner (Lincoln, NE). 3.2.11 Scintillation proximity assays AQX-MN100 was radiolabelled with tritium by GE Healthcare (Piscataway, NJ) to a specific activity of 42 Ci/mmole.  Copper chelate (His-Tag) YSi SPA scintillation beads (GE Healthcare, Piscataway, NJ) were diluted in 0.25% BSA/TBS to 1.5 mg/mL and recombinant, His6-tagged protein added at the indicated concentrations:  wild-type (1 pM), ΔC2 SHIP1 enzyme (1 pM) or C2 domain (10 nM).  BSA or human serum albumin (HSA) (10 nM) were used as controls.  Protein was allowed to bind 1 h at 23°C, and 250 µg of beads were aliquoted per well of a 96-well plate.  5 µCi of  [3H]-AQX-MN100 was added per well, the plate gently agitated for 30 min and the amount of bead associated radioactivity quantified by counting in a Wallac BetaPlate plate scintillation counter.   96 3.3 Results 3.3.1 AQX-MN100 is as biologically active as AQX-016A Previous studies in our lab have demonstratated that the pelorol analogue, AQX- 016A was able to elicit a 3-fold higher activation of SHIP1 enzyme activity than the parent compound and inhibited macrophage production of TNFα in vitro and in vivo (See Figure E.1 in Appendices).  It was observed that SHIP1+/+ cells and mice were far more sensitive to AQX-016A than cells and mice deficient in SHIP1, suggesting that the compound acts by specifically targeting SHIP1.  These observations were corroborated using in vitro phosphatase assays with SHIP1’s most closely related enzyme, SHIP2, where AQX-016A exhibited no enhancement of phosphatase activity (See Figure E.1 in Appendices).  However, the presence of a catechol moiety within AQX-016A (Figure 3.1A) is potentially problematic since catechols can exhibit activities independent of their specific protein pocket-binding interaction 502.  For example, catechols can bind metals or be oxidized to an ortho-quinone which can lead to covalent modification of proteins through redox reactions 516.  To rule out these possibilities a non-catechol version of AQX-016A designated AQX-MN100 was synthesized 510.  Analogous to AQX-016A, AQX-MN100 enhanced SHIP1 enzyme activity in vitro (Figure 3.1B).  Like AQX-016A, AQX-MN100 also selectively inhibited TNFα production from SHIP1+/+ but not SHIP1-/- macrophages (Figure 3.1C).  The EC50 for this inhibition was 0.3 – 0.6 µM. Additionally, AQX-MN100, similarly to IL-10, is able to potently inhibit both peaks of TNFα production in RAW264.7 cells stimulated under continuous flow conditions (Figure 3.1D).   97             Figure 3.1 AQX-MN100 specifically targets SHIP1 to inhibit TNFα production. (A) Structures of Pelorol, AQX-016A and AQX-MN100.  (B) in vitro phosphatase assays were performed with recombinant SHIP1 enzyme with 50 µM IP4 substrate in the presence or absence of AQX-016A (50 µM) or AQX-MN100 (50 µM).  (C) TNFα ELISA of cell supernatants from SHIP1+/+ (n) and SHIP1-/- (Δ) BMDMs stimulated with LPS (10 ng/ml) in the presence or absence of AQX-MN100 at the concentrations indicated for 2 hours.  (D) TNFα ELISA of fractions collected from RAW 264.7 Parental cells treated with AQX-MN100 (10 µM) for 30 minutes prior to continuous flow apparatus stimulation with LPS (10 ng/ml) + Vehicle (n) or LPS + AQX-MN100 (10 µM) (Δ).  Data represent TNFα concentrations of each 5 min fraction over the course of 4 hours stimulation in the continuous flow apparatus.  Data are representative of 2 independent experiments.  98 3.3.2 AQX-MN100 is protective in in vivo models of inflammation We went on to test whether AQX-MN100 would be effective in inhibiting inflammatory reactions in vivo by assessing its ability to confer protection in mouse models or endotoxemia, allergy and colitis.  The mouse model of endotoxic shock involves IP injection of bacterial LPS and measurement of serum TNFα 2 hours post- challenge 517.  As shown in Figure 3.2A, oral administration of AQX-MN100 30 minutes prior to IP injection of LPS markedly inhibited concentrations of serum TNFα to levels comparable to the steroidal drug, dexamethasone. We also tested AQX-MN100’s ability to inhibit cutaneous anaphylaxis. Anaphylactic or allergic responses are mediated by allergen-induced degranulation of pre-sensitized mast cells 518.  The mouse ear edema/cutaneous anaphylaxis model involves pre-sensitization of mice with the haptenizing agent dinitrofluorobenzene (DNFB) 519. One week later, the allergic reaction is elicited by painting DNFB onto the ears of the mice.  The efficacy of potential anti-inflammatory compounds is tested by topical application of the test substance to one ear and comparing the resulting ear edema or inflammation of the two ears.  By measuring the amount of myeloperoxidase (MPO) activity (an abundant enzyme in neutrophils) in ear punch homogenates as an indicator for immune cell recruitment and inflammation, Figure 3.2B shows that topically applied AQX-MN100 dramatically inhibited allergen-induced inflammation compared to the vehicle control-treated ear. As described in Chapter 2, we demonstrated that SHIP1 is required for certain IL- 10 actions but not whether activation of SHIP1 itself might be sufficient to alleviate inflammation resulting from the loss of normal IL-10 function.  One model in which to  99 test this possibility is the IL-10 KO mouse model of colitis.  IL-10-/- mice develop colitis when colonized with normal intestinal flora because the lack of IL-10 eliminates the normal immunosuppressive mechanisms needed to temper the host immune response 234,300,301.  If SHIP1 activation is important in the anti-inflammatory action of IL-10, then SHIP1 activation by the small molecule SHIP1 agonist, AQX-MN100, might reduce disease severity in colitic IL-10 KO animals. We initiated colitis in IL-10 KO mice by inoculating them with freshly isolated colon contents of normal, specific pathogen-free mice and allowed inflammation to develop for 6 weeks 520.  Mice were then treated for an additional 6 weeks with 2 mg/kg AQX-MN100, 0.4 mg/kg dexamethasone or vehicle prior to colon tissue collection for histological and mRNA expression analyses.  Hematoxylin and eosin (H&E) stained sections were prepared from the proximal, medial and distal colons, as well as from mice that were not inoculated with normal fecal contents (no colitis group) (Figure 3.2C).  Two independent blinded investigators scored the sections using a colitis scoring scheme described by Stecher et al. 521 based on submucosal edema, immune cell infiltration, goblet cell ablation and epithelial integrity (Figure 3.2D).  In the three groups in which colitis was induced, the dexamethasone and AQX-MN100 groups had significantly lower pathology scores than the vehicle group.  Total mRNA was isolated from colon sections from all four groups for analysis of TNFα, IL-17 and CCL2 expression.  TNFα mRNA expression was too low for reliable determinations, but IL-17 and CCL2 mRNA were readily detectable.  As shown in Figure 3.2E, both AQX-MN100 and dexamethasone treatment significantly reduced the levels of IL-17 and CCL2 mRNA.  These data suggest  100 that AQX-MN100 treatment can reduce the inflammation in colitis resulting from the loss of IL-10.                  101                   Figure 3.2   102 Figure 3.2 AQX-MN100 inhibits inflammation in in vivo mouse models of inflammation. (A) Mice were administered 20 mg/kg AQX-MN100 or 0.4 mg/kg dexamethasone orally 30 min prior to an IP injection of 2 mg/kg LPS.  Blood was collected 2 hours later for TNFα determination by ELISA.  Each symbol indicates one mouse and data are representative of three independent experiments.  (B) Mice were topically sensitized with DNFB, and vehicle or AQX-MN100 applied to pairs of ears prior to acute DNFB challenge.  Some mice were not challenged with DNFB (no DNFB).  Ears were harvested and MPO levels determined.  P-value <0.05 for the AQX-MN100 vs the vehicle treated groups.  All data are representative of three independent experiments.  (C) Representative H&E stained colon sections and pathological scores (D) of normal (n=6) and colitic IL- 10-/- mice treated with vehicle (Veh, n=9), AQX-MN100 (3 mg/kg) (n=8) or dexamethasone (Dex, 0.4 mg/kg) (n=3) for 3 weeks.  P=proximal colon, M=mid colon, D=distal colon.  (E) RT-qPCR of cDNA prepared from colonic sections of non-colitic and colitic IL-10-/- mice treated with vehicle, AQX-MN100 (3mg/kg), or Dexamethasone (0.4 mg/kg).  Data represent mean IL-17 and CCL2 expression relative to GAPDH.  ** p<0.01, *** p<0.001when comparing drug treatment to vehicle alone (One-way ANOVA)            103 3.3.3 SHIP1 is an allosterically activated enzyme The allosteric regulation of enzymes has remained under-appreciated primarily because allosteric effectors are not easy to find.  While the majority of allosteric regulators have been discovered through serendipity, a few allosteric regulators have been deduced from discovery of activators or inhibitors of enzymes as a result of high- throughput chemical screens (HTS) 522.  For example, the allostery of glucokinase was discovered from an HTS in search of activators for treatment of diabetes 522.  By analogy, our discovery of small molecule activators of SHIP1 led us to postulate that SHIP1 might in fact be allosterically regulated.  To this end, we investigated the molecular mechanism by which AQX-MN100 activates SHIP1, first by performing kinetic analysis of its enzyme activity.  Activity measurements were performed with substrate concentrations ranging from 10 – 100 µM.  Plots of the initial reaction velocity at each substrate concentration should be hyperbolic if SHIP1 obeys conventional Michaelis-Menten kinetics 523.  However, we found SHIP1 displayed sigmoidal reaction kinetics, which suggests allosteric activation by its end-product (Figure 3.3A).  Indeed, addition of the SHIP1 product PI-3,4-P2 to the enzyme reaction activated wild-type SHIP1 enzyme to the same extent as AQX-MN100 (Figure 3.3B). Interestingly, the 3ʹ′ inositol phosphatases PTEN 524 and myotubularin (MTM) 525 have also been recently shown to be allosterically activated by their phosphatidylinositol products (PI-4,5-P2 and PI-5-P respectively). In PTEN and MTM, the allosteric binding sites for their products were mapped to lipid-binding motifs in each protein.  Since our experiments examining the enzymatic properties of SHIP1 revealed that it is allosterically activated by its product PI-3,4-P2 , we searched for potential PI-3,4-P2 binding domains within SHIP1.  Alignment of amino  104 acid sequences of SHIP1 and SHIP2 from multiple species, and secondary structure predictions led to the identification of a predicted C2 domain residing C-terminal of SHIP1’s enzymatic phosphatase domain (Figure 3.3C).  C2 domains were first described in protein kinase C (PKC) where it serves to bind Ca2+, but C2 domains have since been identified in other proteins where they have been shown to bind to a variety of ligands including lipids 526,527.  To test this possibility, we produced SHIP1 enzyme in which the C2 domain (ΔC2 SHIP1) was deleted.   As shown in Figure 3.3B, although the ΔC2 SHIP1 enzyme was as active as the wild-type molecule, its activity could not be enhanced by the addition of either PI-3,4-P2 or AQX-MN100.   This indicates that the C2 domain is required for allosteric activation of SHIP1 activity and that it may be the binding site for its allosteric activators PI-3,4-P2 and AQX-MN100. In order to examine whether the C2 domain could bind PI-3,4-P2, we expressed recombinant, His6-tagged C2 domain and determined its PI-3,4-P2 binding ability using protein lipid overlay assays 515.  Purified C2 domain was incubated with membrane strips spotted with PI-3,4-P2  and bound protein detected using an anti-His6 antibody.  Figure 3.3D shows the C2 domain binds PI-3,4-P2 and that this binding is inhibited by AQX- MN100, consistent with the hypothesis that both AQX-MN100 and PI-3,4-P2 interact with the C2 domain at a common binding site. AQX-MN100 was verified to directly bind the C2 domain using scintillation proximity assays (SPAs) in which SPA beads were coated with either the C2 domain or control protein (BSA) prior to incubation with [3H]-AQX-MN100.  Figure 3.3E shows that the C2 domain does indeed interact with [3H]-AQX-MN100.  In complementary studies, we observed that [3H]-AQX-MN100 bound to wild-type SHIP1 but not to SHIP1  105 lacking its C2 domain (Figure 3.3F).  Together, these data are consistent with AQX- MN100 binding to SHIP1’s C2 domain, resulting in allosteric activation of the enzyme.                  106                  Figure 3.3   107 Figure 3.3 The C2 domain is required for end-product allosteric activation of SHIP1 and binding of AQX-MN100 SHIP1 enzyme initial velocities were determined at the indicated concentration of inositol-1,3,4,5-tetrakisphosphate (IP4) substrate.  (B) The ability of PI-3,4-P2 (20 µM) or AQX-MN100 (30 µM)  to activate wild-type (WT) and C2 domain deleted (ΔC2) SHIP1 enzyme was determined at 30 µM IP4.  (C) ClustalW alignment of all SHIP1 and SHIP2 sequences deposited in the NCBI database corresponding to the regions 160 amino acids C-terminal of the putative phosphatase domains.  (D) Recombinant C2 domain was pre- incubated for 30 min at 23°C with 200 µM AQX-MN100 or Vehicle control (EtOH) and allowed to bind to PI-3,4-P2 immobilized on membrane strips in a protein overlay assay as previously described.  (E) Recombinant C2 domain (10 nM) or Full-length WT or ΔC2 SHIP1 enzyme (1 pM) (F) was coated onto Copper chelate (His-Tag) YSi SPA Scintillation Beads in the presence of 0.25% BSA.  Beads were then incubated with 5 µCi of [3H]-AQX-MN100 and the bead-associated radioactivity measured as described in Materials and Methods.  BSA = bovine serum albumin, HSA = human serum albumin. Data are expressed as mean +/- SEM and are representative of at least three independent experiments. **p<0.01, *** p<0.001 (One-way ANOVA)            108 3.4 Discussion The PI-3 kinase pathway has been the target of intense efforts for the development of therapeutics 499-504,528.  The PI-3 kinase family consists of multiple isoforms which vary in tissue distribution and receptor systems to which they are coupled 501.  The classic PI-3 kinase inhibitors wortmannin and LY292004 have been useful experimental tools for probing PI-3 kinase function, but they have not been successful in clinical development partly because they globally inhibit all members of the PI-3 kinase family and are known to have off-target effects 313,314,338-340,529. Recently however, isoform specific PI-3 kinase inhibitors are emerging as a promising class of therapeutic agents.  For example, a dual PI-3 kinase α/mTOR inhibitor 502 was found to have efficacy in a human glioma xenograft model 509 without any undue toxicities even though PI3Kα is expressed in all tissues and is important in insulin signaling 500,530.  It is postulated that a sufficient therapeutic window exists because cancer cells become very dependent on particular signaling pathways (termed “oncogene addiction” 499) and thus pharmacological inhibitors show selectivity towards cancer vs normal cells 499.  Similarly, PI-3 kinase δ and γ-specific inhibitors are actively being investigated for treatement of inflammatory diseases due to the enrichment of these isoforms in immune cells 307-312. PI-3 kinase γ mediates signaling from G-protein coupled receptors (GPCR) and although its expression can be detected in endothelium, heart and brain, it is mainly expressed in immune cells 529.   PI-3 kinase γ inhibitors thus benefit from its relatively restricted expression compared to the other PI-3 kinase isoforms, and the fact that many (though not all 531) inflammatory processes involve GPCR-dependent steps 529. PI-3 kinase γ inhibitors have been found to be protective in mouse models of rheumatoid arthritis 505  109 and glomerulonephritis 506.  In addition to PI-3 kinase itself, downstream protein kinases are also being targeted with mixed results.  Work is continuing on Akt inhibitors 532-534 with limited success perhaps because of dose limitations due to toxicities 535,536.  The mTORC1 inhibitor rapamycin, on the other hand, is currently approved as an immunosuppressive agent with manageable side effects 537.  The toxicities of both Akt and mTORC1 inhibitors are partly related to the ubiquitous expression of both targets. As an alternative to inhibiting PI-3 kinase and downstream protein kinases, we describe a novel paradigm for inhibiting PI-3 kinase signaling through activation of the phosphatases that negatively regulate this pathway.   The SHIP1 phosphatidylinositol phosphatase is a particularly good target for immune/hemopoietic disorders because of its restricted expression to hemopoietic cells.    Because the relative activity of phosphatases present in a cell will influence the efficacy of kinase inhibitors, as discussed by Knight and Shokat 502, SHIP1 agonists could also be used to potentiate the activition of PI-3 kinase inhibitors and target non-tissue specific PI-3 kinase inhibitors to the hemopoietic/immune cell compartment.  The experiments described in this chapter demonstrate that the small molecule SHIP1 agonist, AQX-MN100, can be used to inhibit immune cell activation in vitro, and in in vivo mouse models of inflammatory disease. Our results suggest that the current model for SHIP1 activation, involving translocation of SHIP1, via its SH2 or its phosphorylated NPXY motifs, from the cytoplasm to the plasma membrane without any change in its intrinsic phosphatase activity 365,495 needs to be modified.  Specifically, we postulate that upon recruitment to the plasma membrane, SHIP1 hydrolyzes a small amount of PIP3 at a low, basal rate. This generates some PI-3,4-P2 which then binds to the C2 domain, leading to a  110 conformation change which enhances its catalytic activity.  Interestingly, end-product activation has also been reported for two 3′inositol lipid phosphatases.  PTEN binds its product (PI-4,5-P2) using an N-terminal lipid binding motif resulting in enhancement of phosphatase activity 524.  Similarly, the MTM phosphatase, binds its product (PI-5-P) via a divergent PH domain, which allosterically activates its function 525.   In the case of PTEN, the requirement for PI-4,5-P2 helps localize PTEN protein to specific regions of the membrane 538-540.  The binding of SHIP1 by its product PI-3,4-P2 may also similarly serve to localize SHIP1 to the plasma membrane in addition to allosterically activating its intrinsic inositol phosphatase activity providing a positive feedback mechanism to rapidly reduce membrane PIP3 levels.   Regardless of whether or not PI-3,4-P2 binding regulates SHIP1 localization, this newly described allosteric activation domain within SHIP1 may be exploited therapeutically by pharmacological agents such as AQX-MN100 that bind to the allosteric activation site to stimulate SHIP1 activity. From their first description, allosteric sites have been considered to be more important as drug targets than active sites and allosteric regulators have been predicted to possess more selectivity than active site modulators 522.  While it remains challenging to prove that a certain drug-target interaction is responsible for mediating its biological effects, our observation that AQX-MN100 had minimal effects on macrophages lacking the SHIP1 target provides compelling support that the PI-3 kinase pathway inhibitory effects observed are mediated by SHIP1 itself.  Furthermore, our observation that AQX- MN100 exhibits efficacy having a submicromolar EC50 suggests that this class of compounds possesses a low likelihood of off-target effects based on calculations by Knight and Shokat 502.  Indeed, it has been determined that AQX-MN100 has minimal  111 off-target effects when screened against 100 other kinases and phosphatases (Figure F.1 in Appendices).  The studies in this chapter offer proof-of-principle that small molecule activators of lipid phosphatases exist and that they provide a new paradigm for inhibition of PI-3 kinase-dependent processes.  Small molecule agonists of the hemopoietic cell-specific SHIP1 enzyme, in particular, represent potential therapeutics for treatment of immune/hemopoietic disorders in which the PI-3 kinase pathway is dysregulated.  Due to their unique target and mechanism of action, these compounds may also be powerful synergistic agents in combination with current therapies.   Agonists of other allosterically regulated phosphatases, such as PTEN 524 and MTM 525, may similarly be useful for diseases in which their impaired activity has been implicated.   112          CHAPTER 4: A PLECKSTRIN HOMOLOGY-RELATED DOMAIN IN SHIP1 MEDIATES MEMBRANE LOCALIZATION IN FCγR-MEDIATED PHAGOCYTOSIS               113 4.1 Introduction Lipid phosphatases have a central role in regulating a vast array of cellular processes induced by extracellular signals, including phagocytosis, cell migration, proliferation, and survival.  In particular, inositol lipid phosphatases such as SHIP1 and SHIP2, PTEN and MTM, are known to be essential for maintaining cellular homeostasis and mutations in these genes are attributed to various hematologic cancers 436,498,541, solid organ tumors 542,543, and skeletal myopathy 544 respectively.  The cellular function of these phosphatases requires their physical recruitment to the intracellular membrane compartments containing their phosphatidyl inositol substrates. Thus, understanding the mechanisms by which these enzymes are recruited to the membrane is important. Phagocytosis is a dynamic process involving the coordinated recruitment and activation of signaling proteins, lipid-modifying enzymes, and components of the cytoskeletal machinery.  How all these elements are spatially and temporally regulated to achieve particle engulfment is still not fully understood.  In FcγR-induced phagocytosis, Ig-opsonized particles bind to FcγRs on the surface of phagocytic cells.  Clustering of ligand-bound FcγRs triggers the activation of Src and Syk family kinases 545-547 and subsequent stimulation of signaling proteins including the PI-3 kinases 471,548 and Rho- family GTPases 549-551.  Together, these pathways facilitate the membrane modifications and actin remodeling required for pseudopod extension and formation of the phagosome around the particle being ingested. One of the principle events committing a cell towards phagocytosis of a particle is generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) by PI-3 kinases through the phosphorylation of PI-4,5-P2.  Pharmacological inhibition of PI-3 kinase by agents such  114 as wortmannin and LY294002 prematurely arrests phagocytosis and demonstrate that PI- 3 kinase is essential for engulfment of particles greater than 2 µm in size 471,548. Synthesis and hydrolysis of PIP3 follows an ordered sequence upon particle binding to and clustering of FcγRs.  Beginning at the base of the phagocytic cup where PI-4,5-P2 is converted to PIP3, the wave of PIP3 generation proceeds up the lateral edges of membrane surrounding the particle, and finally disappears immediately upon phagosome sealing 450,552-554.  Cellular levels of PIP3 are tightly regulated under normal conditions, both by controlling PI-3 kinase activation and by the presence of the 5ʹ-inositol phosphatases (SHIP1, SHIP2, and 72-kDa 5ʹ-phosphatase, 72-5ptase), which hydrolyze PIP3 to PI-3,4-P2, and the 3′-inositol phosphatase PTEN, which generates PI-4,5-P2 492- 494.  Of the four phosphatases, only SHIP1, SHIP2 and 72-5ptase are reported to localize to the phagocytic cup where they are thought to be responsible for the observed hydrolysis of PIP3 to PI-3,4-P2 396,494,555.  Whether these three phosphatases have redundant roles or are regulated independently to perform specific functions during the phagocytic process has yet to be determined. SHIP1 negatively regulates phagocytosis as shown by over-expression 396 and gene knock-out studies 396,555.  FcγRI and FcγRIIa receptors have been reported to recruit SHIP1 to the phagocytic cup via interactions between phosphotyrosines within ITAMs on the receptors and the SHIP1 SH2 domain 349,350.  Once at the membrane, SHIP1 is brought into the vicinity of its substrate where it negatively regulates FcγRI/FcγRIIa signaling and phagocytosis through degradation of PIP3.  Imaging of inositol lipids during phagosome formation has shown that membrane recruitment of SHIP1 to the leading edge correlates with a decreasing gradient of PIP3 and a corresponding increase  115 of the SHIP1 product PI-3,4-P2 along the developing phagosome 450,554,556.  Additionally, SHIP1 has also been reported to be recruited to the phagocytic cup via the inhibitory FcγRIIb receptor through interactions between its SH2 domain and phosphotyrosines within immunoreceptor tyrosine inhibitory motifs (ITIMs) on FcγRIIb 352,414,557.  The membrane proximal SHIP1 then negatively regulates the activating signals generated by FcγRI/FcγRIIa. This chapter describes a previously unrecognized domain in SHIP1 that mediates direct binding to membrane lipids and that this lipid mediated interaction is the major mechanism by which SHIP1 is recruited to the phagocytic cup.   Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that this segment of SHIP1 adopts an independently folded structure predicted to have pleckstrin homology (PH) domain-like topology.  This PH-related (PH-R) domain binds PIP3 and is required for SHIP1 localization of SHIP1 to the phagocytic cup to inhibit FcγR-mediated phagocytosis.  Site directed mutagenesis of candidate amino acid residues reveals two critical lysine residues involved in the binding.  Replacement of these lysines with alanines abrogates the ability of recombinant PH-R domain to interact with PIP3 in in vitro lipid binding assays and the ability of SHIP1 protein to translocate to the phagocytic cup in macrophages to negatively regulate particle uptake.  These studies provide further insight into mechanisms regulating SHIP1 function in cells and indicate that phosphoinositol lipid- mediated recruitment of proteins is an important step in phagosome maturation.  These findings also suggest that small molecules, which alter PH-R domain interactions with PIP3, may be another means of modifying SHIP1 activity for therapeutic purposes.   116 4.2 Materials and methods 4.2.1 SHIP1 sequence domain identification Secondary structure and order/disorder prediction algorithms were used to identify a potentially folded region in SHIP1 adjacent to the phosphatase domain (Figure 4.2). Alignments performed using ClustalW 558 confirmed that this region is conserved amongst all deposited SHIP1 sequences of the various species represented in the National Center for Biotechnology Information database (Figure 4.1).  To define the boundaries of the structured region more precisely, truncation and extension constructs were expressed and tested for their lipid binding ability as described below. 4.2.2 Expression and purification of SHIP1 PH-R domain and K397A/K370A (KAKA) mutant domain  A cDNA encoding residues 292-401 of SHIP1 (mouse) was generated by PCR and cloned into pET28c (Novagen, Madison, WI).  The cDNA was inserted such that a 21 amino acid segment containing a 6×His epitope tag was fused to the N-terminus of the 111-residue protein (full sequence in Supplemental Methods).  Constructs were transformed into E. coli BL21 (DE3) competent cells (Promega, Madison, WI).  Protein was expressed and purified using a method as described previously 559.  Standard site- directed mutagenesis methodologies were used to generate mutant PH-R constructs in which candidate PIP binding residues were replaced with alanines.  Recombinant protein from these constructs, including the KAKA variant with K370A/K397A were expressed and purified as described above.   117 4.2.3 Sequence of 6×His affinity tagged SHIP1 PH-R domain MGSSHHHHHHSSGLVPRGSHMSTNRRSLIPPVTFEVKSESLGIPQKMHLKVDV ESGKLIVKKSKDGSEDKFYSHKKILQLIKSQKFLNKLVILVETEKEKILRKEYVFA DSKKREGFCQLLQQMKNKHSEQ The 21 amino acid segment containing the 6×His epitope tag is indicated in bold face. 4.2.4 Expression and purification of the isotopically labeled PH-R domain Uniformly labeled SHIP1 PH-R domain was expressed in E. coli Rosetta (λDE3) cells (Novagen, Madison, WI) in M9 minimal medium supplemented with 15NH4Cl and 13C6- D-glucose (Cambridge Isotope Laboratories Inc., Andover, MA). The expression protocol was modified from the methods of Marley et al. 560,561 to reduce the consumption of isotopically-labeled ingredients. Briefly, the cells were initially grown in 4×1 L terrific broth (TB) at 37 °C to an OD600 of 0.7 and then harvested at 2,600×g at 4 °C for 15 min. Pelleted cells were resuspended and pooled in a wash solution of the M9 buffer salts (50 mL) and allowed to stand at 4 °C for 20 min followed by centrifuging again at 2,600×g at 4 °C for 15 min. This cleaning step was repeated once more to remove any residual rich media. The washed pellet was resuspended in 10 mL of the M9 salt solution and added to 500 mL of pre-incubated (at 37 °C) 2×M9 media with 0.7 g 15NH4Cl and 2 g 13C6-D-glucose as the sole sources of nitrogen and carbon. The M9 media was also supplemented with nutrients and vitamins according to Neidhardt et al. 562. Expression of the recombinant PH-R domain was initiated after 30 min by the addition of 240 mg of isopropyl-β-D-thiogalactopyranoside (IPTG) (BioShop, Burlington, ON). Expression was halted after 16 hours by chilling the cells on ice for 30  118 min, followed by centrifugation at 2,600×g at 4 °C for 30 min. Harvested cells were flash-frozen in liquid nitrogen and stored at -80 °C.  The majority of the expressed PH-R domain was found in the insoluble fraction. Protein purified from the soluble fraction under non-denaturing conditions produced the same 2-dimensional 1H–15N HSQC correlation spectrum as the PH-R domain purified from the inclusion bodies under strong denaturing and reducing conditions. For this reason all further purifications of the PH-R domain were done under denaturing conditions to maximize the protein yield. Pellets from the protein expression (approximately 10 g wet weight) were thawed and resuspended in 40 mL of extraction buffer (6 M guanidine hydrochloride (Gdn-HCl), 100 mM Tris-HCl, 500 mM NaCl, 5 mM tris(2-carboxyethyl) phosphine (TCEP) at pH 8.2). The suspended cells were microprobe sonicated (3 mm tapered tip) on a Branson Sonifier 250 ultra sonic cell disruptor for 15 min at power 4 and a 50% duty cycle to reduce sample heating. The sonicated sample was then flushed through a 22 gauge, hypodermic needle twice and then a 26 gauge needle once to shear DNA and reduce the sample viscosity. The lysed cells were centrifuged at 30,600×g for 30 min to remove the insoluble material. The supernatant was then injected onto 5 mL HisTrap column (GE Heathcare, Piscataway, NJ) that was pre-equilibrated with the extraction buffer on a ÄKTA purifier (GE Healthcare, Piscataway, NJ) running at a flow rate of 4 mL/min. Once the entire sample was injected (in 10 mL increments), the column was washed with an additional 40 mL of the extraction buffer. A two-step imidazole gradient was used to wash and elute the protein from the column by combing the extraction buffer and the elution buffer (6 M Gdn, 100 mM Tris-HCl, 300 mM NaCl, 5 mM TCEP and 200 mM  119 imidazole). The first gradient had an increasing imidazole concentration rate of 4 mM/min for 15 min (12 column volumes), followed by a second gradient increase at a rate of 20 mM/min for 7 min reaching 100% of the elution buffer with 200 mM imidazole. The column was then washed with an additional 8 column volumes of the 100% elution buffer. The PH-R domain began eluting with approximately 50 mM imidazole. The appropriate fractions were pooled, concentrated with an Amicon Ultra-15 (Millipore, Billerica, MA) centrifugal filter to a final volume of 10 mL. The sample was then serially dialyzed at room temperature (5 hours each) in 3,500 MWCO SnakeSkin (Pierce, Rockford, IL) to remove the Gdn and salts against 0.1, 0.05, and 0.01 M ammonium acetate at pH 4. A final dialysis step was done against water. The dialysate was then frozen and lyophilized. The protein yield was in general between 15 and 20 mg by this method.  The K370A/K397A double mutant was transformed and expressed under identical conditions as the wild-type PH-R domain except that the M9 media was isotopically enriched with only 15NH4Cl. All other aspects of the expression and purification of the K370A/K397A mutant are identical. 4.2.5 NMR sample preparation Samples of the His6-tagged PH-R domain were initially prepared at 0.3 mM concentration at pH 7 in 20 mM Tris-HCl, with 150 NaCl and 10 mM β-mercaptoethanol (βME). Although showing well dispersed spectra, there were a number of missing or weak resonances. At 0.3 mM, the PH-R samples also seemed to reach a critical concentration as the sample would show signs of precipitation within a week. To overcome this stability issue, subsequent samples were prepared by dissolving the  120 lyophilized protein at pH 5.8 in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer with 200 mM Gdn-HCl, and 5 mM TCEP. Under these conditions protein samples at 0.5 mM remained soluble for months. 4.2.6 NMR spectral assignments Spectra of the 131-residue 13C/15N-labeled His6-tagged PH-R domain were recorded at 25 °C on a 600 MHz Varian INOVA spectrometer equipped with a cryogenic triple resonance probe head, using standard sensitivity-enhanced gradient Varian BioPack pulse sequences 563-567 including 1H-15N HSQC, HNCACB 564, CBCA(CO)NH 565 , HNCO 566 and HN(CA)CO 567. Spectra were processed with NMRPipe 568 and subsequently exported to Sparky for analysis 569. 1H chemical shifts were referenced to external 2,2- dimethyl-2-silapentane-5-sulfonate (DSS) at 25 °C 570. 15N and 13C referenced using recommended magnetogyric ratios 571. 4.2.7 NMR-monitored titrations The interaction of phosphatidylinositol-3,4,5-trisphosphate diC8 (Echelon Biosciences, Salt Lake City, UT) with the PH-R domain was monitored by 1H-15N HSQC spectroscopy. Aliquots of saturated PIP3 solution were titrated to a final concentration of 0.17 mM into ~0.1 mM 15N-labeled PH-R domain. The protein was in 20 mM Tris-HCl pH 7 with 150 mM NaCl, as initial studies of the PH-R domain with PIP3 in PLO assays showed that the ligand did not interact in the presence of Gdn-HCl and at pH 5.8. 4.2.8 Phosphoinositol binding (PLO assay) diC16 lipids (Echelon Biosciences, Salt Lake City, UT) reconstituted in a 2:1.8 solution of methanol and water were spotted onto dry PVDF membranes at the indicated  121 quantities.  Membranes were blocked in blocking buffer (3% BSA in TBS with 0.05% NaN3) for 1 hour at 23°C with gentle agitation.  Membranes were then incubated with 625 nM of recombinant PH-R domain protein in a 6 ml volume of blocking buffer for 6 hours, washed 10 times over 50 minutes in TBST buffer (20 mM Tris-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween-20), followed by incubation with 1 µg/mL anti-His6 mouse IgG (Sigma, Mississauga, ON) for 1 hour at 23°C.  Membranes were washed an additional 10 times with TBST buffer, and incubated with AlexaFluor660 goat anti-mouse IgG (Invitrogen, Burlington, ON) for 1 hour at room temperature.  After washing, bound domain was detected and quantified on a LiCor Odyssey Infrared Scanner (Lincoln, NE) 4.2.9 Cells and reagents RAW264.7 cells were obtained from the American Type Culture Collection.  Cells were maintained in Dulbecco’s modified eagle’s medium (Thermo Scientific, Logan, UT) supplemented with 9% (v/v) fetal bovine serum (FBS) (Thermo Scientific, Logan, UT). J16 (SHIP1+/+) and J17 (SHIP1-/-) cell lines were generated by infecting BMMϕs from C57Bl/6 SHIP1+/+ and SHIP1-/- mice respectively, with the J2 virus 572 and maintained in Iscove’s Modified Dulbecco’s Medium (Thermo Scientific, Logan, UT), supplemented with 10% FBS, 10 µM β-mercaptoethanol, 150 µM monothioglycolate, and 1 mM L- glutamine.  J17 cells were transduced with lentiviruses harbouring expression constructs for wild-type (WT) SHIP1 or SHIP1 with residues K370 and K397 mutated to alanines (KAKA) under the control of a tetracycline-responsive promoter.  Transduced cells were separated from non-transduced cells by fluorescence-activated cell sorting based on the co-expression of mCherry on the lentiviral transfer plasmid.  Transduced cells were treated with 2 µg/ml of doxycycline (Sigma, Mississauga, ON) for 48 hours to induce  122 WT or KAKA SHIP1 expression prior to use.  Expression of WT and KAKA SHIP1 were confirmed by resolving cell lysates by SDS-PAGE and immunoblotting using anti- SHIP1 (P1C1) antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). 4.2.10 GFP-tagged SHIP1 PH-R domains eGFP fusion constructs were generated by inserting PCR-generated cDNA fragments corresponding to SHIP1 residues 292-401 and AKT1 residues 1-165 into the pEGFP-C1 vector (Clontech, Mountain View, CA) using EcoRI and BamHI restriction cut sites. Plasmid constructs were then transiently transfected into RAW264.7 cells using FuGENE HD (Roche, Laval, QC) as per the manufacturer’s protocol. 4.2.11 Scintillation proximity assays (SPA) Copper chelate YSi SPA Scintillation Beads (GE Healthcare, Piscataway, NJ) were diluted in 0.25% BSA in TBS to 1.5 mg/ml.  10 nM of each His6-tagged SHIP1 C2 domain or PH-R domain were allowed to bind to the beads by incubating at 23°C for 1 hour.  250 µg of domain-coated beads were aliquoted per well of a 96-well plate.  0.185 MBq (5 µCi) of [3H]-AQX-MN100 was then added to each well and incubated for 30 minutes with gentle agitation.  The amount of bead-associated radioactivity was quantified by counting in a Wallac (Perkin-Elmer, Waltham, MA) Betaplate scintillation counter. 4.2.12 Phagocytosis assays Bead preparation - 3 µm or 15 µm latex beads (Polysciences Inc, Warrington, PA) were opsonized with 100 µg/ml human IgG (Sigma, Mississauga, ON), or 100 µg/ml of human IgG labeled with a Dylight 680 conjugating kit (Thermo Scientific, Rockford, IL),  123 respectively, overnight at 37°C with gentle agitation.  Beads were washed extensively with PBS containing 3% FBS and resuspended in cold DMEM supplemented with 9% FBS.  Cell Preparation – Transfected RAW264.7 cells, J16 SHIP1+/+ (SHIP1+/+), J17 SHIP1-/- (SHIP1-/-), J17 SHIP1-/- cells reconstituted with SHIP1 WT (SHIP1-/-:WT) or J17 SHIP1-/- cells reconstituted with SHIP1 KAKA (SHIP1-/-:KAKA) were plated onto poly- L-Lysine treated glass coverslips and allowed to adhere overnight at 37°C, 5% CO2. Bead stimulation – Overnight supernatants were removed from the cells and replaced with opsonized beads at a 10 beads:1 cell ratio.  Plates were centrifuged at 4°C to allow beads to settle onto the coverslips, then plates were immediately placed in a 37°C, 5% CO2 incubator for phagocytosis to proceed.  Slide preparation – Stimulated cells were washed thoroughly with cold PBS, fixed in 2% paraformaldehyde in PBS for 30 minutes at 37°C, washed again with PBS, then mounted onto glass slides using Prolong® Gold antifade reagent with DAPI (Invitrogen, Burlington, ON).  Images were acquired using a Zeiss Axioplan2 Fluorescence microscope equipped with a 63X oil-immersion lens and analyzed using Zeiss AxioVision4.8 software. Phagocytic indices were calculated where the phagocytic index was defined as the average number of beads phagocytosed by one cell.  For experiments involving localization of SHIP1 by immunocytochemistry, cells on coverslips were stimulated with 15 µm beads prior to 2% paraformaldehyde in PBS fixation and permeabilization with 0.5% TritonX-100 in PBS.  The coverslips were blocked with 3% BSA, stained with primary mouse anti-SHIP1 (P1C1) antibody (SantaCruz Biotechnologies, SantaCruz, CA), followed by FITC-conjugated goat anti- mouse secondary antibodies (Jackson Immunoresearch Laboratories, Westgrove, PA). The coverslips were then mounted onto glass slides and images acquired using a Zeiss  124 LSM780 confocal microscope equipped with a 63X oil-immersion lens.  Confocal images were analyzed using Zeiss Zen 2009 software.  Images were scored using a 4-point grading system by 3 independent, blinded investigators. 4.2.13 Production of recombinant SHIP1 WT and SHIP1 KAKA Full-length N-terminal His6-tagged WT or KAKA SHIP1 were produced in Sf9 cells using a baculovirus expression system (Invitrogen, Burlington, ON).  Cells were pelleted, lysed, and recombinant enzyme was purified from lysates using TALON cobalt affinity chromatography resin (Clontech, Mountain View, CA) and determined to be more than 95% pure as described above. 4.2.14 in vitro phosphatase assays SHIP1 enzyme assays were performed by diluting 10 ng of recombinant WT of KAKA SHIP1 enzyme in 25 µl of dilution buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2) in each well of a 96-well microtiter plate.  Enzymes were incubated with vehicle, PI-3,4-P2 or AQX-MN100 for 15 minutes at 23°C prior to the addition of 100 µM inositol 1,3,4,5-tetrakisphosphate (IP4, Echelon Biosciences, Salt Lake City, UT). After 10 minutes incubation at 37°C, malachite green 573 reagent was added and the amount of inorganic phosphate released was measured by an absorbance reading at 650 nm.  For enzyme kinetics determinations, enzyme reactions with IP4 substrate concentrations ranging from 10-200 µM IP4 were sampled at 1 minute intervals for 10 minutes.  Enzyme initial rates were determined from the slope of the linear portion of the resulting time courses and plotted against IP4 concentrations.   125 4.3 Results 4.3.1 Identification of a PH related (PH-R) domain in SHIP1 Since our study of the enzymatic properties of SHIP1 described in Chapter 3 revealed that it is allosterically activated by its product PI-3,4-P2 , we searched for potential PI-3,4-P2  binding domains within SHIP1 559.  In addition to the C2 domain, amino acid alignments (Figure 4.1) and secondary structure predictions revealed a second 110 amino acid region N-terminal to the phosphatase domain that had features of a PH domain (Figure 4.2).   In order to characterize this newly recognized region of SHIP1, residues 290-410 were cloned and expressed for NMR spectroscopic analysis.  As shown in Figure 4.3A (red), the resulting protein fragment yielded a well-dispersed 1H-15N heteronuclear single quantum coherence (HSQC) spectrum, confirming that this segment of SHIP1 indeed adopts an independently folded structure.  After assigning the signals from the mainchain 1H, 13C and 15N nuclei of this species (Figure 4.3B), the chemical shift-based secondary structure propensity (SSP) algorithm 574 was used to identify its secondary structural elements (Figure 4.3C).  Consistent with the sequence-based secondary structure predictions, the SHIP1 domain contains several β-strands as well as a clear C-terminal helix.  Unfortunately, although having an assignable 1H-15N HSQC spectrum, residues 290-410 of SHIP1 also exhibited unfavorable dynamic properties leading to extensive conformational exchange broadening of the NMR signals from sidechain nuclei.  This prevented us from determining its tertiary structure using NMR measured restraints. Therefore, we used the THRIFTY webserver 575 to predict the fold of the SHIP1 domain  126 based upon chemical shift-guided homology threading.  This program essentially finds residues in the database of NMR derived structures that have similar chemical shifts to a query protein.  With two perpendicular β-sheets followed by a C-terminal α-helix, the resulting model has the overall fold characteristic of a PH domain (Figure 4.3D). However, it is important to stress that PH domains show significant variation in sequence, structure and function, reviewed in 576,577.  Accordingly, in the absence of a more detailed structural analysis, we conservatively denote this region of SHIP1 as a PH related (PH-R) domain.             127                     Figure 4.1 Sequence alignment and phylogram of the PH-R domain. (A) ClustalW alignment and (B) phylogram of all SHIP1 and SHIP2 sequences deposited in the NCBI database corresponding to the regions 120 amino acids N-terminal of the putative phosphatase domains   128 0 300 600 1200900 SH2 ,QRVLWRO·3KRVSKDWDVH 13;<PRWLIV 3;;3PRWLIV 3,3 ELQGLQJGRPDLQV 3+5 C2 A B C D  0  0.2  0.4  0.6  0.8  1  200  400  600  800  1000 PO ND R  sc or e  0  0.2  0.4  0.6  0.8  1  320  360  400 Residue HNN 292 401 GOR4 PSIPRED SOPMA Jpred3 PORTER PROF SSpro JUFO consensus SSP -1 -0.5  0  0.5  1  300  320  340  360  380  400 M ea n Sc or e Residue                   Figure 4.2  129 Figure 4.2 SHIP1 contains an independently folded Pleckstrin Homolgy-Related (PH-R) domain. (A) Schematic of the domain structure of SHIP1.  Following the N-terminal SH2 domain, there is a 300-residue segment leading up to the catalytic phosphatase domain that contains the newly recognized PH-R domain.  Following the phosphatase domain, the C- terminal region is a stretch of approximately 350 residues that contains a calcium- dependent binding domain (C2) 559, 2 phosphotyrosine motifs (NPXY) and 3 potential SH3 domain binding motifs (PXXP).  (B) To identify the PIP3 lipid-binding region more precisely in order to clone the domain, the program PONDR 578-580 was used to predict the ordered and disordered regions of SHIP1. Shown are PONDR L-XT scores for the full length 1191 residue murine SHIP1 sequence (solid green line) and for the isolated putative PH-R domain (residues 292-401; dashed magenta line). The lower panel is an expanded view for residues 290-410. Regions above/below the threshold of 0.5 are considered disordered/ordered.  (C) Secondary structure predictions (helices, red cylinders; strands, blue arrows) for residues 292-401 were obtained using the webserver algorithms HNN 581, GOR4 582, PSIPRED 583,584, SOPMA 585, Jpred3 586, PORTER 587, PROF 588, Sspro 589, and JUFO 590,591.  (D) A consensus score was generated for each residue with predicted helices (red) being valued as +1, strands (blue) as -1, and coils as 0. Also shown for comparison in panel B are the SSP (Secondary Structure Propensity 574) scores based upon the observed 13Cα and 13Cβ chemical shifts of the isolated PH-R domain.           130                   Figure 4.3  131 Figure 4.3 SHIP1 contains an independently folded PH-R domain. (A) Overlaid 1H-15N HSQC spectra of the His6-tagged PH-R in the absence (red) and presence of 170 µM PIP3 (blue).  The excellent dispersion of the signals from the amide 1H-15N groups indicates that the isolated PH-R domain is folded and the spectral changes upon addition of PIP3 confirm the binding of this ligand.  (B) Assigned amide backbone region of the 1H–15N HSQC spectrum of His6-tagged PH-R domain from SHIP1 (0.5 mM) at pH 5.8 in 20 mM MES, 5 mM TCEP, and 200 mM Gdn-HCl. The spectrum was recorded at 25°C on a 600 MHz Varian INOVA spectrometer equipped with a cryogenic triple resonance probehead using 612×128 complex points (1H sw = 12 ppm; 15N sw = 32 ppm) and 16 scans per increment. The dashed and solid ellipses encompass the signals from sidechains of Asn/Gln and Arg (aliased), respectively. The high degree of signal intensity variation is attributed to a range of conformational dynamic exhibited the isolated PH-R domain.  (C) The SSP (Secondary Structure Propensity) scores determined from 13Cα and 13Cβ chemical shifts identifies experimentally the helical (+1) and strand (- 1) regions in the PH-R domain 574.  (D) A model generated with THRIFTY 575 using chemical shift information to guide molecular homology threading, demonstrates that residues 292-401 of SHIP1 adopt a PH domain-like fold.  Highlighted in yellow is a possible PIP binding motif similar to that proposed in 592.  Also shown in magenta are the side chains of K370 and K397.            132 4.3.2 Functional characterization of the PH-R domain To test the ability of this PH-R domain within SHIP1 to bind PIPs, we expressed and purified the N-terminal His6-tagged PH-R protein in E. coli for in vitro studies. Much of the PH-R protein was found as insoluble inclusion bodies but ~10% existed in a soluble form that we purified by cobalt-chelating affinity chromatography from Nonidet P-40 lysates. We tested the ability of the PH-R domain to bind PIPs in a PLO assay 559. PVDF membranes were spotted with 0 to 50 pmoles of PI-4,5-P2, PIP3, or PI-3,4-P2 and incubated with 0 to 625 nM of His6-PH-R protein.  Figure 4.4A shows a representative PLO blot and the quantification is summarized in Figure 4.4B.  The SHIP1 PH-R domain bound to PIP3 more strongly than either PI-4,5-P2 or PI-3,4-P2.  To calculate the binding affinities of the PH-R domain to each of the phosphatidyl inositol lipids, we plotted double-reciprocal graphs 593 of the data shown in Figure 4.4B  (Figure 4.4C).  Based on this analysis, the equilibrium dissociation constant, KD, of the PH-R domain/PIP3 complex was found to be 1.9 ± 0.2 nM.  The KD values for the PI-4,5-P2 and PI-3,4-P2 could not be determined with precision by this approach giving estimates of ~10 nM for both lipids.  In parallel, we also used NMR spectroscopy to confirm that the isolated PH- R domain binds PIP3 (Figure 4.3A, blue).   As described in Chapter 3, SHIP1 is allosterically activated by its product, PI-3,4- P2, and by the small molecule agonist, AQX-MN100 via SHIP1’s C2 domain.  Because of the observed PI-3,4-P2  binding of the PH-R domain, we tested whether it might also bind AQX-MN100.   We compared the ability of the PH-R and C2 domains to bind [3H]- AQX-MN100 using SPA beads coated with recombinant PH-R or C2 protein.  Figure 4.4D shows that the C2 domain binds AQX-MN100 as previously described.  In contrast,  133 the PH-R domain and BSA negative control protein exhibit similar levels of [3H]-AQX- MN100 binding.  Thus, the PH-R domain does not measurably bind this compound.  We next determined whether the PH-R domain behaved like other PIP3-binding PH domains in cell-based assays.  FcγR-mediated phagocytosis is associated with activation of PI-3 kinase, which causes a rapid and transient accumulation of PIP3 at the phagocytic cup 471,548. The Akt-PH domain preferentially interacts with 3′ - phosphoinositides and binding of PIP3 and PI-3,4-P2 by an N-terminal GFP fusion of this domain has been well characterized in cells 554,594.  We constructed a similar N-terminal fusion protein of the SHIP1 PH-R domain and transiently expressed either Akt-PH domain-GFP, SHIP1 PH-R domain-GFP, or GFP alone in RAW264.7 macrophage cells. These transfected cells were plated onto coverslips and exposed to Ig-coated 3 µm latex beads (Ig-opsonized beads) for the indicated length of time (Figures 4.5B and 4.5C). GFP fluorescence associated with fusion domain recruitment from the cytoplasm to the phagocytic cup was then quantified by scoring micrographs with a 4 point scoring key (Figure 4.5A).  As shown in Figure 4.5C, the SHIP1-PH-R domain translocated to the forming phagocytic cup with similar kinetics to the Akt-PH domain whereas GFP alone did not exhibit any defined localization.      134                 Figure 4.4   135 Figure 4.4 SHIP1’s PH-R domain preferentially binds PIP3 but does not bind the allosteric activator AQX-MN100. (A) Ability of recombinant PH-R domain to bind to PI-4,5-P2 , PI-3,4-P2  or PIP3 in protein lipid overlay (PLO) assays.  PVDF membranes were spotted with the indicated amount of PIP and incubated PH-R domain as described in Materials and Methods.  The PLO membrane scan presented is representative of 3 independent experiments.  (B) The spots in (A) were quantified and data are expressed as mean intensities ± standard deviations (n=3)  ***P <0.001 when comparing PIP3 binding to PI-4,5-P2 or PI-3,4-P2 [Two-way ANOVA].  (C) Reciprocal of the mean intensities in (B) were plotted against the reciprocal of the amount of lipid spotted.  KD values were calculated from the slope of the lines 593 as determined by linear regression (GraphPad Prism, San Diego, CA).  (D) Ability of PH-R domain to bind [3H]-AQX-MN100.  Recombinant SHIP1 PH-R or C2 domains were coated onto copper chelating (His-Tag) YSi SPA Scintillation Beads in the presence of 0.25% BSA.  Beads were incubated with 0.185 MBq (5 µCi) of [3H]-AQX- MN100 and the bead-associated radioactivity measured.  Data are expressed as scintillation count means ± standard deviations (n=3). **P <0.01 [One-way ANOVA]             136                  Figure 4.5  137 Figure 4.5 The SHIP1 PH-R domain localizes to the phagocytic cup. RAW264.7 cells transfected with SHIP1 PH-R or AKT1 PH domain GFP fusion constructs or GFP alone were stimulated with human IgG opsonized 3 µm Latex Beads for the times indicated.  An asterisk indicates the position of the latex bead on the micrographs.  Scale bars = 10 µm.  Micrographs were scored by 3 independent, blinded investigators according to the four-point scoring key depicted (A).  Representative brightfield and confocal micrographs are shown in (B) and the mean scores of 100 phagocytosis events at each time point are presented ± standard deviations (C). **P <0.01, ***P <0.001 when comparing Akt-PH-GFP or SHIP1-PH-R-GFP to GFP alone [Two-way ANOVA]               138 4.3.3 Identification of amino acid residues involved in PIP3 binding To identify the specific amino acid residues involved in PIP3 binding, we expressed PH-R mutant proteins in which alanines were substituted for candidate residues (Figures 4.6A and Figure G.1 in Appendices).  These residues were chosen based upon their conservation among the 14 SHIP1 orthologs and their charge and/or sequence proximity to positively-charged residues, which could potentially mediate an interaction with the negatively-charged PIP3 inositol headgroup.  Unfortunately, few clues were provided from the NMR-monitored titrations shown in Figure 4.3A, as residues showing chemical shift perturbations upon PIP3 binding were distributed broadly over the model of the PH-R domain.  The lack of a clearly defined binding site could reflect indirect conformational changes of the rather dynamic protein domain (Figure 4.3B), as well as the possibility of multiple binding interfaces. As before, His6-tagged proteins were expressed in E. coli, purified, and tested for their ability to bind PIPs in a PLO assay (Figure 4.6).  Of the 10 mutant domains analyzed, two lysine to alanine mutants, K370A and K397A, exhibited an impaired ability to bind PIP3 and PI-3,4-P2 (Figure 4.6A and Figure G.1 in Appendices). The KD values for the mutant domains were indeterminable as they bound PIP3 and PI-3,4-P2 with such low affinity that we were not able to saturate binding (Figure 4.6A and Figure H.1 in Appendices).  We then constructed a double mutant domain where both K370 and K397 residues were substituted with alanines (KAKA).  The resulting KAKA PH-R mutant exhibited a further reduction in PIP3 and PI-3,4-P2 binding as compared to either of the individual point mutation domains (Figure 4.6B).  Interestingly, the wild-type (WT) and KAKA mutant proteins bound PI-4,5-P2 equally well.   The 1H-15N-HSQC  139 spectrum of the KAKA PH-R domain was also well-dispersed, indicating that the loss of PIP3 and PI-3,4-P2 binding ability is not due to a disruption of global protein structure (Figure 4.7).  Dowler et al. suggest a PIP binding motif for PH domains of the form K-X- small-X(6-11)-R/K-X-R-hydrophobic-hydrophobic 592.  The SHIP1 PH-R domain has a region including one of the critical lysines (K370) and spanning residues 334-372 that has a similar form of KDG-X(31)-JEKIL (Figure 4.3D).  The second critical lysine residue, K397 is not involved in this motif, but rather is in the C-terminal helix 592.  It is possible that K397 is part of a second binding interaction that binds PIP3 via basic residues between the C-terminal helix and the adjacent β-strand in a manner similar to phox homology (PX) domain binding 595. 4.3.4 The KAKA PH-R protein has impaired localization to the phagocytic cup We then examined the ability of the KAKA PH-R domain to be recruited to the phagocytic cup in RAW264.7 macrophages stimulated with Ig-opsonized beads.  Using the same scoring key described in Figure 4.5A, we found that the KAKA PH-R protein was unable to translocate to the phagocytic cup, suggesting that residues K370 and K397 were important in mediating the translocation and/or association of the PH-R domain with the membrane (Figure 4.6D).      140                    Figure 4.6  141 Figure 4.6 Mutation of SHIP1 PH-R domain residues K370 and K397 to alanines impairs its ability to interact with PIP3. PLO assays were performed and quantified as previously described for each of PI-(4,5)- P2, PIP3, and PI-(3,4)-P2 (spotted at the amounts indicated) with 625 nM of (A) recombinant WT SHIP1 PH-R domain, K370A, or K397A mutant PH-R domains or (B) the double-mutant KAKA PH-R domain.   Data are expressed as mean intensities ± standard deviations and are representative of 3 independent experiments. *P <0.05, **P <0.01, ***P <0.001 [Two-way ANOVA].  (C) RAW264.7 cells were transiently transfected with WT PH-R-GFP or KAKA PH-R-GFP fusion constructs and imaged as in Figure 4.5.  Micrographs were scored by 3 independent, blinded investigators according to the four-point scoring key depicted Figure 4A.  *P <0.05, ***P <0.001 [Two-way ANOVA].              142              Figure 4.7 Overlaid 1H–15N HSQC spectra of the His6-tagged wild type (blue) and K370A/K397A double mutant (red) PH-R domains at pH 5.8 and 25oC in 20 mM MES, 5 mM TCEP, and 200 mM Gdn-HCl. The wild type sample was 0.5 mM, whereas the less soluble mutant was ~0.4 mM. Both domains yield well-dispersed spectra and hence adopt defined global, tertiary structures. However, the spectral differences suggest that the mutations cause changes in the conformation of the PH-R domain.   143 4.3.5 SHIP1 with the KAKA substitution is still subject to allosteric regulation To further define the consequence of K370/K397 mutations on SHIP1 function, we expressed full-length WT SHIP1 and SHIP1 containing the KAKA mutations and compared their ability to bind PIP3 in vitro.  His6-tagged proteins were expressed and purified from Sf9 cells and their PIP3 binding ability assessed by PLO as previously described.  As seen in Figure 4.8A, KAKA SHIP1 had significantly lower PIP3 binding than WT SHIP1.  This confirms that the PH-R domain and the K370/K397 residues in particular, contribute to SHIP1’s interaction with PIP3.  We next compared the enzymatic properties of WT and KAKA SHIP1 and found their catalytic rates and specific activities indistinguishable (Figure 4.8B).  We also compared the ability of the KAKA SHIP1 enzyme to be stimulated by SHIP1’s allosteric activators, PI-3,4-P2 and AQX-MN100.  Addition of either PI-3,4-P2 or AQX-MN100 enhanced the phosphatase activity of both WT and KAKA SHIP1 (Figure 4.8C). Altogether, while K370A/K397A mutations interfere with SHIP1’s ability to interact with PIP3, its catalytic activity and allosteric activation remain unaltered, suggesting that these two residues only facilitate SHIP1’s localization to PIP3 and that this process is independent of SHIP1’s catalytic domain.      144                  Figure 4.8  145 Figure 4.8 Mutation of residues K370 and K397 to alanines in full-length SHIP1 abrogates in vitro PIP3 binding ability but does not affect its phosphatase activity. (A) PLO assays were preformed and quantified as previously described for PIP3 spotted at 50 pmols with a titration of recombinant full-length WT or KAKA SHIP1 enzyme. Data are expressed as mean intensities ± standard deviations (n=3)  *P <0.05.  (B) Initial rate enzyme kinetics for full-length WT or KAKA SHIP1 enzyme were determined using an in vitro phosphatase assay and increasing concentrations of inositol-1,3,4,5- tetrakisphosphate (IP4).  Data are representative of 3 independent experiments.  (C) in vitro phosphatase assays were performed with recombinant, full-length WT or KAKA SHIP1 with 50 µM IP4 substrate in the presence of increasing concentrations of the allosteric activators PI-(3,4)-P2 and AQX-MN100.  Data are expressed as the mean fold- increases of enzyme activity ± standard deviations (n=3).              146 4.3.6 SHIP1 with the KAKA substitution is not able to inhibit Fcγ-R mediated phagocytosis We next investigated the impact of the KAKA substitutions on the function of SHIP1 protein in FcγR-mediated phagocytosis.  Using lentivirus mediated gene transfer, we reconstituted the J2M SHIP1-/- macrophages with either full length WT or KAKA SHIP1 protein.  SHIP1 expression in the SHIP1+/+, SHIP1-/-, and SHIP1-/- cells reconstituted with WT and KAKA SHIP1 is shown in Figure 4.9A.   Note that the expression level of WT and KAKA SHIP1 are similar, but both are ~50% less than that of endogenous SHIP1 in the SHIP1+/+ cell line.  Difficulty in obtaining high-level expression of SHIP1 has been reported previously 345 and is attributed to the fact that ectopic/over-expression of SHIP1 inhibits growth and survival. Using immunoflourescence confocal microscopy, we tested the ability of full length WT and KAKA SHIP to be recruited to the phagocytic cup in the reconstituted cell lines.  Cells were incubated with latex beads opsonized with fluorescently labeled human IgG (red) for the times indicated, fixed, permeabilized and stained with anti- SHIP1 antibody followed by a fluorescently labeled secondary detection antibody (green) (Figure 4.9B).  Similar to SHIP1+/+ cells, cells reconstituted with WT enzyme exhibited pronounced fluorescence at the phagocytic cup consistent with SHIP1’s translocation from the cytoplasm to accumulating levels of PIP3 at the cell membrane.  However, cells reconstituted with KAKA enzyme had significantly lower mean fluorescence scores than SHIP1+/+ cells or cells reconstituted with WT enzyme, indicating that K370A and K397A mutations impair SHIP1’s localization to PIP3 being produced at the phagocytic cup.  147 As described by others, SHIP1-/- cells phagocytose more rapidly and ingest more latex beads per cell than SHIP1+/+ cells 396,555.  This suggests that SHIP1 has a role in the negative regulation of FcγR-mediated phagocytosis.  To correlate our observations of impaired SHIP1 recruitment in KAKA SHIP1 reconstituted cells with phagocytic function, we measured the phagocytic index (defined as the average number of beads phagocytosed per cell) of our SHIP1+/+, SHIP1-/- cells, and WT and KAKA SHIP1 reconstituted cells.  As seen in the representative micrographs and quantification in Figure 4.10, reconstitution of SHIP1-/- cells with KAKA SHIP1 failed to reduce phagocytic activity to WT levels.  In comparison, cells reconstituted with WT SHIP1 had a partial reduction of phagocytic activity to WT levels.  This partial phenotype is likely attributed to the fact that reconstituted cells had overall lower expression levels of SHIP1 than SHIP1+/+ cells.          148                  Figure 4.9  149 Figure 4.9 SHIP1 with residues K370 and K397 mutated to alanines has impaired recruitment to the phagocytic cup. (A) Lysates from J16 SHIP+/+ (SHIP+/+), J17 SHIP-/- (SHIP-/-), and cells transduced with lentivirus encoding WT SHIP1 (SHIP1-/-:WT) or mutant SHIP 1 (SHIP1-/-:KAKA) were subjected to immunoblot analysis with anti-SHIP1 (P1C1) primary antibody and anti- STAT3 as a loading control.  Band intensities were determined using Biorad Quantity One Software.  (B) SHIP1+/+, SHIP1-/-, SHIP1-/-: WT and SHIP1-/-:KAKA cells were stimulated with AlexaFluor680-labelled, human IgG-opsonized 15 µm Latex Beads (Red) for the times indicated.  Cells were fixed, permeabilized and stained with anti-SHIP1 (P1C1) primary followed by FITC-labelled secondary detection antibody (green) and counterstained with the DNA binding dye DAPI (blue). Scale bars = 10 µm. Micrographs were scored by 3 independent, blinded investigators according to the four- point scoring key previously described.  Representative micrographs are presented (left) and the mean scores of 100 phagocytosis events at each time point are presented ± standard deviations (right). *P <0.05 when comparing to either SHIP1+/+ or SHIP1-/-: WT cells [Two-way ANOVA].             150             Figure 4.10 SHIP1 with residues K370 and K397 mutated to alanines cannot restore normal regulation of FcγR-mediated phagocytosis. SHIP1+/+, SHIP1-/-, SHIP1-/-: WT and SHIP1-/-:KAKA cells were stimulated with human IgG-opsonized 3 µm latex beads for the times indicated.  Representative micrographs are presented (upper) and phagocytic indices at each time point quantified ± standard deviations (lower) Scale bars = 10 µm.  Micrographs were scored by 3 independent, blinded investigators.  *P <0.05, **P <0.01, ***P <0.001 [Two-way ANOVA].    151 4.4 Discussion FcγR-mediated phagocytosis is essential for removal of antibody-opsonized foreign particles.  Upon ligation of FcγR on the macrophage cell membrane, ITAMs on the cytoplasmic tail of the FcγR become phosphorylated and provide docking sites for SH2-domain containing signaling proteins including Syk 596,597, PI-3 kinase 471,597, and PKC 598.  Once recruited to the membrane, these proteins initiate dynamic changes in membrane lipid composition and the actin cytoskeleton necessary for phagocytic cup formation, particle internalization, and phagosome maturation.  SHIP1 has also been demonstrated to be recruited to the phagocytic cup via interaction of its SH2 domain with phosphotyrosines within ITAMs where it negatively regulates phagocytosis, presumably by degrading PIP3 349,432 though there may also be contributions of other signaling proteins recruited by virtue of SHIP1’s capacity to act as an adapter protein via its multiple protein interaction domains.  SHIP1 has additionally been shown to interact with the tyrosine phosphorylated ITIMs on the inhibitory FcγRIIb 352,414,557. We have characterized a new domain in SHIP1, termed the PH-R domain, which possesses structural and functional similarities with members of the PH domain family. Although PH domains have been described to bind a variety of ligands, their interaction with phosphoinositol lipids are the best characterized 599.  As shown herein, SHIP1’s PH- R domain binds to PIP3 and also to PI-3,4-P2 and PI-4,5-P2, albeit with lower affinity, and the interaction with PIP3 is mediated either directly or indirectly by at least 2 key lysine residues at positions 370 and 397.  The 1H-15N-HSQC NMR spectra of wild-type and KAKA PH-R domains confirmed that mutation of these two lysines does not interfere with global conformation or proper folding.  However, both we and Edlich et al. 600 have  152 observed numerous spectral differences between wild-type and lysine residue substituted domains which suggest that the substitutions themselves cause some conformational perturbations.  Interestingly, while mutation of these two residues to alanines significantly reduces the ability of the PH-R domain to interact with PIP3 and PI-3,4-P2, binding to PI-4,5-P2 remains relatively unaltered.  If K370 and K397 are directly involved in lipid binding, then these positively-charged lysine residues could possibly facilitate stable interaction with PIP3 via co-ordination with the negatively-charged phosphate at the 3′ position of the inositol headgroup whereas other residues in the PH- R domain are required for binding to PI-4,5-P2. The binding of PIP3 and PI-3,4-P2 by the PH-R domain could serve several functions.  PIP3 is a SHIP1 substrate so the PH-R domain might recruit PIP3 to the enzyme and transfer it to the phosphatase domain.  However, this seems unlikely since although substituting lysine residues 370 and 397 with alanine significantly impairs the ability of the domain to bind PIP3, this does not alter SHIP1’s basal phosphatase activity. PI-3,4-P2, a product of SHIP1 hydrolysis of PIP3, is an allosteric activator of SHIP1 phosphatase activity 559 so the PH-R domain might be involved in PI-3,4-P2-mediated activation of SHIP1’s enzymatic activity.  However, this also seems unlikely since although the KAKA mutant is impaired in its ability to bind PI-3,4-P2, it retains its ability to be activated by both PI-3,4-P2  and AQX-MN100. The observation that mutations in the PH-R domain have no impact on SHIP1’s phosphatase activity is consistent with the literature reports on mutations, deletions, and truncations made in regions outside of the core 5′-phosphatase domain 360,387,601-604.  In  153 a study by Aman et al. 604 in which a series of SHIP1 truncations and deletions were expressed, they found the non-catalytic C-terminal region extending beyond amino acid residue 900 was necessary for FcγRIIB-mediated inhibition of calcium flux in DT40 chicken B-cells.  Interestingly, reconstitution of SHIP1-/- cells with a SHIP1 C-terminal truncation at residue 900 (SHIP1-900) was able to partially restore inhibition of calcium flux whereas a construct lacking both the presently characterized PH-R domain and the C-terminal domain (SH2-18aa-401-900) was not.  These results support our identification of SHIP1’s PH-R domain and suggest that in addition to the non-catalytic C-terminal region, SHIP1’s PH-R domain may also contribute to stabilizing the interaction between SHIP1 and FcγRIIB indirectly via binding to membrane PIP3. We have additionally demonstrated that the PH-R domain contributes to SHIP1’s localization to the phagocytic cup during FcγR-mediated phagocytosis.  SHIP1 transiently localizes to the phagocytic cup at the early stages of cup formation and is sequestered at the leading edge of the phagosome 396,399,554.  Our data show that despite having a functional SH2 domain capable of interacting with FcγR ITAM, KAKA SHIP1 has a significantly impaired ability to translocate to the cell membrane in cells stimulated with Ig-opsonized beads.  These findings suggest that SHIP1’s SH2 domain is not sufficient to mediate SHIP1’s recruitment to the phagocytic cup.  Zhang et al. 605 have recently proposed a model whereby the density of FcγR activation determines early- stage signals such as the recruitment of Syk, while later stages of FcγR signaling, i.e. those that ultimately commit the cell to complete phagocytosis of a particle, are dependent on 3′ PI levels (e.g. PIP3 and PI-3,4-P2) in unclosed phagocytic cups.  It is thus possible that, similarly to Syk, relatively few molecules of SHIP1 are recruited to the  154 FcγR upon ligand binding via SHIP1’s SH2 domain interacting with tyrosine phosphorylated ITAM/ITIM motifs.  The number of SHIP1 molecules recruited during the early stage is thus directly related to the number of FcγRs engaged, which in turn is determined by the density of IgG opsonization on the particles.  Subsequently, only phagocytic cups having accumulated a threshold number of PIP3 molecules are able to successfully commit to the formation of a phagosome around a particle and concomitantly recruit large amounts of SHIP1 to the phagocytic cup possibly via its newly identified PH-R domain. Our current understanding of phagocytosis has moved beyond the classical “zipper” model where engulfment occurs as receptors bind ligands sequentially along the surface of the particle being ingested.  A review by Jaumouillé and Grinstein 606 eloquently described a model that considers the actin cytoskeletal contributions to the regulation of phagocytosis by partitioning or “fencing-off” regions of membrane to limit the lateral movement of FcγRs.  While actin polymerization is required for the initial filopodial extensions that are necessary for the identification and attachment of opsonized particles, their model suggests that underlying cortical actin barriers must be broken down in order for FcγR to move laterally and form the receptor clusters responsible for initiating the signaling cascade and actin remodeling that drives particle internalization. Applying this model to our own observations of the PH-R domain dependent translocation of SHIP1 to the plasma membrane, we suggest that SHIP1 can be recruited to the membrane through both SHIP1-SH2/FcγR phosphotyrosine and SHIP1-PH-R/PIP3 interactions.   However, the actin partitions, which restrict lateral movement of transmembrane proteins, prevent the aggregation of FcγR molecules and thus limit the  155 amount of FcγR ITAM/ITIM-bound SHIP1 to densities too low to be detected by conventional immunofluorescence microscopy.  On the other hand, it is possible that similar to what has been reported for PI-4,5-P2 607, PIP3 may not be subject to the same restrictions in lateral motion by actin and thus the density of PIP3 would be high enough such that the SHIP1 recruited (via its PH-R domain) is easily visualized at the phagocytic cup. Identification and characterization of SHIP1’s PH-R domain now provides a more detailed illustration of the mechanism whereby SHIP1 translocates to the membrane upon FcγR ligation and lends further insight into how SHIP1 contributes to modifications in membrane lipid composition and actin rearrangements that collectively promote particle engulfment.  By directly interacting with PIP3 being generated at the phagocytic cup, the PH-R domain facilitates SHIP1’s rapid enrichment at the leading edge where it can hydrolyze PIP3 to PI-3,4-P2 and thus regulate the progression of stages through phagosome maturation. The degradation of PIP3 at the phagocytic cup may be only one of many mechanisms by which SHIP1 negatively regulates FcγR mediated signaling and phagocytosis.  A study by Ganesan et al. has shown that SHIP1’s non-catalytic function regulates Ras/Erk dependent induction of Interleukin-1β upon FcγR stimulation 397. Mehta et al. recently described the association of SHIP1 with LyGD1, a Rho guanidine dissociation inhibitor that prevents the membrane association of the small GTPase Rac and thus negatively influences actin assembly needed for phagosome formation 608.  In the same study, the authors reported the identification of several other proteins that uniquely interacted with SHIP1 and that many had known functions in regulating actin  156 re-organization.  The PIP3–mediated membrane localization of SHIP1 may not be limited solely to FcγR signaling and future studies will seek to determine whether the PH-R domain also mediates SHIP1’s recruitment to other cellular compartments and in response to other cell stimuli 420,609.   It will also be interesting to find small molecules which modify PH-R domain interactions with PIP3 as these may have potential therapeutic applications.          157       CHAPTER 5: CONCLUSION                   158 5.1 Conclusion  The work presented in this thesis describes hitherto unappreciated mechanisms by which SHIP1 mediates IL-10’s anti-inflammatory action and how SHIP1 activity itself is regulated within immune cells.  Both IL-10 and SHIP1 are known to have significant contributions to proper immune cell function as exemplified by animal knock-out models and in human pathologies associated with mutations within the loci of these genes.  Thus, a better understanding of how IL-10 signals in cells, the nature of the role SHIP1 plays in IL-10 signaling, and how SHIP1 activity can be modulated, will provide insights towards developing targeted therapeutics with application in treating of a variety of inflammatory diseases. In the current literature, all of IL-10’s signaling is believed to be mediated by STAT3, which then upregulates the expression of specific gene products.  These IL-10 induced products, in turn, negatively regulate immune cell activation at the level of transcription.  However, expanding upon work initiated by previous graduate students in our lab, the experiments described in Chapter 2 characterize a STAT3-independent signaling pathway utilized by IL-10 whereby IL-10 activates SHIP1, the predominant lipid phosphatase in immune cells responsible for countering PI-3 kinase-generated PIP3 via degradation of PIP3 to PI-3,4-P2.  By opposing the PI-3 kinase pathway, IL-10 induced activation of SHIP1 negatively regulates the activation of downstream proteins, such as Akt, and inhibits the transcription of a number of PRGs, which are among the earliest genes whose expressions are upregulated in response to immune cell stimulation by TLR-agonists.  159   The role of the PI-3 kinase pathway in immune cell activation - macrophages especially - has been a point of contention for many researchers.  Data from our lab strongly supports a positive role for the PI-3 kinase pathway in macrophage activation and we propose that differences in experimental design account for the contrary observations made by other groups.  One of the major factors contributing to this discrepancy is the fact that LPS stimulation of macrophages, in addition to up-regulating the expression of PRGs, also induces the expression of IL-10 within 2 hours.  The autocrine action of IL-10 can potentially confound interpretation of pro-inflammatory cytokine production and the role of PI-3 kinase signaling in regulating their production (particularly evident at longer timepoints).  To address the issue of autocrine cytokine production and signaling, we have adapted a perifusion system frequently employed in diabetes research to observe glucose-induced changes in pancreatic beta-cell insulin production, to supply continuous stimulation to macrophage monolayers and effectively remove cytokines that they produce from the extracellular milieu.  This continuous-flow apparatus allows us to profile changes in cytokine production over time. Use of the continuous-flow apparatus revealed several fascinating features of LPS-stimulated TNFα production.  Firstly, within the first 2 hours of stimulation, TNFα production occurs in 2 phases with peak concentrations at approximately 50 and 110 minutes post-stimulation.  Secondly, in conjunction with SHIP1 KO cells and inducible siRNA knockdown cell lines, we observed that IL-10 was able to inhibit the initial phase of TNFα production only in the presence of SHIP1.  STAT3 siRNA knockdown cells, however, were fully responsive to IL-10 mediated inhibition of this first peak indicating that STAT3 is not required for inhibition of this first influx of TNFα.  While, we concede  160 that the ~57% knockdown of STAT3 achieved using the inducible siRNAs is modest, it nevertheless appears to be sufficient to significantly impair IL-10’s ability to inhibit the second peak of TNFα production.  Thus, IL-10 appears to inhibit the first and second phases of TNFα differentially via a SHIP1-dependent and STAT3-dependent pathways respectively. In the second chapter, we have also shown that via the SHIP1-dependent pathway, IL-10 inhibits the phosphorylation and activation of CDK9, and the subsequent phosphorylation of RNAPolII at Ser2.  Phosphorylation at Ser2 is necessary for switching RNAPolII from synthesizing low levels of full-length unspliced transcripts to producing high levels of mature, spliced, protein-coding mRNAs.  However, IL-10 was unable to inhibit CDK9 phosphorylation or subsequent RNAPolII Ser2 phosphorylation in cells expressing SHIP1 siRNA.  SHIP1 thus appears to be necessary for mediating IL-10’s inhibition of transcriptional elongation of PRGs like TNFα.  Interestingly, we made the additional observation that in SHIP1 KO PMΦs, and SHIP1 siRNA expressing cells, low- dose IL-10 treatment of LPS-stimulated cells enhanced rather than inhibited the mRNA expression of 11/17 PRGs.  These findings suggest that IL-10 signaling through SHIP1 is not only necessary for mediating IL-10’s early phase anti-inflammatory action but is required for suppressing IL-10’s augmentation of pro-inflammatory gene expression as well.  The enhanced production of TNFα mRNA in SHIP1 deficient cells is reflected by enhanced TNFα protein production during the first peak of expression detected in continuous-flow culture macrophages.   Our observations of the requirement for SHIP1 in IL-10 action in vitro were recapitulated in vivo in the mouse endotoxemia model.   IL-10  161 inhibited LPS induced production of TNFα and CCL2 levels in wild-type but not SHIP1 KO mice. Taken together, these results recommend a revision of the current, prevailing opinion in the literature for the absolute requirement of STAT3 in IL-10 signaling.  While our results are consistent with STAT3 being necessary for mediating IL-10’s inhibition of TNFα at longer timepoints post-LPS stimulation - corresponding with the second phase of TNFα production – we have demonstrated that STAT3 is dispensable for IL-10’s inhibition of the first peak of TNFα production within the first hour post-stimulation. However, the presence of SHIP1 is required for IL-10 mediated inhibition of this first phase of TNFα production, and in SHIP1’s absence, IL-10 actually has the capacity to enhance the expression of TNFα and other PRGs. The results described in Chapter 2 open a number of avenues for future investigation.  Firstly, of the potential sources of discrepancy with regards to differing accounts for the role of PI-3 kinase signaling in macrophage activation, we have yet to investigate the contribution of the individual PI-3 kinase p110 isoforms.  It is entirely possible that specific p110 isoforms predominate in response to LPS stimulation as has been previously reported by other groups who have employed the new isoform specific PI-3 kinase inhibitors to delineate their respective roles 401,610.  It is equally feasible that SHIP1 may specifically target different cellular pools of PIP3 being generated by a particular p110 isoform.  In this manner, SHIP1 activity may serve to negatively regulate some PI-3 kinase signaling events which positively contribute to macrophage activation, while others are independent of SHIP1 regulation.  Combinatorial use of the isoform specific inhibitors and/or siRNAs in conjunction with SHIP1 KO or siRNA expressing  162 cells may facilitate a better understanding of the relationship between p110 isoforms and SHIP1 activity.  Secondly, by using the continuous-flow culture apparatus, we were able to collect culture supernatant fractions at specific time points and apply them to naïve cultures.  We observed that there is a soluble factor produced within the first phase of TNFα production that is necessary for maximal induction of the second peak. Identification of this factor may provide further insight into the dynamic sequence of events during the early stages of LPS stimulation.  Thirdly, we discovered that a subset of PRGs as described by Hargreaves et al. 213,214 are induced by low-dose treatment of IL-10 in SHIP1 KO cells rather than inhibited as they are in WT cells.  These data lead to the question of whether there are shared elements amongst the promoters of these PRGs – a shared sequence that recruits a “master” transcription factor, for example, that in the absence of SHIP1, mediates a pro-inflammatory response to IL-10 instead of a deactivating response.  Bioinformatic analysis of the promoter regions of the PRGs and luciferase reporter assays may help to elucidate these possibilities.  The studies proposed are in line with the growing appreciation for the organization of innate immune responses into transcriptional programs 210,213,214,611,612.  Such programs allow for rapid and elegant control of genes that together perform similar or related functions.  Thus, it is possible that SHIP1 may serve as a key regulator of an arm of the TLR-induced transcriptional program.  Alternatively, these PRGs may be differentially regulated at the post- transcriptional level.  miRNAs are known to have integral roles in controlling the inflammatory response 613-618.  High through-put microarray analysis of the mRNA and microRNA profiles of LPS +/- IL-10 treated macrophages from SHIP1+/+ and SHIP1-/-  163 mice may reveal if IL-10 is capable of regulating these immunoregulatory molecules in a SHIP1-dependent manner. Having observed that IL-10 does indeed signal through STAT3-independent pathways and that SHIP1is necessary for IL-10’s early phase signal transduction, in Chapter 3 I investigated whether it was possible to activate SHIP1 using small molecule agonists and whether by doing so, we could mimic the biological activity of IL-10 to inhibit inflammation in mouse models where IL-10 is lacking.  Pelorol, a compound that enhanced SHIP1 activity in a high throughput in vitro SHIP1 assay, was identified from a marine invertebrate extract library as a SHIP1 agonist and used as a starting point for derivatization to produce more active compounds.    One of these derivatives, AQX- MN100 exhibited approximately 8-fold higher enhancement of SHIP1 phosphatase activity than its parent compound and inhibited inflammatory cell activation in vitro. AQX-MN100 is specific for SHIP1 as it does not inhibit TNFα production in macrophages lacking SHIP1 nor does it exhibit any appreciable effects when screened against an in vitro panel of 100 other kinases and phosphatases.  AQX-MN100 was also effective at reducing inflammation in several in vivo murine models including LPS- induced septic shock, DNFB-induced cutaneous anaphylaxis, and in IL-10-/- mouse colitis. Through the course of the in vitro studies described in Chapter 3, we noticed that SHIP1 exhibits a unique enzyme kinetics profile.  Unlike most enzymes, which typically display standard Michaelis-Menton saturation kinetics, SHIP1 exhibited a sigmoidal profile, which is the hallmark of enzymes subject to end-product allosteric activation. Indeed, when we added exogenous PI-3,4-P2, SHIP1’s product, to the in vitro enzyme  164 reaction, SHIP1’s phosphatase activity is increased to the same degree as the enhancement observed when AQX-MN100.  Bioinformatic analysis of SHIP1’s sequence revealed a previously unidentified structured region immediately C-terminal of SHIP1’s central catalytic domain with predicted features similar to lipid-binding C2 domains.  We determined that the C2 domain binds both SHIP1’s end-product, PI-3,4-P2, and AQX- MN100 in a manner similar to the allosteric activation mechanisms described for other lipid phosphatases like PTEN and MTM, The data presented in Chapter 3 illustrate an additional means of regulating SHIP1’s activity that has not previously been appreciated.  In the current literature, it is believed that SHIP1’s activity is constitutively active and is regulated solely by its subcellular localization.  In response to activation of membrane receptors, SHIP1 is believed to translocate to the membrane via protein-protein interactions mediated by its SH2 domain and C-terminal PRR with adaptor proteins or membrane receptors directly. At the membrane, SHIP1 then has access to its PIP3 substrate.  However, we have discovered that SHIP1 can be allosterically activated by PI-3,4-P2 and by small molecule SHIP1 agonists thus providing an additional level of regulation.  We propose a model whereby SHIP1 is recruited by phosphorylated membrane receptors and adaptor proteins to the localized site of PIP3 accumulation.  At this stage, SHIP1 activity is modest and is only capable of converting small amounts of PIP3 to PI-3,4-P2.  These small amounts of PI-3,4-P2 however, are capable of binding to the C2 domain, inducing a conformational change that makes the enzyme active site more accessible to substrate.  SHIP1 is then able to rapidly degrade the remaining PIP3 from the membrane.  165 Identification of SHIP1’s C2 domain and allosteric activation mechanism has exciting implications towards the development of a new class of anti-inflammatory compounds similar to AQX-MN100 that manipulate SHIP1’s activity in cells.  Not only can AQX-MN100 effectively suppress immune cell activation and pro-inflammatory cytokine production in inflammatory disease models, but they would also possess an added advantage over conventional immunsuppressive agents.  By specifically targeting SHIP1, an enzyme primarily expressed in hemopoietic cells, SHIP1 agonists would avoid many of the side-effects attributed with the more conventional immunsuppressive agents such as glucocorticoids and mTOR inhibitors (e.g. Rapamycin/Sirolimus) whose drug targets are ubiquitously expressed throughout the body.  Further, targeting an allosteric regulatory site confers even greater drug specificity as most small molecule drugs are enzyme inhibitors, which target enzyme active sites that are required to be more structurally conserved amongst related enzymes in order to perform a catalytic function. Allosteric regulatory sites, on the other hand, are not as structurally constrained. While our PLO and SPA assays provide strong support for direct binding of PI- 3,4-P2 and AQX-MN100 to SHIP1’s C2 domain, we still lack structural information as to the exact nature of these interactions.  We have made several attempts to analyze the C2 domain’s structure using NMR spectroscopy, however, the domain is insoluble under the buffer conditions required for NMR analysis.  Thus, future studies may investigate the structure of the C2 domain using other techniques such as X-ray crystallography.  By co- crystallizing the C2 domain with and without PI-3,4-P2 or AQX-MN100, we may identify the specific amino acid residues required for mediating binding between SHIP1 and its allosteric activators.  Further, we could express the C2 domain in context with SHIP1’s  166 phosphatase domain to characterize the conformational changes induced by allosteric binding of PI-3,4-P2 or AQX-MN100 and how these structural modications lead to enhanced phosphatase activity.  These data in turn may allow us to perform rational drug design to generate more efficacious small molecule SHIP1 activators. However, characterization of AQX-MN100 as a SHIP1 allosteric activator itself opens many opportunities for future investigation.  Having a specific small molecule SHIP1 agonist allows us to further dissect the role of SHIP1 in inflammatory and immune cell processes.  Of note, the immune defects associated with SHIP1 KO mice are primarily attributed to perturbations in the myeloid cell compartment 405,406.  Therefore, we can employ AQX-MN100 as a tool to further characterize the function of SHIP1 in myeloid cell development and its function in response to various inflammatory stimuli. One area of particular interest is SHIP1’s role in the development of myeloid suppressor cells (aka MSCs, Myeloid-Derived Suppressor Cells, Myeloid Immune Suppressor Cells, MIR).  Several reports have provided evidence that SHIP1 deficiency gives rise to increased numbers of MSCs 395,402,403.  In the IL-10-/- mouse colitis experiments described in Chapter 3, in addition to colon sections, we collected lymph nodes and spleens and preliminary flow cytometry experiments indicated that MSC numbers (defined by gating on CD11b+Gr1+ events) were modulated upon treatment with AQX-MN100 (data not shown).  However, further experimentation needs to be performed to confirm these observations and to confirm the suppressive function of these cells.  Intriguingly, it has also been reported that SHIP1 is required for TH17 cell development, a T-cell sub-type known to have a positive role in the development of IBD 430,619.  Because of our data indicating that SHIP1 activation via treatment with AQX-MN100 is protective in colitis  167 and inhibits production of IL-17, it would be of great interest to determine if, similarly to its action on myeloid suppressor cells, AQX-MN100 modulates TH17 populations as well. In addition to the C2 domain, I have characterized a previously unrecognized PH- like domain residing N-terminal of SHIP1’s phosphatase domain.  Results described in Chapter 4 indicate that SHIP1’s PH-like domain exhibits a binding specificity for PIP3 and that it is necessary for regulating SHIP1’s membrane localization to the phagocytic cup during FcγR-mediated phagocytosis.  Generation of SHIP1 enzyme where point mutations were made that abrogated PIP3 binding activity further demonstrated that despite having intact, functional, SH2 domains and PRRs, mutant SHIP1 recruitment to the phagocytic cup was still impaired.  These data suggest that SHIP1’s PH-like domain is one of the primary contributors to its rapid recruitment to the PIP3-rich phagocytic cup. Thus, similarly to the C2 domain, it may be possible to develop small molecule drugs targeting the PH-like domain to regulate SHIP1 activity by modulating its membrane recruitment to membrane PIP3 pools. Although PH domains have been best characterized for their interactions with lipids, the PH domains of several other signaling proteins have been reported to mediate protein-protein interactions as well (reviewed in 620).  In particular, the PH domains of Btk and PLCβ have been reported to interact with filamentous actin 621,622.  Initial studies with SHIP1’s PH-R domain have similarly demonstrated an ability to bind actin (data not shown).  Future NMR studies of SHIP1’s PH-R domain in the presence and absence of filamentous actin could further characterize this interaction.  Perhaps of greater interest, however, is investigation into whether PH-R domain/Actin interactions are required for  168 proper SHIP1 activity.  Co-localization studies using confocal microscopy may yield added insight into an actin-dependent regulatory mechanism of SHIP1 function possibly by coordinating its shuttling from cytoplasmic compartments to the membrane or by influencing its lateral movement in membrane corrals as described by Jamouillé and Grinstein 606. In summary, the contents of this thesis describe a number of previously unrecognized mechanisms of immune cell regulation namely: IL-10’s inhibition of the early phase of pro-inflammatory cytokine production via SHIP1, allosteric activation of SHIP1 by binding of its newly identified C2 domain with its natural end-product, and membrane recruitment of SHIP1 mediated by direct interactions with its newly characterized PH-R domain and membrane lipids.  Data presented also demonstrate that some of these mechanisms can be exploited by small molecule drug agonists.  SHIP1 activators thus represent an exciting new class of anti-inflammatory compounds, which would have obvious clinical applications but could additionally be used as a molecular tool to further characterize the role of this important regulatory phosphatase in immune cell function.         169 BIBLIOGRAPHY 1. Majno, G. The healing hand : man and wound in the ancient world, (Harvard University Press, Cambridge, Mass., 1975). 2. Rather, L.J. Disturbance of function (functio laesa): the legendary fifth cardinal sign of inflammation, added by Galen to the four cardinal signs of Celsus. Bull N Y Acad Med 47, 303-322 (1971). 3. Janeway, C.A., Jr. & Medzhitov, R. Innate immune recognition. Annu Rev Immunol 20, 197-216 (2002). 4. Medzhitov, R. & Janeway, C.A., Jr. Innate immune recognition and control of adaptive immune responses. Semin Immunol 10, 351-353 (1998). 5. Medzhitov, R. & Janeway, C.A., Jr. Self-defense: the fruit fly style. Proc Natl Acad Sci U S A 95, 429-430 (1998). 6. Medzhitov, R., Preston-Hurlburt, P. & Janeway, C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394-397 (1997). 7. Gallucci, S. & Matzinger, P. Danger signals: SOS to the immune system. Curr Opin Immunol 13, 114-119 (2001). 8. Matzinger, P. Tolerance, danger, and the extended family. Annu Rev Immunol 12, 991-1045 (1994). 9. Matzinger, P. The danger model: a renewed sense of self. Science 296, 301-305 (2002). 10. Medzhitov, R. Inflammation 2010: new adventures of an old flame. Cell 140, 771-776 (2010). 11. McEver, R.P. & Zhu, C. Rolling cell adhesion. Annu Rev Cell Dev Biol 26, 363- 396. 12. Wang, F. The signaling mechanisms underlying cell polarity and chemotaxis. Cold Spring Harb Perspect Biol 1, a002980 (2009). 13. Rose, D.M., Alon, R. & Ginsberg, M.H. Integrin modulation and signaling in leukocyte adhesion and migration. Immunol Rev 218, 126-134 (2007). 14. Wong, C.H., Heit, B. & Kubes, P. Molecular regulators of leucocyte chemotaxis during inflammation. Cardiovasc Res 86, 183-191. 15. Imhof, B.A. & Aurrand-Lions, M. Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 4, 432-444 (2004). 16. Serhan, C.N. & Savill, J. Resolution of inflammation: the beginning programs the end. Nat Immunol 6, 1191-1197 (2005). 17. Marshall, J.C. Inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome. Crit Care Med 29, S99-106 (2001). 18. Papadakis, K.A. & Targan, S.R. Role of cytokines in the pathogenesis of inflammatory bowel disease. Annu Rev Med 51, 289-298 (2000). 19. Itzkowitz, S.H. & Yio, X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol 287, G7-17 (2004). 20. Gordon, S. The macrophage: past, present and future. Eur J Immunol 37 Suppl 1, S9-17 (2007). 21. Taylor, P.R., et al. Macrophage receptors and immune recognition. Annu Rev Immunol 23, 901-944 (2005).  170 22. Gordon, S. The role of the macrophage in immune regulation. Res Immunol 149, 685-688 (1998). 23. Koh, T.J. & DiPietro, L.A. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med 13, e23. 24. Van Furth, R., Diesselhoff-den Dulk, M.C. & Mattie, H. Quantitative study on the production and kinetics of mononuclear phagocytes during an acute inflammatory reaction. J Exp Med 138, 1314-1330 (1973). 25. Passlick, B., Flieger, D. & Ziegler-Heitbrock, H.W. Identification and characterization of a novel monocyte subpopulation in human peripheral blood. Blood 74, 2527-2534 (1989). 26. Ziegler-Heitbrock, H.W., et al. The novel subset of CD14+/CD16+ blood monocytes exhibits features of tissue macrophages. Eur J Immunol 23, 2053-2058 (1993). 27. Ziegler-Heitbrock, H.W., et al. Small (CD14+/CD16+) monocytes and regular monocytes in human blood. Pathobiology 59, 127-130 (1991). 28. Palframan, R.T., et al. Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues. J Exp Med 194, 1361-1373 (2001). 29. Ziegler-Heitbrock, L., et al. Nomenclature of monocytes and dendritic cells in blood. Blood 116, e74-80. 30. Ziegler-Heitbrock, L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 81, 584-592 (2007). 31. Ziegler-Heitbrock, H.W. Definition of human blood monocytes. J Leukoc Biol 67, 603-606 (2000). 32. Ziegler-Heitbrock, H.W. Heterogeneity of human blood monocytes: the CD14+ CD16+ subpopulation. Immunol Today 17, 424-428 (1996). 33. Raffetseder, U., et al. Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1. Kidney Int 75, 185- 196 (2009). 34. Weber, C., et al. Differential chemokine receptor expression and function in human monocyte subpopulations. J Leukoc Biol 67, 699-704 (2000). 35. Grage-Griebenow, E., et al. Identification of a novel dendritic cell-like subset of CD64(+) / CD16(+) blood monocytes. Eur J Immunol 31, 48-56 (2001). 36. Grage-Griebenow, E., Flad, H.D. & Ernst, M. Heterogeneity of human peripheral blood monocyte subsets. J Leukoc Biol 69, 11-20 (2001). 37. Geissmann, F., Jung, S. & Littman, D.R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71-82 (2003). 38. Saha, P. & Geissmann, F. Toward a functional characterization of blood monocytes. Immunol Cell Biol 89, 2-4. 39. Auffray, C., Sieweke, M.H. & Geissmann, F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol 27, 669- 692 (2009). 40. Geissmann, F., et al. Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses. Immunol Cell Biol 86, 398-408 (2008).  171 41. Auffray, C., et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317, 666-670 (2007). 42. McCusker, K. & Hoidal, J. Characterization of scavenger receptor activity in resident human lung macrophages. Exp Lung Res 15, 651-661 (1989). 43. Palecanda, A., et al. Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles. J Exp Med 189, 1497-1506 (1999). 44. Taylor, P.R., et al. The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J Immunol 169, 3876-3882 (2002). 45. Newman, S.L., Musson, R.A. & Henson, P.M. Development of functional complement receptors during in vitro maturation of human monocytes into macrophages. J Immunol 125, 2236-2244 (1980). 46. Stout, R.D., et al. Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences. J Immunol 175, 342-349 (2005). 47. Stout, R.D. & Suttles, J. Functional plasticity of macrophages: reversible adaptation to changing microenvironments. J Leukoc Biol 76, 509-513 (2004). 48. Quinn, J.M. & Gillespie, M.T. Modulation of osteoclast formation. Biochem Biophys Res Commun 328, 739-745 (2005). 49. Smith, P.D., Ochsenbauer-Jambor, C. & Smythies, L.E. Intestinal macrophages: unique effector cells of the innate immune system. Immunol Rev 206, 149-159 (2005). 50. Smythies, L.E., et al. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. J Clin Invest 115, 66-75 (2005). 51. Heinsbroek, S.E. & Gordon, S. The role of macrophages in inflammatory bowel diseases. Expert Rev Mol Med 11, e14 (2009). 52. Gordon, S. & Taylor, P.R. Monocyte and macrophage heterogeneity. Nat Rev Immunol 5, 953-964 (2005). 53. Mantovani, A., Sica, A. & Locati, M. New vistas on macrophage differentiation and activation. Eur J Immunol 37, 14-16 (2007). 54. Martinez, F.O. Regulators of macrophage activation. Eur J Immunol 41, 1531- 1534. 55. Benoit, M., Desnues, B. & Mege, J.L. Macrophage polarization in bacterial infections. J Immunol 181, 3733-3739 (2008). 56. Gordon, S. & Martinez, F.O. Alternative activation of macrophages: mechanism and functions. Immunity 32, 593-604. 57. Martinez, F.O., Helming, L. & Gordon, S. Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol 27, 451-483 (2009). 58. Mantovani, A., et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677-686 (2004). 59. Hume, D.A. Differentiation and heterogeneity in the mononuclear phagocyte system. Mucosal Immunol 1, 432-441 (2008). 60. Wang, X. & Quinn, P.J. Lipopolysaccharide: Biosynthetic pathway and structure modification. Prog Lipid Res 49, 97-107.  172 61. Fujihara, M., et al. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol Ther 100, 171-194 (2003). 62. Fujihara, M., Muroi, M., Muroi, Y., Ito, N. & Suzuki, T. Mechanism of lipopolysaccharide-triggered junB activation in a mouse macrophage-like cell line (J774). J Biol Chem 268, 14898-14905 (1993). 63. Magalhaes, P.O., et al. Methods of endotoxin removal from biological preparations: a review. J Pharm Pharm Sci 10, 388-404 (2007). 64. Barton, G.M. & Medzhitov, R. Toll-like receptors and their ligands. Curr Top Microbiol Immunol 270, 81-92 (2002). 65. Kawai, T. & Akira, S. TLR signaling. Cell Death Differ 13, 816-825 (2006). 66. Lang, T. & Mansell, A. The negative regulation of Toll-like receptor and associated pathways. Immunol Cell Biol 85, 425-434 (2007). 67. Pasare, C. & Medzhitov, R. Toll-like receptors: linking innate and adaptive immunity. Adv Exp Med Biol 560, 11-18 (2005). 68. Barton, G.M. & Medzhitov, R. Toll-like receptor signaling pathways. Science 300, 1524-1525 (2003). 69. Peri, F., Piazza, M., Calabrese, V., Damore, G. & Cighetti, R. Exploring the LPS/TLR4 signal pathway with small molecules. Biochem Soc Trans 38, 1390- 1395. 70. Yamamoto, M. & Akira, S. Lipid A receptor TLR4-mediated signaling pathways. Adv Exp Med Biol 667, 59-68 (2009). 71. Kenny, E.F. & O'Neill, L.A. Signalling adaptors used by Toll-like receptors: an update. Cytokine 43, 342-349 (2008). 72. Jin, M.S. & Lee, J.O. Structures of the toll-like receptor family and its ligand complexes. Immunity 29, 182-191 (2008). 73. McGettrick, A.F. & O'Neill, L.A. Regulators of TLR4 signaling by endotoxins. Subcell Biochem 53, 153-171. 74. Lu, Y.C., Yeh, W.C. & Ohashi, P.S. LPS/TLR4 signal transduction pathway. Cytokine 42, 145-151 (2008). 75. Kitchens, R.L. & Thompson, P.A. Modulatory effects of sCD14 and LBP on LPS- host cell interactions. J Endotoxin Res 11, 225-229 (2005). 76. Wolfs, T.G., et al. Increased release of sMD-2 during human endotoxemia and sepsis: a role for endothelial cells. Mol Immunol 45, 3268-3277 (2008). 77. Visintin, A., Mazzoni, A., Spitzer, J.A. & Segal, D.M. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll- like receptor 4. Proc Natl Acad Sci U S A 98, 12156-12161 (2001). 78. Iwami, K.I., et al. Cutting edge: naturally occurring soluble form of mouse Toll- like receptor 4 inhibits lipopolysaccharide signaling. J Immunol 165, 6682-6686 (2000). 79. Mitsuzawa, H., et al. Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice. J Immunol 177, 8133-8139 (2006).  173 80. Hyakushima, N., et al. Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling. J Immunol 173, 6949-6954 (2004). 81. Dunn-Siegrist, I., et al. Pivotal involvement of Fcgamma receptor IIA in the neutralization of lipopolysaccharide signaling via a potent novel anti-TLR4 monoclonal antibody 15C1. J Biol Chem 282, 34817-34827 (2007). 82. Hamerman, J.A., Tchao, N.K., Lowell, C.A. & Lanier, L.L. Enhanced Toll-like receptor responses in the absence of signaling adaptor DAP12. Nat Immunol 6, 579-586 (2005). 83. Turnbull, I.R. & Colonna, M. Activating and inhibitory functions of DAP12. Nat Rev Immunol 7, 155-161 (2007). 84. Divanovic, S., et al. Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor homolog RP105. Nat Immunol 6, 571-578 (2005). 85. Divanovic, S., et al. Inhibition of TLR-4/MD-2 signaling by RP105/MD-1. J Endotoxin Res 11, 363-368 (2005). 86. Kropf, P., Herath, S., Klemenz, R. & Muller, I. Signaling through the T1/ST2 molecule is not necessary for Th2 differentiation but is important for the regulation of type 1 responses in nonhealing Leishmania major infection. Infect Immun 71, 1961-1971 (2003). 87. Bergers, G., Reikerstorfer, A., Braselmann, S., Graninger, P. & Busslinger, M. Alternative promoter usage of the Fos-responsive gene Fit-1 generates mRNA isoforms coding for either secreted or membrane-bound proteins related to the IL- 1 receptor. EMBO J 13, 1176-1188 (1994). 88. Tominaga, S. A putative protein of a growth specific cDNA from BALB/c-3T3 cells is highly similar to the extracellular portion of mouse interleukin 1 receptor. FEBS Lett 258, 301-304 (1989). 89. Brint, E.K., et al. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 5, 373-379 (2004). 90. Wald, D., et al. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol 4, 920-927 (2003). 91. Veliz Rodriguez, T., et al. Role of TIR8/SIGIRR, a Negative Regulator of IL- 1R/TLR Signalling, in Resistance to Acute Pseudomonas aeruginosa Lung Infection. Infect Immun. 92. Garlanda, C., et al. Intestinal inflammation in mice deficient in Tir8, an inhibitory member of the IL-1 receptor family. Proc Natl Acad Sci U S A 101, 3522-3526 (2004). 93. Diehl, G.E., et al. TRAIL-R as a negative regulator of innate immune cell responses. Immunity 21, 877-889 (2004). 94. Krikos, A., Laherty, C.D. & Dixit, V.M. Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements. J Biol Chem 267, 17971-17976 (1992). 95. Opipari, A.W., Jr., Boguski, M.S. & Dixit, V.M. The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein. J Biol Chem 265, 14705-14708 (1990).  174 96. Boone, D.L., et al. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nat Immunol 5, 1052-1060 (2004). 97. Gilchrist, M., et al. Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 441, 173-178 (2006). 98. Gao, H., et al. Identification of beta-arrestin2 as a G protein-coupled receptor- stimulated regulator of NF-kappaB pathways. Mol Cell 14, 303-317 (2004). 99. Witherow, D.S., Garrison, T.R., Miller, W.E. & Lefkowitz, R.J. beta-Arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaB inhibitor IkappaBalpha. Proc Natl Acad Sci U S A 101, 8603-8607 (2004). 100. Wang, Y., et al. Association of beta-arrestin and TRAF6 negatively regulates Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol 7, 139-147 (2006). 101. Chen, P., et al. Restraint of proinflammatory cytokine biosynthesis by mitogen- activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J Immunol 169, 6408-6416 (2002). 102. Zhao, Q., et al. The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38. J Biol Chem 280, 8101-8108 (2005). 103. Shepherd, E.G., et al. The function of mitogen-activated protein kinase phosphatase-1 in peptidoglycan-stimulated macrophages. J Biol Chem 279, 54023-54031 (2004). 104. Salojin, K.V., et al. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol 176, 1899-1907 (2006). 105. Wang, T., et al. Flightless I homolog negatively modulates the TLR pathway. J Immunol 176, 1355-1362 (2006). 106. Mashima, R., et al. FLN29, a novel interferon- and LPS-inducible gene acting as a negative regulator of toll-like receptor signaling. J Biol Chem 280, 41289-41297 (2005). 107. Rao, N., Nguyen, S., Ngo, K. & Fung-Leung, W.P. A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling. Mol Cell Biol 25, 6521-6532 (2005). 108. Wesche, H., et al. IRAK-M is a novel member of the Pelle/interleukin-1 receptor- associated kinase (IRAK) family. J Biol Chem 274, 19403-19410 (1999). 109. Kobayashi, K., et al. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110, 191-202 (2002). 110. Hardy, M.P. & O'Neill, L.A. The murine IRAK2 gene encodes four alternatively spliced isoforms, two of which are inhibitory. J Biol Chem 279, 27699-27708 (2004). 111. Janssens, S., Burns, K., Vercammen, E., Tschopp, J. & Beyaert, R. MyD88S, a splice variant of MyD88, differentially modulates NF-kappaB- and AP-1- dependent gene expression. FEBS Lett 548, 103-107 (2003). 112. Burns, K., et al. Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J Exp Med 197, 263-268 (2003).  175 113. Janssens, S., Burns, K., Tschopp, J. & Beyaert, R. Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88. Curr Biol 12, 467-471 (2002). 114. Saitoh, T., et al. Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1. Nat Immunol 7, 598-605 (2006). 115. Suizu, F., Ryo, A., Wulf, G., Lim, J. & Lu, K.P. Pin1 regulates centrosome duplication, and its overexpression induces centrosome amplification, chromosome instability, and oncogenesis. Mol Cell Biol 26, 1463-1479 (2006). 116. Ryo, A., et al. Regulation of NF-kappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 12, 1413- 1426 (2003). 117. Meylan, E., et al. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation. Nat Immunol 5, 503-507 (2004). 118. Wang, Y., et al. Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood 110, 962-971 (2007). 119. Mink, M., Fogelgren, B., Olszewski, K., Maroy, P. & Csiszar, K. A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans. Genomics 74, 234-244 (2001). 120. Carty, M., et al. The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling. Nat Immunol 7, 1074-1081 (2006). 121. O'Neill, L.A. DisSARMing Toll-like receptor signaling. Nat Immunol 7, 1023- 1025 (2006). 122. An, H., et al. SHP-2 phosphatase negatively regulates the TRIF adaptor protein- dependent type I interferon and proinflammatory cytokine production. Immunity 25, 919-928 (2006). 123. Mansell, A., et al. Suppressor of cytokine signaling 1 negatively regulates Toll- like receptor signaling by mediating Mal degradation. Nat Immunol 7, 148-155 (2006). 124. Palsson-McDermott, E.M., et al. TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88-independent TLR4 pathway. Nat Immunol 10, 579-586 (2009). 125. Burns, K., et al. Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol 2, 346-351 (2000). 126. Zhang, G. & Ghosh, S. Negative regulation of toll-like receptor-mediated signaling by Tollip. J Biol Chem 277, 7059-7065 (2002). 127. Takeshita, F., et al. TRAF4 acts as a silencer in TLR-mediated signaling through the association with TRAF6 and TRIF. Eur J Immunol 35, 2477-2485 (2005). 128. Chuang, T.H. & Ulevitch, R.J. Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors. Nat Immunol 5, 495-502 (2004). 129. Fearns, C., Pan, Q., Mathison, J.C. & Chuang, T.H. Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding. J Biol Chem 281, 34592-34600 (2006).  176 130. Shi, M., et al. TRIM30 alpha negatively regulates TLR-mediated NF-kappa B activation by targeting TAB2 and TAB3 for degradation. Nat Immunol 9, 369-377 (2008). 131. Carswell, E.A., et al. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A 72, 3666-3670 (1975). 132. Michlewska, S., Dransfield, I., Megson, I.L. & Rossi, A.G. Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and anti-inflammatory agents: key role for TNF- alpha. FASEB J 23, 844-854 (2009). 133. Feldmann, M., Brennan, F.M., Elliott, M., Katsikis, P. & Maini, R.N. TNF alpha as a therapeutic target in rheumatoid arthritis. Circ Shock 43, 179-184 (1994). 134. Maini, R.N., Elliott, M.J., Brennan, F.M. & Feldmann, M. Beneficial effects of tumour necrosis factor-alpha (TNF-alpha) blockade in rheumatoid arthritis (RA). Clin Exp Immunol 101, 207-212 (1995). 135. Vassalli, P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol 10, 411-452 (1992). 136. Clark, I.A. How TNF was recognized as a key mechanism of disease. Cytokine Growth Factor Rev 18, 335-343 (2007). 137. Parameswaran, N. & Patial, S. Tumor necrosis factor-alpha signaling in macrophages. Crit Rev Eukaryot Gene Expr 20, 87-103. 138. Spriggs, D.R., Deutsch, S. & Kufe, D.W. Genomic structure, induction, and production of TNF-alpha. Immunol Ser 56, 3-34 (1992). 139. Collart, M.A., Baeuerle, P. & Vassalli, P. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Mol Cell Biol 10, 1498-1506 (1990). 140. Rhoades, K.L., Golub, S.H. & Economou, J.S. The regulation of the human tumor necrosis factor alpha promoter region in macrophage, T cell, and B cell lines. J Biol Chem 267, 22102-22107 (1992). 141. Guha, M., et al. Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 98, 1429-1439 (2001). 142. Guha, M. & Mackman, N. LPS induction of gene expression in human monocytes. Cell Signal 13, 85-94 (2001). 143. Means, T.K., Pavlovich, R.P., Roca, D., Vermeulen, M.W. & Fenton, M.J. Activation of TNF-alpha transcription utilizes distinct MAP kinase pathways in different macrophage populations. J Leukoc Biol 67, 885-893 (2000). 144. Anderson, P. Post-transcriptional control of cytokine production. Nat Immunol 9, 353-359 (2008). 145. Caput, D., et al. Identification of a common nucleotide sequence in the 3'- untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A 83, 1670-1674 (1986). 146. Blackshear, P.J., et al. Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates. J Biol Chem 278, 19947-19955 (2003).  177 147. Worthington, M.T., et al. RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein. J Biol Chem 277, 48558- 48564 (2002). 148. Lai, W.S., et al. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol 19, 4311-4323 (1999). 149. Carballo, E., Lai, W.S. & Blackshear, P.J. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science 281, 1001-1005 (1998). 150. Sun, L., et al. Tristetraprolin (TTP)-14-3-3 complex formation protects TTP from dephosphorylation by protein phosphatase 2a and stabilizes tumor necrosis factor- alpha mRNA. J Biol Chem 282, 3766-3777 (2007). 151. Buxade, M., et al. The Mnks are novel components in the control of TNF alpha biosynthesis and phosphorylate and regulate hnRNP A1. Immunity 23, 177-189 (2005). 152. Stoecklin, G., Lu, M., Rattenbacher, B. & Moroni, C. A constitutive decay element promotes tumor necrosis factor alpha mRNA degradation via an AU-rich element-independent pathway. Mol Cell Biol 23, 3506-3515 (2003). 153. Mahtani, K.R., et al. Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mRNA stability. Mol Cell Biol 21, 6461-6469 (2001). 154. Lopez de Silanes, I., et al. Identification and functional outcome of mRNAs associated with RNA-binding protein TIA-1. Mol Cell Biol 25, 9520-9531 (2005). 155. Saito, K., Chen, S., Piecyk, M. & Anderson, P. TIA-1 regulates the production of tumor necrosis factor alpha in macrophages, but not in lymphocytes. Arthritis Rheum 44, 2879-2887 (2001). 156. Piecyk, M., et al. TIA-1 is a translational silencer that selectively regulates the expression of TNF-alpha. EMBO J 19, 4154-4163 (2000). 157. Kontoyiannis, D., et al. Interleukin-10 targets p38 MAPK to modulate ARE- dependent TNF mRNA translation and limit intestinal pathology. EMBO J 20, 3760-3770 (2001). 158. Taylor, G.A., et al. A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4, 445-454 (1996). 159. Vasudevan, S., Tong, Y. & Steitz, J.A. Cell-cycle control of microRNA-mediated translation regulation. Cell Cycle 7, 1545-1549 (2008). 160. Vasudevan, S., Tong, Y. & Steitz, J.A. Switching from repression to activation: microRNAs can up-regulate translation. Science 318, 1931-1934 (2007). 161. Black, R.A., et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385, 729-733 (1997). 162. Armstrong, L., Godinho, S.I., Uppington, K.M., Whittington, H.A. & Millar, A.B. Contribution of TNF-alpha converting enzyme and proteinase-3 to TNF-alpha processing in human alveolar macrophages. Am J Respir Cell Mol Biol 34, 219- 225 (2006).  178 163. Ermert, M., et al. In situ localization of TNFalpha/beta, TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue. Cytokine 22, 89-100 (2003). 164. Lieu, Z.Z., et al. A trans-Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo. Proc Natl Acad Sci U S A 105, 3351-3356 (2008). 165. Shurety, W., Merino-Trigo, A., Brown, D., Hume, D.A. & Stow, J.L. Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages. J Interferon Cytokine Res 20, 427-438 (2000). 166. Rosendahl, M.S., et al. Identification and characterization of a pro-tumor necrosis factor-alpha-processing enzyme from the ADAM family of zinc metalloproteases. J Biol Chem 272, 24588-24593 (1997). 167. Macchia, D., et al. Membrane tumour necrosis factor-alpha is involved in the polyclonal B-cell activation induced by HIV-infected human T cells. Nature 363, 464-466 (1993). 168. Pellegrini, J.D., Puyana, J.C., Lapchak, P.H., Kodys, K. & Miller-Graziano, C.L. A membrane TNF-alpha/TNFR ratio correlates to MODS score and mortality. Shock 6, 389-396 (1996). 169. Birkland, T.P., Sypek, J.P. & Wyler, D.J. Soluble TNF and membrane TNF expressed on CD4+ T lymphocytes differ in their ability to activate macrophage antileishmanial defense. J Leukoc Biol 51, 296-299 (1992). 170. Allenbach, C., et al. Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major. J Immunol 176, 6656-6664 (2006). 171. Peck, R., Brockhaus, M. & Frey, J.R. Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells. Cell Immunol 122, 1-10 (1989). 172. Grell, M., et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 83, 793-802 (1995). 173. Eissner, G., et al. Critical involvement of transmembrane tumor necrosis factor- alpha in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin. Blood 86, 4184-4193 (1995). 174. Grell, M. Tumor necrosis factor (TNF) receptors in cellular signaling of soluble and membrane-expressed TNF. J Inflamm 47, 8-17 (1995). 175. Tartaglia, L.A., et al. The two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci U S A 88, 9292-9296 (1991). 176. Carpentier, I., Coornaert, B. & Beyaert, R. Function and regulation of tumor necrosis factor receptor type 2. Curr Med Chem 11, 2205-2212 (2004). 177. Pimentel-Muinos, F.X. & Seed, B. Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity 11, 783-793 (1999). 178. Faustman, D. & Davis, M. TNF receptor 2 pathway: drug target for autoimmune diseases. Nat Rev Drug Discov 9, 482-493.  179 179. Grell, M., Becke, F.M., Wajant, H., Mannel, D.N. & Scheurich, P. TNF receptor type 2 mediates thymocyte proliferation independently of TNF receptor type 1. Eur J Immunol 28, 257-263 (1998). 180. Krippner-Heidenreich, A., et al. Control of receptor-induced signaling complex formation by the kinetics of ligand/receptor interaction. J Biol Chem 277, 44155- 44163 (2002). 181. Tartaglia, L.A., Ayres, T.M., Wong, G.H. & Goeddel, D.V. A novel domain within the 55 kd TNF receptor signals cell death. Cell 74, 845-853 (1993). 182. Hsu, H., Xiong, J. & Goeddel, D.V. The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell 81, 495-504 (1995). 183. Rothe, M., Sarma, V., Dixit, V.M. & Goeddel, D.V. TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40. Science 269, 1424-1427 (1995). 184. Rothe, M., Wong, S.C., Henzel, W.J. & Goeddel, D.V. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681-692 (1994). 185. Wesemann, D.R. & Benveniste, E.N. STAT-1 alpha and IFN-gamma as modulators of TNF-alpha signaling in macrophages: regulation and functional implications of the TNF receptor 1:STAT-1 alpha complex. J Immunol 171, 5313- 5319 (2003). 186. Breese, E.J., et al. Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 106, 1455-1466 (1994). 187. Murch, S.H., Braegger, C.P., Walker-Smith, J.A. & MacDonald, T.T. Location of tumour necrosis factor alpha by immunohistochemistry in chronic inflammatory bowel disease. Gut 34, 1705-1709 (1993). 188. Kontoyiannis, D., Pasparakis, M., Pizarro, T.T., Cominelli, F. & Kollias, G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 10, 387-398 (1999). 189. Fisher, C.J., Jr., et al. Influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. The CB0006 Sepsis Syndrome Study Group. Crit Care Med 21, 318-327 (1993). 190. Damas, P., et al. Tumor necrosis factor and interleukin-1 serum levels during severe sepsis in humans. Crit Care Med 17, 975-978 (1989). 191. Calvano, S.E., et al. Monocyte tumor necrosis factor receptor levels as a predictor of risk in human sepsis. Arch Surg 131, 434-437 (1996). 192. van der Poll, T. & Lowry, S.F. Tumor necrosis factor in sepsis: mediator of multiple organ failure or essential part of host defense? Shock 3, 1-12 (1995). 193. Fong, Y. & Lowry, S.F. Tumor necrosis factor in the pathophysiology of infection and sepsis. Clin Immunol Immunopathol 55, 157-170 (1990). 194. Gortz, B., et al. Tumour necrosis factor activates the mitogen-activated protein kinases p38alpha and ERK in the synovial membrane in vivo. Arthritis Res Ther 7, R1140-1147 (2005). 195. Voulgari, P.V., et al. Role of cytokines in the pathogenesis of anemia of chronic disease in rheumatoid arthritis. Clin Immunol 92, 153-160 (1999).  180 196. Catrina, A.I., et al. Evidence that anti-tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages, but not lymphocytes, in rheumatoid arthritis joints: extended report. Arthritis Rheum 52, 61-72 (2005). 197. Taylor, P.C., et al. Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor alpha blockade in patients with rheumatoid arthritis. Arthritis Rheum 43, 38-47 (2000). 198. Aukrust, P., et al. Tumor necrosis factor superfamily molecules in acute coronary syndromes. Ann Med 43, 90-103. 199. Gustafson, B. Adipose tissue, inflammation and atherosclerosis. J Atheroscler Thromb 17, 332-341. 200. McKellar, G.E., McCarey, D.W., Sattar, N. & McInnes, I.B. Role for TNF in atherosclerosis? Lessons from autoimmune disease. Nat Rev Cardiol 6, 410-417 (2009). 201. Kleemann, R., Zadelaar, S. & Kooistra, T. Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc Res 79, 360-376 (2008). 202. Weinblatt, M.E., et al. A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. N Engl J Med 340, 253-259 (1999). 203. Silva, L.C., Ortigosa, L.C. & Benard, G. Anti-TNF-alpha agents in the treatment of immune-mediated inflammatory diseases: mechanisms of action and pitfalls. Immunotherapy 2, 817-833. 204. Assasi, N., et al. Patient outcomes after anti TNF-alpha drugs for Crohn's disease. Expert Rev Pharmacoecon Outcomes Res 10, 163-175. 205. Hanauer, S.B., et al. Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial. Lancet 359, 1541-1549 (2002). 206. Sands, B.E., et al. Infliximab maintenance therapy for fistulizing Crohn's disease. N Engl J Med 350, 876-885 (2004). 207. Oussalah, A., Danese, S. & Peyrin-Biroulet, L. Efficacy of TNF antagonists beyond one year in adult and pediatric inflammatory bowel diseases: a systematic review. Curr Drug Targets 11, 156-175. 208. Willert, R.P. & Lawrance, I.C. Use of infliximab in the prevention and delay of colectomy in severe steroid dependant and refractory ulcerative colitis. World J Gastroenterol 14, 2544-2549 (2008). 209. Antoni, C. & Braun, J. Side effects of anti-TNF therapy: current knowledge. Clin Exp Rheumatol 20, S152-157 (2002). 210. Ramsey, S.A., et al. Uncovering a macrophage transcriptional program by integrating evidence from motif scanning and expression dynamics. PLoS Comput Biol 4, e1000021 (2008). 211. Ravasi, T., Wells, C.A. & Hume, D.A. Systems biology of transcription control in macrophages. Bioessays 29, 1215-1226 (2007). 212. Hume, D.A., Wells, C.A. & Ravasi, T. Transcriptional regulatory networks in macrophages. Novartis Found Symp 281, 2-18; discussion 18-24, 50-13, 208-209 (2007). 213. Medzhitov, R. & Horng, T. Transcriptional control of the inflammatory response. Nat Rev Immunol 9, 692-703 (2009).  181 214. Hargreaves, D.C., Horng, T. & Medzhitov, R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell 138, 129-145 (2009). 215. Smallie, T., et al. IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages. J Exp Med 207, 2081-2088. 216. Fiorentino, D.F., Bond, M.W. & Mosmann, T.R. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 170, 2081-2095 (1989). 217. Mosmann, T.R. & Coffman, R.L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7, 145-173 (1989). 218. Fiorentino, D.F., et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J Immunol 146, 3444-3451 (1991). 219. Vieira, P., et al. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: homology to Epstein-Barr virus open reading frame BCRFI. Proc Natl Acad Sci U S A 88, 1172-1176 (1991). 220. Moore, K.W., et al. Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI. Science 248, 1230-1234 (1990). 221. Ouyang, W., Rutz, S., Crellin, N.K., Valdez, P.A. & Hymowitz, S.G. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev Immunol 29, 71-109. 222. Moore, K.W., de Waal Malefyt, R., Coffman, R.L. & O'Garra, A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19, 683-765 (2001). 223. Boonstra, A., et al. Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF- dependent TLR signals, and TLR-independent signals. J Immunol 177, 7551-7558 (2006). 224. Fiorentino, D.F., Zlotnik, A., Mosmann, T.R., Howard, M. & O'Garra, A. IL-10 inhibits cytokine production by activated macrophages. J Immunol 147, 3815- 3822 (1991). 225. Chang, E.Y., Guo, B., Doyle, S.E. & Cheng, G. Cutting edge: involvement of the type I IFN production and signaling pathway in lipopolysaccharide-induced IL-10 production. J Immunol 178, 6705-6709 (2007). 226. Brightbill, H.D., Plevy, S.E., Modlin, R.L. & Smale, S.T. A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages. J Immunol 164, 1940-1951 (2000). 227. Tone, M., Powell, M.J., Tone, Y., Thompson, S.A. & Waldmann, H. IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3. J Immunol 165, 286-291 (2000). 228. Liu, Y.W., Tseng, H.P., Chen, L.C., Chen, B.K. & Chang, W.C. Functional cooperation of simian virus 40 promoter factor 1 and CCAAT/enhancer-binding protein beta and delta in lipopolysaccharide-induced gene activation of IL-10 in mouse macrophages. J Immunol 171, 821-828 (2003). 229. Liu, Y.W., Chen, C.C., Tseng, H.P. & Chang, W.C. Lipopolysaccharide-induced transcriptional activation of interleukin-10 is mediated by MAPK- and NF-  182 kappaB-induced CCAAT/enhancer-binding protein delta in mouse macrophages. Cell Signal 18, 1492-1500 (2006). 230. Powell, M.J., Thompson, S.A., Tone, Y., Waldmann, H. & Tone, M. Posttranscriptional regulation of IL-10 gene expression through sequences in the 3'-untranslated region. J Immunol 165, 292-296 (2000). 231. Liu, Y., Wei, S.H., Ho, A.S., de Waal Malefyt, R. & Moore, K.W. Expression cloning and characterization of a human IL-10 receptor. J Immunol 152, 1821- 1829 (1994). 232. Ho, A.S., et al. A receptor for interleukin 10 is related to interferon receptors. Proc Natl Acad Sci U S A 90, 11267-11271 (1993). 233. Kotenko, S.V., et al. Identification and functional characterization of a second chain of the interleukin-10 receptor complex. EMBO J 16, 5894-5903 (1997). 234. Spencer, S.D., et al. The orphan receptor CRF2-4 is an essential subunit of the interleukin 10 receptor. J Exp Med 187, 571-578 (1998). 235. Donnelly, R.P., Dickensheets, H. & Finbloom, D.S. The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes. J Interferon Cytokine Res 19, 563-573 (1999). 236. Williams, L.M., Ricchetti, G., Sarma, U., Smallie, T. & Foxwell, B.M. Interleukin-10 suppression of myeloid cell activation--a continuing puzzle. Immunology 113, 281-292 (2004). 237. Riley, J.K., Takeda, K., Akira, S. & Schreiber, R.D. Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor- derived signals for anti-inflammatory action. J Biol Chem 274, 16513-16521 (1999). 238. Takeda, K., et al. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10, 39-49 (1999). 239. Murray, P.J., Wang, L., Onufryk, C., Tepper, R.I. & Young, R.A. T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J Immunol 158, 315-321 (1997). 240. Yoon, S.I., et al. Structure and mechanism of receptor sharing by the IL-10R2 common chain. Structure 18, 638-648. 241. Murray, P.J. Understanding and exploiting the endogenous interleukin- 10/STAT3-mediated anti-inflammatory response. Curr Opin Pharmacol 6, 379- 386 (2006). 242. Williams, L., Bradley, L., Smith, A. & Foxwell, B. Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages. J Immunol 172, 567-576 (2004). 243. Rodig, S.J., et al. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 93, 373-383 (1998). 244. O'Farrell, A.M., Liu, Y., Moore, K.W. & Mui, A.L. IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3- dependent and -independent pathways. EMBO J 17, 1006-1018 (1998).  183 245. El Kasmi, K.C., et al. Cutting edge: A transcriptional repressor and corepressor induced by the STAT3-regulated anti-inflammatory signaling pathway. J Immunol 179, 7215-7219 (2007). 246. Kuwata, H., et al. IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF- alpha production in macrophages. Blood 102, 4123-4129 (2003). 247. Lang, R., Patel, D., Morris, J.J., Rutschman, R.L. & Murray, P.J. Shaping gene expression in activated and resting primary macrophages by IL-10. J Immunol 169, 2253-2263 (2002). 248. Cassatella, M.A., et al. Interleukin-10 (IL-10) selectively enhances CIS3/SOCS3 mRNA expression in human neutrophils: evidence for an IL-10-induced pathway that is independent of STAT protein activation. Blood 94, 2880-2889 (1999). 249. Berlato, C., et al. Involvement of suppressor of cytokine signaling-3 as a mediator of the inhibitory effects of IL-10 on lipopolysaccharide-induced macrophage activation. J Immunol 168, 6404-6411 (2002). 250. Wang, Y. & Rice, A.P. Interleukin-10 inhibits HIV-1 LTR-directed gene expression in human macrophages through the induction of cyclin T1 proteolysis. Virology 352, 485-492 (2006). 251. Bromberg, J.F., et al. Stat3 as an oncogene. Cell 98, 295-303 (1999). 252. Ricchetti, G.A., Williams, L.M. & Foxwell, B.M. Heme oxygenase 1 expression induced by IL-10 requires STAT-3 and phosphoinositol-3 kinase and is inhibited by lipopolysaccharide. J Leukoc Biol 76, 719-726 (2004). 253. D'Andrea, A., et al. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 178, 1041-1048 (1993). 254. de Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C.G. & de Vries, J.E. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174, 1209-1220 (1991). 255. de Waal Malefyt, R., et al. Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparison with IL-4 and modulation by IFN-gamma or IL-10. J Immunol 151, 6370-6381 (1993). 256. Gruber, M.F., Williams, C.C. & Gerrard, T.L. Macrophage-colony-stimulating factor expression by anti-CD45 stimulated human monocytes is transcriptionally up-regulated by IL-1 beta and inhibited by IL-4 and IL-10. J Immunol 152, 1354- 1361 (1994). 257. Berkman, N., et al. Inhibition of macrophage inflammatory protein-1 alpha expression by IL-10. Differential sensitivities in human blood monocytes and alveolar macrophages. J Immunol 155, 4412-4418 (1995). 258. Rossi, D.L., Vicari, A.P., Franz-Bacon, K., McClanahan, T.K. & Zlotnik, A. Identification through bioinformatics of two new macrophage proinflammatory human chemokines: MIP-3alpha and MIP-3beta. J Immunol 158, 1033-1036 (1997). 259. Marfaing-Koka, A., Maravic, M., Humbert, M., Galanaud, P. & Emilie, D. Contrasting effects of IL-4, IL-10 and corticosteroids on RANTES production by human monocytes. Int Immunol 8, 1587-1594 (1996).  184 260. Kopydlowski, K.M., et al. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J Immunol 163, 1537-1544 (1999). 261. Inoue, Y., et al. Novel regulatory mechanisms of CD40-induced prostanoid synthesis by IL-4 and IL-10 in human monocytes. J Immunol 172, 2147-2154 (2004). 262. Niiro, H., et al. MAP kinase pathways as a route for regulatory mechanisms of IL- 10 and IL-4 which inhibit COX-2 expression in human monocytes. Biochem Biophys Res Commun 250, 200-205 (1998). 263. Niho, Y., Niiro, H., Tanaka, Y., Nakashima, H. & Otsuka, T. Role of IL-10 in the crossregulation of prostaglandins and cytokines in monocytes. Acta Haematol 99, 165-170 (1998). 264. Niiro, H., et al. IL-10 inhibits prostaglandin E2 production by lipopolysaccharide- stimulated monocytes. Int Immunol 6, 661-664 (1994). 265. Mertz, P.M., DeWitt, D.L., Stetler-Stevenson, W.G. & Wahl, L.M. Interleukin 10 suppression of monocyte prostaglandin H synthase-2. Mechanism of inhibition of prostaglandin-dependent matrix metalloproteinase production. J Biol Chem 269, 21322-21329 (1994). 266. Lacraz, S., Nicod, L.P., Chicheportiche, R., Welgus, H.G. & Dayer, J.M. IL-10 inhibits metalloproteinase and stimulates TIMP-1 production in human mononuclear phagocytes. J Clin Invest 96, 2304-2310 (1995). 267. Stearns, M.E., Rhim, J. & Wang, M. Interleukin 10 (IL-10) inhibition of primary human prostate cell-induced angiogenesis: IL-10 stimulation of tissue inhibitor of metalloproteinase-1 and inhibition of matrix metalloproteinase (MMP)-2/MMP-9 secretion. Clin Cancer Res 5, 189-196 (1999). 268. de Waal Malefyt, R., et al. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J Exp Med 174, 915-924 (1991). 269. Ding, L., Linsley, P.S., Huang, L.Y., Germain, R.N. & Shevach, E.M. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up- regulation of B7 expression. J Immunol 151, 1224-1234 (1993). 270. Kubin, M., Kamoun, M. & Trinchieri, G. Interleukin 12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J Exp Med 180, 211-222 (1994). 271. Willems, F., et al. Interleukin-10 inhibits B7 and intercellular adhesion molecule- 1 expression on human monocytes. Eur J Immunol 24, 1007-1009 (1994). 272. Muzio, M., et al. Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164, 5998-6004 (2000). 273. te Velde, A.A., de Waal Malefijt, R., Huijbens, R.J., de Vries, J.E. & Figdor, C.G. IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10. J Immunol 149, 4048-4052 (1992). 274. Calzada-Wack, J.C., Frankenberger, M. & Ziegler-Heitbrock, H.W. Interleukin- 10 drives human monocytes to CD16 positive macrophages. J Inflamm 46, 78-85 (1996).  185 275. Schottelius, A.J., Mayo, M.W., Sartor, R.B. & Baldwin, A.S., Jr. Interleukin-10 signaling blocks inhibitor of kappaB kinase activity and nuclear factor kappaB DNA binding. J Biol Chem 274, 31868-31874 (1999). 276. Murray, P.J. The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription. Proc Natl Acad Sci U S A 102, 8686-8691 (2005). 277. Denys, A., et al. Evidence for a dual mechanism for IL-10 suppression of TNF- alpha production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-kappa B in primary human macrophages. J Immunol 168, 4837- 4845 (2002). 278. Schaljo, B., et al. Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10. J Immunol 183, 1197-1206 (2009). 279. Nakagawa, R., et al. SOCS-1 participates in negative regulation of LPS responses. Immunity 17, 677-687 (2002). 280. Stoiber, D., et al. Lipopolysaccharide induces in macrophages the synthesis of the suppressor of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-gamma. J Immunol 163, 2640-2647 (1999). 281. Docke, W.D., et al. Monocyte deactivation in septic patients: restoration by IFN- gamma treatment. Nat Med 3, 678-681 (1997). 282. Randow, F., et al. In vitro prevention and reversal of lipopolysaccharide desensitization by IFN-gamma, IL-12, and granulocyte-macrophage colony- stimulating factor. J Immunol 158, 2911-2918 (1997). 283. Grutz, G. New insights into the molecular mechanism of interleukin-10-mediated immunosuppression. J Leukoc Biol 77, 3-15 (2005). 284. Tonks, N.K. PTP1B: from the sidelines to the front lines! FEBS Lett 546, 140-148 (2003). 285. Driessler, F., Venstrom, K., Sabat, R., Asadullah, K. & Schottelius, A.J. Molecular mechanisms of interleukin-10-mediated inhibition of NF-kappaB activity: a role for p50. Clin Exp Immunol 135, 64-73 (2004). 286. Lenardo, M. & Siebenlist, U. Bcl-3-mediated nuclear regulation of the NF-kappa B trans-activating factor. Immunol Today 15, 145-147 (1994). 287. Chen, C.Y. & Shyu, A.B. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci 20, 465-470 (1995). 288. Rajasingh, J., et al. IL-10-induced TNF-alpha mRNA destabilization is mediated via IL-10 suppression of p38 MAP kinase activation and inhibition of HuR expression. FASEB J 20, 2112-2114 (2006). 289. Jang, S., Uematsu, S., Akira, S. & Salgame, P. IL-6 and IL-10 induction from dendritic cells in response to Mycobacterium tuberculosis is predominantly dependent on TLR2-mediated recognition. J Immunol 173, 3392-3397 (2004). 290. Redford, P.S., Murray, P.J. & O'Garra, A. The role of IL-10 in immune regulation during M. tuberculosis infection. Mucosal Immunol 4, 261-270. 291. Netea, M.G., et al. Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol 172, 3712- 3718 (2004).  186 292. van der Kleij, D., et al. A novel host-parasite lipid cross-talk. Schistosomal lyso- phosphatidylserine activates toll-like receptor 2 and affects immune polarization. J Biol Chem 277, 48122-48129 (2002). 293. Gazzinelli, R.T., et al. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J Immunol 157, 798-805 (1996). 294. Khan, I.A., Matsuura, T. & Kasper, L.H. IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice. Parasite Immunol 17, 185-195 (1995). 295. Kane, M.M. & Mosser, D.M. The role of IL-10 in promoting disease progression in leishmaniasis. J Immunol 166, 1141-1147 (2001). 296. Ejrnaes, M., et al. Resolution of a chronic viral infection after interleukin-10 receptor blockade. J Exp Med 203, 2461-2472 (2006). 297. Slobedman, B., Barry, P.A., Spencer, J.V., Avdic, S. & Abendroth, A. Virus- encoded homologs of cellular interleukin-10 and their control of host immune function. J Virol 83, 9618-9629 (2009). 298. Franke, A., et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 40, 1319-1323 (2008). 299. Glocker, E.O., et al. Inflammatory bowel disease and mutations affecting the interleukin-10 receptor. N Engl J Med 361, 2033-2045 (2009). 300. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K. & Muller, W. Interleukin-10- deficient mice develop chronic enterocolitis. Cell 75, 263-274 (1993). 301. Yen, D., et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 116, 1310-1316 (2006). 302. Tomoyose, M., Mitsuyama, K., Ishida, H., Toyonaga, A. & Tanikawa, K. Role of interleukin-10 in a murine model of dextran sulfate sodium-induced colitis. Scand J Gastroenterol 33, 435-440 (1998). 303. Duchmann, R., Schmitt, E., Knolle, P., Meyer zum Buschenfelde, K.H. & Neurath, M. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. Eur J Immunol 26, 934-938 (1996). 304. Braat, H., Peppelenbosch, M.P. & Hommes, D.W. Interleukin-10-based therapy for inflammatory bowel disease. Expert Opin Biol Ther 3, 725-731 (2003). 305. Braat, H., et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 4, 754-759 (2006). 306. Tilg, H., et al. Treatment of Crohn's disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma. Gut 50, 191-195 (2002). 307. Fruman, D.A. & Cantley, L.C. Phosphoinositide 3-kinase in immunological systems. Semin Immunol 14, 7-18 (2002). 308. Koyasu, S. The role of PI3K in immune cells. Nat Immunol 4, 313-319 (2003). 309. Vanhaesebroeck, B., Guillermet-Guibert, J., Graupera, M. & Bilanges, B. The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol 11, 329-341.  187 310. Wymann, M.P. & Pirola, L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1436, 127-150 (1998). 311. Fruman, D.A., Meyers, R.E. & Cantley, L.C. Phosphoinositide kinases. Annu Rev Biochem 67, 481-507 (1998). 312. Vanhaesebroeck, B., et al. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem 70, 535-602 (2001). 313. Gharbi, S.I., et al. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem J 404, 15-21 (2007). 314. Davies, S.P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95-105 (2000). 315. Ward, S., Sotsios, Y., Dowden, J., Bruce, I. & Finan, P. Therapeutic potential of phosphoinositide 3-kinase inhibitors. Chem Biol 10, 207-213 (2003). 316. Vanhaesebroeck, B., Ali, K., Bilancio, A., Geering, B. & Foukas, L.C. Signalling by PI3K isoforms: insights from gene-targeted mice. Trends Biochem Sci 30, 194- 204 (2005). 317. Bhattacharyya, S., et al. Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and of IkappaB kinase activity. Blood 104, 1100-1109 (2004). 318. Hattori, Y., Hattori, S. & Kasai, K. Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation. Eur J Pharmacol 481, 153-158 (2003). 319. Hebeis, B.J., Vigorito, E. & Turner, M. The p110delta subunit of phosphoinositide 3-kinase is required for the lipopolysaccharide response of mouse B cells. Biochem Soc Trans 32, 789-791 (2004). 320. Jones, B.W., Heldwein, K.A., Means, T.K., Saukkonen, J.J. & Fenton, M.J. Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages. Ann Rheum Dis 60 Suppl 3, iii6-12 (2001). 321. Re, F. & Strominger, J.L. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells. J Biol Chem 276, 37692-37699 (2001). 322. Su, S.C., et al. LTA and LPS mediated activation of protein kinases in the regulation of inflammatory cytokines expression in macrophages. Clin Chim Acta 374, 106-115 (2006). 323. Utsugi, M., et al. PI3K p110beta positively regulates lipopolysaccharide-induced IL-12 production in human macrophages and dendritic cells and JNK1 plays a novel role. J Immunol 182, 5225-5231 (2009). 324. Weinstein, S.L., et al. Phosphatidylinositol 3-kinase and mTOR mediate lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-beta. J Leukoc Biol 67, 405-414 (2000). 325. An, H., et al. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism. Blood 105, 4685-4692 (2005). 326. Bowling, W.M., Flye, M.W., Qiu, Y.Y. & Callery, M.P. Inhibition of phosphatidylinositol-3'-kinase prevents induction of endotoxin tolerance in vitro. J Surg Res 63, 287-292 (1996).  188 327. Diaz-Guerra, M.J., Castrillo, A., Martin-Sanz, P. & Bosca, L. Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages. J Immunol 162, 6184-6190 (1999). 328. Fang, H., et al. Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP. J Immunol 173, 360-366 (2004). 329. Guha, M. & Mackman, N. The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem 277, 32124- 32132 (2002). 330. Keck, S., Freudenberg, M. & Huber, M. Activation of murine macrophages via TLR2 and TLR4 is negatively regulated by a Lyn/PI3K module and promoted by SHIP1. J Immunol 184, 5809-5818. 331. Laird, R.M. & Hayes, S.M. Profiling of the early transcriptional response of murine gammadelta T cells following TCR stimulation. Mol Immunol 46, 2429- 2438 (2009). 332. Luyendyk, J.P., et al. Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages. J Immunol 180, 4218-4226 (2008). 333. Monick, M.M., et al. Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and Akt activity in human alveolar macrophages. J Immunol 167, 5977-5985 (2001). 334. Pahan, K., Raymond, J.R. & Singh, I. Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells. J Biol Chem 274, 7528-7536 (1999). 335. Park, Y.C., Lee, C.H., Kang, H.S., Chung, H.T. & Kim, H.D. Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, enhances LPS-induced NO production from murine peritoneal macrophages. Biochem Biophys Res Commun 240, 692-696 (1997). 336. Schabbauer, G., Tencati, M., Pedersen, B., Pawlinski, R. & Mackman, N. PI3K- Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler Thromb Vasc Biol 24, 1963-1969 (2004). 337. Zhang, W.J., Wei, H., Hagen, T. & Frei, B. Alpha-lipoic acid attenuates LPS- induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway. Proc Natl Acad Sci U S A 104, 4077-4082 (2007). 338. Nakanishi, S., et al. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem 267, 2157-2163 (1992). 339. Liu, Y., et al. Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian polo-like kinase. Chem Biol 12, 99-107 (2005). 340. Bain, J., et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 408, 297-315 (2007). 341. Williams, O., et al. Discovery of dual inhibitors of the immune cell PI3Ks p110delta and p110gamma: a prototype for new anti-inflammatory drugs. Chem Biol 17, 123-134. 342. Uno, J.K., et al. Altered macrophage function contributes to colitis in mice defective in the phosphoinositide-3 kinase subunit p110delta. Gastroenterology 139, 1642-1653, 1653 e1641-1646.  189 343. Krystal, G. Lipid phosphatases in the immune system. Semin Immunol 12, 397- 403 (2000). 344. Damen, J.E., et al. The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase. Proc Natl Acad Sci U S A 93, 1689-1693 (1996). 345. Lioubin, M.N., et al. p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity. Genes Dev 10, 1084-1095 (1996). 346. Kavanaugh, W.M., et al. Multiple forms of an inositol polyphosphate 5- phosphatase form signaling complexes with Shc and Grb2. Curr Biol 6, 438-445 (1996). 347. Valderrama-Carvajal, H., et al. Activin/TGF-beta induce apoptosis through Smad- dependent expression of the lipid phosphatase SHIP. Nat Cell Biol 4, 963-969 (2002). 348. Huber, M., et al. The role of SHIP in growth factor induced signalling. Prog Biophys Mol Biol 71, 423-434 (1999). 349. Huang, Z.Y., Hunter, S., Kim, M.K., Indik, Z.K. & Schreiber, A.D. The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcgamma receptors FcgammaRIIB and FcgammaRIIA. J Leukoc Biol 73, 823- 829 (2003). 350. Maresco, D.L., Osborne, J.M., Cooney, D., Coggeshall, K.M. & Anderson, C.L. The SH2-containing 5'-inositol phosphatase (SHIP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes. J Immunol 162, 6458-6465 (1999). 351. Osborne, M.A., et al. The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation. J Biol Chem 271, 29271-29278 (1996). 352. Fong, D.C., et al. Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated Fc gammaRIIB during negative regulation of IgE-dependent mouse mast cell activation. Immunol Lett 54, 83-91 (1996). 353. Pearse, R.N., et al. SHIP recruitment attenuates Fc gamma RIIB-induced B cell apoptosis. Immunity 10, 753-760 (1999). 354. Famiglietti, S.J., Nakamura, K. & Cambier, J.C. Unique features of SHIP, SHP-1 and SHP-2 binding to FcgammaRIIb revealed by surface plasmon resonance analysis. Immunol Lett 68, 35-40 (1999). 355. Tridandapani, S., Phee, H., Shivakumar, L., Kelley, T.W. & Coggeshall, K.M. Role of SHIP in FcgammaRIIb-mediated inhibition of Ras activation in B cells. Mol Immunol 35, 1135-1146 (1998). 356. Harmer, S.L. & DeFranco, A.L. The src homology domain 2-containing inositol phosphatase SHIP forms a ternary complex with Shc and Grb2 in antigen receptor-stimulated B lymphocytes. J Biol Chem 274, 12183-12191 (1999). 357. Kepley, C.L., et al. Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes. J Biol Chem 279, 35139-35149 (2004). 358. Liu, L., et al. The Src homology 2 (SH2) domain of SH2-containing inositol phosphatase (SHIP) is essential for tyrosine phosphorylation of SHIP, its  190 association with Shc, and its induction of apoptosis. J Biol Chem 272, 8983-8988 (1997). 359. Lamkin, T.D., et al. Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP. J Biol Chem 272, 10396- 10401 (1997). 360. Gupta, N., et al. The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1- mediated inhibition of B cell receptor signaling. J Biol Chem 274, 7489-7494 (1999). 361. Wolf, I., Lucas, D.M., Algate, P.A. & Rohrschneider, L.R. Cloning of the genomic locus of mouse SH2 containing inositol 5-phosphatase (SHIP) and a novel 110-kDa splice isoform, SHIPdelta. Genomics 69, 104-112 (2000). 362. Lucas, D.M. & Rohrschneider, L.R. A novel spliced form of SH2-containing inositol phosphatase is expressed during myeloid development. Blood 93, 1922- 1933 (1999). 363. Geier, S.J., et al. The human SHIP gene is differentially expressed in cell lineages of the bone marrow and blood. Blood 89, 1876-1885 (1997). 364. March, M.E., Lucas, D.M., Aman, M.J. & Ravichandran, K.S. p135 src homology 2 domain-containing inositol 5'-phosphatase (SHIPbeta ) isoform can substitute for p145 SHIP in fcgamma RIIB1-mediated inhibitory signaling in B cells. J Biol Chem 275, 29960-29967 (2000). 365. Kalesnikoff, J., et al. The role of SHIP in cytokine-induced signaling. Rev Physiol Biochem Pharmacol 149, 87-103 (2003). 366. Bai, L. & Rohrschneider, L.R. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev 24, 1882-1892. 367. Desponts, C., Ninos, J.M. & Kerr, W.G. s-SHIP associates with receptor complexes essential for pluripotent stem cell growth and survival. Stem Cells Dev 15, 641-646 (2006). 368. Rohrschneider, L.R., Custodio, J.M., Anderson, T.A., Miller, C.P. & Gu, H. The intron 5/6 promoter region of the ship1 gene regulates expression in stem/progenitor cells of the mouse embryo. Dev Biol 283, 503-521 (2005). 369. Tu, Z., et al. Embryonic and hematopoietic stem cells express a novel SH2- containing inositol 5'-phosphatase isoform that partners with the Grb2 adapter protein. Blood 98, 2028-2038 (2001). 370. Pan, H., et al. SMAD4 is required for development of maximal endotoxin tolerance. J Immunol 184, 5502-5509. 371. Sly, L.M., Rauh, M.J., Kalesnikoff, J., Song, C.H. & Krystal, G. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity 21, 227-239 (2004). 372. Costinean, S., et al. Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice. Blood 114, 1374-1382 (2009). 373. Cremer, T.J., et al. MiR-155 induction by F. novicida but not the virulent F. tularensis results in SHIP down-regulation and enhanced pro-inflammatory cytokine response. PLoS One 4, e8508 (2009).  191 374. O'Connell, R.M., Chaudhuri, A.A., Rao, D.S. & Baltimore, D. Inositol phosphatase SHIP1 is a primary target of miR-155. Proc Natl Acad Sci U S A 106, 7113-7118 (2009). 375. O'Connell, R.M., et al. Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder. J Exp Med 205, 585-594 (2008). 376. O'Connell, R.M., Taganov, K.D., Boldin, M.P., Cheng, G. & Baltimore, D. MicroRNA-155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci U S A 104, 1604-1609 (2007). 377. Ruschmann, J., et al. Tyrosine phosphorylation of SHIP promotes its proteasomal degradation. Exp Hematol 38, 392-402, 402 e391. 378. Aman, M.J., Tosello-Trampont, A.C. & Ravichandran, K. Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts. J Biol Chem 276, 46371-46378 (2001). 379. Kalesnikoff, J., et al. SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-kappa B activity. J Immunol 168, 4737- 4746 (2002). 380. Kalesnikoff, J., Lam, V. & Krystal, G. SHIP represses mast cell activation and reveals that IgE alone triggers signaling pathways which enhance normal mast cell survival. Mol Immunol 38, 1201-1206 (2002). 381. Robson, J.D., Davidson, D. & Veillette, A. Inhibition of the Jun N-terminal protein kinase pathway by SHIP-1, a lipid phosphatase that interacts with the adaptor molecule Dok-3. Mol Cell Biol 24, 2332-2343 (2004). 382. Tomlinson, M.G., Heath, V.L., Turck, C.W., Watson, S.P. & Weiss, A. SHIP family inositol phosphatases interact with and negatively regulate the Tec tyrosine kinase. J Biol Chem 279, 55089-55096 (2004). 383. Tridandapani, S., Wang, Y., Marsh, C.B. & Anderson, C.L. Src homology 2 domain-containing inositol polyphosphate phosphatase regulates NF-kappa B- mediated gene transcription by phagocytic Fc gamma Rs in human myeloid cells. J Immunol 169, 4370-4378 (2002). 384. Aman, M.J., Lamkin, T.D., Okada, H., Kurosaki, T. & Ravichandran, K.S. The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells. J Biol Chem 273, 33922-33928 (1998). 385. Astoul, E., Watton, S. & Cantrell, D. The dynamics of protein kinase B regulation during B cell antigen receptor engagement. J Cell Biol 145, 1511-1520 (1999). 386. Carver, D.J., Aman, M.J. & Ravichandran, K.S. SHIP inhibits Akt activation in B cells through regulation of Akt membrane localization. Blood 96, 1449-1456 (2000). 387. Ono, M., et al. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90, 293-301 (1997). 388. Bolland, S., Pearse, R.N., Kurosaki, T. & Ravetch, J.V. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8, 509- 516 (1998). 389. Scharenberg, A.M., et al. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5- P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J 17, 1961-1972 (1998).  192 390. Koncz, G., et al. Co-clustering of Fcgamma and B cell receptors induces dephosphorylation of the Grb2-associated binder 1 docking protein. Eur J Biochem 268, 3898-3906 (2001). 391. Song, M., et al. Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C- gamma1 and modulates EGF-induced PLC activity. Exp Mol Med 37, 161-168 (2005). 392. Wahle, J.A., et al. Inappropriate recruitment and activity by the Src homology region 2 domain-containing phosphatase 1 (SHP1) is responsible for receptor dominance in the SHIP-deficient NK cell. J Immunol 179, 8009-8015 (2007). 393. Peng, Q., et al. TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1. Sci Signal 3, ra38. 394. Sly, L.M., et al. The role of SHIP in macrophages. Front Biosci 12, 2836-2848 (2007). 395. Rauh, M.J., et al. SHIP represses the generation of alternatively activated macrophages. Immunity 23, 361-374 (2005). 396. Cox, D., Dale, B.M., Kashiwada, M., Helgason, C.D. & Greenberg, S. A regulatory role for Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) in phagocytosis mediated by Fc gamma receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18). J Exp Med 193, 61-71 (2001). 397. Ganesan, L.P., et al. FcgammaR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways. Blood 108, 718-725 (2006). 398. Tiwari, S., Choi, H.P., Matsuzawa, T., Pypaert, M. & MacMicking, J.D. Targeting of the GTPase Irgm1 to the phagosomal membrane via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) promotes immunity to mycobacteria. Nat Immunol 10, 907-917 (2009). 399. Kamen, L.A., Levinsohn, J. & Swanson, J.A. Differential association of phosphatidylinositol 3-kinase, SHIP-1, and PTEN with forming phagosomes. Mol Biol Cell 18, 2463-2472 (2007). 400. Kamen, L.A., Levinsohn, J., Cadwallader, A., Tridandapani, S. & Swanson, J.A. SHIP-1 increases early oxidative burst and regulates phagosome maturation in macrophages. J Immunol 180, 7497-7505 (2008). 401. Sly, L.M., et al. SHIP prevents lipopolysaccharide from triggering an antiviral response in mice. Blood 113, 2945-2954 (2009). 402. Ghansah, T., et al. Expansion of myeloid suppressor cells in SHIP-deficient mice represses allogeneic T cell responses. J Immunol 173, 7324-7330 (2004). 403. Paraiso, K.H., Ghansah, T., Costello, A., Engelman, R.W. & Kerr, W.G. Induced SHIP deficiency expands myeloid regulatory cells and abrogates graft-versus-host disease. J Immunol 178, 2893-2900 (2007). 404. Weisser, S.B., et al. SHIP-deficient, alternatively activated macrophages protect mice during DSS-induced colitis. J Leukoc Biol 90, 483-492. 405. Kerr, W.G. Inhibitor and activator: dual functions for SHIP in immunity and cancer. Ann N Y Acad Sci 1217, 1-17. 406. Leung, W.H., Tarasenko, T. & Bolland, S. Differential roles for the inositol phosphatase SHIP in the regulation of macrophages and lymphocytes. Immunol Res 43, 243-251 (2009).  193 407. Freeburn, R.W., et al. Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors. J Immunol 169, 5441-5450 (2002). 408. Tessmer, M.S., et al. KLRG1 binds cadherins and preferentially associates with SHIP-1. Int Immunol 19, 391-400 (2007). 409. Wang, J.W., et al. Influence of SHIP on the NK repertoire and allogeneic bone marrow transplantation. Science 295, 2094-2097 (2002). 410. Liu, Q., et al. The inositol polyphosphate 5-phosphatase ship is a crucial negative regulator of B cell antigen receptor signaling. J Exp Med 188, 1333-1342 (1998). 411. Helgason, C.D., et al. Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes Dev 12, 1610- 1620 (1998). 412. Karlsson, M.C., et al. Macrophages control the retention and trafficking of B lymphocytes in the splenic marginal zone. J Exp Med 198, 333-340 (2003). 413. Kashiwada, M., et al. Downstream of tyrosine kinases-1 and Src homology 2- containing inositol 5'-phosphatase are required for regulation of CD4+CD25+ T cell development. J Immunol 176, 3958-3965 (2006). 414. Ono, M., Bolland, S., Tempst, P. & Ravetch, J.V. Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc(gamma)RIIB. Nature 383, 263-266 (1996). 415. Helgason, C.D., et al. A dual role for Src homology 2 domain-containing inositol- 5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice. J Exp Med 191, 781-794 (2000). 416. Nitschke, L., Carsetti, R., Ocker, B., Kohler, G. & Lamers, M.C. CD22 is a negative regulator of B-cell receptor signalling. Curr Biol 7, 133-143 (1997). 417. Tridandapani, S., et al. Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc gamma immunoreceptor tyrosine- based inhibition motif peptide motif. Mol Cell Biol 17, 4305-4311 (1997). 418. Poe, J.C., Fujimoto, M., Jansen, P.J., Miller, A.S. & Tedder, T.F. CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux. J Biol Chem 275, 17420- 17427 (2000). 419. Wahle, J.A., et al. Cutting edge: dominance by an MHC-independent inhibitory receptor compromises NK killing of complex targets. J Immunol 176, 7165-7169 (2006). 420. Nishio, M., et al. Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1. Nat Cell Biol 9, 36-44 (2007). 421. Huber, M., et al. The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation. Proc Natl Acad Sci U S A 95, 11330- 11335 (1998). 422. Huber, M., et al. Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells. EMBO J 17, 7311-7319 (1998). 423. Haddon, D.J., et al. SHIP1 is a repressor of mast cell hyperplasia, cytokine production, and allergic inflammation in vivo. J Immunol 183, 228-236 (2009). 424. Kuroda, E., et al. SHIP represses Th2 skewing by inhibiting IL-4 production from basophils. J Immunol 186, 323-332.  194 425. Kuroda, E., et al. SHIP represses the generation of IL-3-induced M2 macrophages by inhibiting IL-4 production from basophils. J Immunol 183, 3652-3660 (2009). 426. Baran, C.P., et al. The inositol 5'-phosphatase SHIP-1 and the Src kinase Lyn negatively regulate macrophage colony-stimulating factor-induced Akt activity. J Biol Chem 278, 38628-38636 (2003). 427. Bronte, V., et al. Apoptotic death of CD8+ T lymphocytes after immunization: induction of a suppressive population of Mac-1+/Gr-1+ cells. J Immunol 161, 5313-5320 (1998). 428. Dong, S., et al. T cell receptor for antigen induces linker for activation of T cell- dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2. J Exp Med 203, 2509-2518 (2006). 429. Collazo, M.M., et al. SHIP limits immunoregulatory capacity in the T-cell compartment. Blood 113, 2934-2944 (2009). 430. Locke, N.R., et al. SHIP regulates the reciprocal development of T regulatory and Th17 cells. J Immunol 183, 975-983 (2009). 431. Tarasenko, T., et al. T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses. Proc Natl Acad Sci U S A 104, 11382-11387 (2007). 432. Nakamura, K., Malykhin, A. & Coggeshall, K.M. The Src homology 2 domain- containing inositol 5-phosphatase negatively regulates Fcgamma receptor- mediated phagocytosis through immunoreceptor tyrosine-based activation motif- bearing phagocytic receptors. Blood 100, 3374-3382 (2002). 433. Galandrini, R., et al. SH2-containing inositol phosphatase (SHIP-1) transiently translocates to raft domains and modulates CD16-mediated cytotoxicity in human NK cells. Blood 100, 4581-4589 (2002). 434. Parihar, R., et al. Src homology 2-containing inositol 5'-phosphatase 1 negatively regulates IFN-gamma production by natural killer cells stimulated with antibody- coated tumor cells and interleukin-12. Cancer Res 65, 9099-9107 (2005). 435. Fortenbery, N.R., et al. SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation. J Immunol 184, 5065-5074 (2010). 436. Luo, J.M., et al. Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia. Leukemia 17, 1-8 (2003). 437. Wisniewski, D., et al. A novel SH2-containing phosphatidylinositol 3,4,5- trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells. Blood 93, 2707-2720 (1999). 438. Sattler, M., et al. The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors. Oncogene 15, 2379-2384 (1997). 439. Jiang, X., et al. Evidence for a positive role of SHIP in the BCR-ABL-mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia. Blood 102, 2976-2984 (2003).  195 440. Vanderwinden, J.M., et al. Differences in signaling pathways and expression level of the phosphoinositide phosphatase SHIP1 between two oncogenic mutants of the receptor tyrosine kinase KIT. Cell Signal 18, 661-669 (2006). 441. Hocking, L.J., et al. Genomewide search in familial Paget disease of bone shows evidence of genetic heterogeneity with candidate loci on chromosomes 2q36, 10p13, and 5q35. Am J Hum Genet 69, 1055-1061 (2001). 442. Vonakis, B.M., et al. Basophil FcepsilonRI histamine release parallels expression of Src-homology 2-containing inositol phosphatases in chronic idiopathic urticaria. J Allergy Clin Immunol 119, 441-448 (2007). 443. Vonakis, B.M., Gibbons, S., Jr., Sora, R., Langdon, J.M. & MacDonald, S.M. Src homology 2 domain-containing inositol 5' phosphatase is negatively associated with histamine release to human recombinant histamine-releasing factor in human basophils. J Allergy Clin Immunol 108, 822-831 (2001). 444. Muthukuru, M. & Cutler, C.W. Upregulation of immunoregulatory Src homology 2 molecule containing inositol phosphatase and mononuclear cell hyporesponsiveness in oral mucosa during chronic periodontitis. Infect Immun 74, 1431-1435 (2006). 445. Kerr, W.G., Park, M.Y., Maubert, M. & Engelman, R.W. SHIP deficiency causes Crohn's disease-like ileitis. Gut 60, 177-188. 446. McLarren, K.W., et al. SHIP-deficient mice develop spontaneous intestinal inflammation and arginase-dependent fibrosis. Am J Pathol 179, 180-188. 447. Arijs, I., et al. Intestinal expression of SHIP in inflammatory bowel diseases. Gut. 448. May, R.C. & Machesky, L.M. Phagocytosis and the actin cytoskeleton. J Cell Sci 114, 1061-1077 (2001). 449. Yeung, T. & Grinstein, S. Lipid signaling and the modulation of surface charge during phagocytosis. Immunol Rev 219, 17-36 (2007). 450. Swanson, J.A. & Hoppe, A.D. The coordination of signaling during Fc receptor- mediated phagocytosis. J Leukoc Biol 76, 1093-1103 (2004). 451. Murray, R.Z., Kay, J.G., Sangermani, D.G. & Stow, J.L. A role for the phagosome in cytokine secretion. Science 310, 1492-1495 (2005). 452. Huynh, K.K., Kay, J.G., Stow, J.L. & Grinstein, S. Fusion, fission, and secretion during phagocytosis. Physiology (Bethesda) 22, 366-372 (2007). 453. van Egmond, M., Hanneke van Vuuren, A.J. & van de Winkel, J.G. The human Fc receptor for IgA (Fc alpha RI, CD89) on transgenic peritoneal macrophages triggers phagocytosis and tumor cell lysis. Immunol Lett 68, 83-87 (1999). 454. Yokota, A., et al. Two forms of the low-affinity Fc receptor for IgE differentially mediate endocytosis and phagocytosis: identification of the critical cytoplasmic domains. Proc Natl Acad Sci U S A 89, 5030-5034 (1992). 455. Garcia-Garcia, E. & Rosales, C. Signal transduction during Fc receptor-mediated phagocytosis. J Leukoc Biol 72, 1092-1108 (2002). 456. Cassel, D.L., et al. Differential expression of Fc gamma RIIA, Fc gamma RIIB and Fc gamma RIIC in hematopoietic cells: analysis of transcripts. Mol Immunol 30, 451-460 (1993). 457. Indik, Z.K., Park, J.G., Hunter, S. & Schreiber, A.D. The molecular dissection of Fc gamma receptor mediated phagocytosis. Blood 86, 4389-4399 (1995).  196 458. Sobota, A., et al. Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation. J Immunol 175, 4450-4457 (2005). 459. Gu, H., Botelho, R.J., Yu, M., Grinstein, S. & Neel, B.G. Critical role for scaffolding adapter Gab2 in Fc gamma R-mediated phagocytosis. J Cell Biol 161, 1151-1161 (2003). 460. Cox, D. & Greenberg, S. Phagocytic signaling strategies: Fc(gamma)receptor- mediated phagocytosis as a model system. Semin Immunol 13, 339-345 (2001). 461. Coppolino, M.G., et al. Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor signalling during phagocytosis. J Cell Sci 114, 4307-4318 (2001). 462. Patel, J.C., Hall, A. & Caron, E. Vav regulates activation of Rac but not Cdc42 during FcgammaR-mediated phagocytosis. Mol Biol Cell 13, 1215-1226 (2002). 463. Castellano, F., Montcourrier, P. & Chavrier, P. Membrane recruitment of Rac1 triggers phagocytosis. J Cell Sci 113 ( Pt 17), 2955-2961 (2000). 464. Machesky, L.M. & Insall, R.H. Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr Biol 8, 1347-1356 (1998). 465. Lorenzi, R., Brickell, P.M., Katz, D.R., Kinnon, C. & Thrasher, A.J. Wiskott- Aldrich syndrome protein is necessary for efficient IgG-mediated phagocytosis. Blood 95, 2943-2946 (2000). 466. Kitamura, Y., et al. Possible involvement of Wiskott-Aldrich syndrome protein family in aberrant neuronal sprouting in Alzheimer's disease. Neurosci Lett 346, 149-152 (2003). 467. Kitamura, Y., et al. Involvement of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) and Rac1 in the phagocytosis of amyloid- beta(1-42) in rat microglia. J Pharmacol Sci 92, 115-123 (2003). 468. Etienne-Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature 420, 629- 635 (2002). 469. Bishop, A.L. & Hall, A. Rho GTPases and their effector proteins. Biochem J 348 Pt 2, 241-255 (2000). 470. Dharmawardhane, S., Brownson, D., Lennartz, M. & Bokoch, G.M. Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils. J Leukoc Biol 66, 521-527 (1999). 471. Araki, N., Johnson, M.T. & Swanson, J.A. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J Cell Biol 135, 1249-1260 (1996). 472. Botelho, R.J., et al. Localized biphasic changes in phosphatidylinositol-4,5- bisphosphate at sites of phagocytosis. J Cell Biol 151, 1353-1368 (2000). 473. Kusner, D.J., Hall, C.F. & Jackson, S. Fc gamma receptor-mediated activation of phospholipase D regulates macrophage phagocytosis of IgG-opsonized particles. J Immunol 162, 2266-2274 (1999).  197 474. Kusner, D.J., Hall, C.F. & Schlesinger, L.S. Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages. J Exp Med 184, 585-595 (1996). 475. Larsen, E.C., et al. Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells. J Immunol 165, 2809- 2817 (2000). 476. Lennartz, M.R., et al. Phospholipase A2 inhibition results in sequestration of plasma membrane into electronlucent vesicles during IgG-mediated phagocytosis. J Cell Sci 110 ( Pt 17), 2041-2052 (1997). 477. Ninomiya, N., et al. Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling. J Biol Chem 269, 22732-22737 (1994). 478. Ramirez-Carrozzi, V.R., et al. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell 138, 114-128 (2009). 479. Saunders, A., Core, L.J. & Lis, J.T. Breaking barriers to transcription elongation. Nat Rev Mol Cell Biol 7, 557-567 (2006). 480. Sims, R.J., 3rd, Belotserkovskaya, R. & Reinberg, D. Elongation by RNA polymerase II: the short and long of it. Genes Dev 18, 2437-2468 (2004). 481. Kohn, A.D., et al. Construction and characterization of a conditionally active version of the serine/threonine kinase Akt. J Biol Chem 273, 11937-11943 (1998). 482. Mitchell, C.A., Brown, S., Campbell, J.K., Munday, A.D. & Speed, C.J. Regulation of second messengers by the inositol polyphosphate 5-phosphatases. Biochem Soc Trans 24, 994-1000 (1996). 483. Ooms, L.M., et al. The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease. Biochem J 419, 29-49 (2009). 484. Hokin, M.R. Effect of norepinephrine on 32P incorporation into individual phosphatides in slices from different areas of the guinea pig brain. J Neurochem 16, 127-134 (1969). 485. Rana, R.S. & Hokin, L.E. Role of phosphoinositides in transmembrane signaling. Physiol Rev 70, 115-164 (1990). 486. Serunian, L.A., Auger, K.R. & Cantley, L.C. Identification and quantification of polyphosphoinositides produced in response to platelet-derived growth factor stimulation. Methods Enzymol 198, 78-87 (1991). 487. Weber-Nordt, R.M., et al. Stat3 recruitment by two distinct ligand-induced, tyrosine-phosphorylated docking sites in the interleukin-10 receptor intracellular domain. J Biol Chem 271, 27954-27961 (1996). 488. Pritchard, C.A., Samuels, M.L., Bosch, E. & McMahon, M. Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells. Mol Cell Biol 15, 6430- 6442 (1995). 489. Khan, A.A., Martin, S. & Saha, B. SEB-induced signaling in macrophages leads to biphasic TNF-alpha. J Leukoc Biol 83, 1363-1369 (2008). 490. Yasukawa, H., et al. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat Immunol 4, 551-556 (2003). 491. Deane, J.A. & Fruman, D.A. Phosphoinositide 3-kinase: diverse roles in immune cell activation. Annu Rev Immunol 22, 563-598 (2004).  198 492. Sly, L.M., Rauh, M.J., Kalesnikoff, J., Buchse, T. & Krystal, G. SHIP, SHIP2, and PTEN activities are regulated in vivo by modulation of their protein levels: SHIP is up-regulated in macrophages and mast cells by lipopolysaccharide. Exp Hematol 31, 1170-1181 (2003). 493. Vivanco, I. & Sawyers, C.L. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2, 489-501 (2002). 494. Horan, K.A., et al. Regulation of FcgammaR-stimulated phagocytosis by the 72- kDa inositol polyphosphate 5-phosphatase: SHIP1, but not the 72-kDa 5- phosphatase, regulates complement receptor 3 mediated phagocytosis by differential recruitment of these 5-phosphatases to the phagocytic cup. Blood 110, 4480-4491 (2007). 495. Coggeshall, K.M., Nakamura, K. & Phee, H. How do inhibitory phosphatases work? Mol Immunol 39, 521-529 (2002). 496. Liang, X., et al. Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells. Proteomics 6, 4554-4564 (2006). 497. Fukuda, R., et al. Alteration of phosphatidylinositol 3-kinase cascade in the multilobulated nuclear formation of adult T cell leukemia/lymphoma (ATLL). Proc Natl Acad Sci U S A 102, 15213-15218 (2005). 498. Luo, J.M., et al. Mutation analysis of SHIP gene in acute leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi 12, 420-426 (2004). 499. Workman, P., Clarke, P.A., Guillard, S. & Raynaud, F.I. Drugging the PI3 kinome. Nat Biotechnol 24, 794-796 (2006). 500. Simon, J.A. Using isoform-specific inhibitors to target lipid kinases. Cell 125, 647-649 (2006). 501. Hennessy, B.T., Smith, D.L., Ram, P.T., Lu, Y. & Mills, G.B. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4, 988-1004 (2005). 502. Knight, Z.A., et al. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell 125, 733-747 (2006). 503. Feldman, M.E. & Shokat, K.M. New inhibitors of the PI3K-Akt-mTOR pathway: insights into mTOR signaling from a new generation of Tor Kinase Domain Inhibitors (TORKinibs). Curr Top Microbiol Immunol 347, 241-262. 504. Lindsley, C.W. The Akt/PKB family of protein kinases: a review of small molecule inhibitors and progress towards target validation: a 2009 update. Curr Top Med Chem 10, 458-477. 505. Camps, M., et al. Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis. Nat Med 11, 936-943 (2005). 506. Barber, D.F., et al. PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus. Nat Med 11, 933-935 (2005). 507. Ameriks, M.K. & Venable, J.D. Small molecule inhibitors of phosphoinositide 3- kinase (PI3K) delta and gamma. Curr Top Med Chem 9, 738-753 (2009). 508. Park, S.J., Min, K.H. & Lee, Y.C. Phosphoinositide 3-kinase delta inhibitor as a novel therapeutic agent in asthma. Respirology 13, 764-771 (2008). 509. Fan, Q.W., et al. A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 9, 341-349 (2006).  199 510. Yang, L., et al. Synthesis of pelorol and analogues: activators of the inositol 5- phosphatase SHIP. Org Lett 7, 1073-1076 (2005). 511. Ng, D.H.W., Harder, K.W., Clark-Lewis, I., Jirik, F. & Johnson, P. Non- radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells. Journal of Immunological Methods 179, 177-185 (1995). 512. Hyun, E., et al. Anti-inflammatory effects of nitric oxide-releasing hydrocortisone NCX 1022, in a murine model of contact dermatitis. Br J Pharmacol 143, 618- 625 (2004). 513. Fukushima, K., et al. Colonization of microflora in mice: mucosal defense against luminal bacteria. J Gastroenterol 34, 54-60 (1999). 514. Madsen, K., et al. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 121, 580-591 (2001). 515. Dowler, S., Kular, G. & Alessi, D.R. Protein lipid overlay assay. Sci STKE 2002, PL6 (2002). 516. Bindoli, A., Rigobello, M.P. & Deeble, D.J. Biochemical and toxicological properties of the oxidation products of catecholamines. Free Radic Biol Med 13, 391-405 (1992). 517. Galanos, C. & Freudenberg, M.A. Mechanisms of endotoxin shock and endotoxin hypersensitivity. Immunobiology 187, 346-356 (1993). 518. Kemp, S.F. & Lockey, R.F. Anaphylaxis: a review of causes and mechanisms. J Allergy Clin Immunol 110, 341-348 (2002). 519. Young, J.M., et al. The mouse ear inflammatory response to topical arachidonic acid. J Invest Dermatol 82, 367-371 (1984). 520. Sellon, R.K., et al. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect Immun 66, 5224-5231 (1998). 521. Stecher, B., et al. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect Immun 72, 4138-4150 (2004). 522. Lindsley, J.E. & Rutter, J. Whence cometh the allosterome? Proc Natl Acad Sci U S A 103, 10533-10535 (2006). 523. Fersht, A. Enzyme Structure and Mechanism, (W.H. Freeman and Company, New York, 1985). 524. Campbell, R.B., Liu, F. & Ross, A.H. Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate. J Biol Chem 278, 33617-33620 (2003). 525. Schaletzky, J., et al. Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases. Curr Biol 13, 504-509 (2003). 526. Duncan, R.R., Shipston, M.J. & Chow, R.H. Double C2 protein. A review. Biochimie 82, 421-426 (2000). 527. Sondermann, H. & Kuriyan, J. C2 can do it, too. Cell 121, 158-160 (2005). 528. Ward, S.G. & Finan, P. Isoform-specific phosphoinositide 3-kinase inhibitors as therapeutic agents. Curr Opin Pharmacol 3, 426-434 (2003). 529. Ruckle, T., Schwarz, M.K. & Rommel, C. PI3Kgamma inhibition: towards an 'aspirin of the 21st century'? Nat Rev Drug Discov 5, 903-918 (2006).  200 530. Foukas, L.C., et al. Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation. Nature 441, 366-370 (2006). 531. Ohashi, P.S. & Woodgett, J.R. Modulating autoimmunity: pick your PI3 kinase. Nat Med 11, 924-925 (2005). 532. Argiris, A., et al. A phase II trial of perifosine, an oral alkylphospholipid, in recurrent or metastatic head and neck cancer. Cancer Biol Ther 5, 766-770 (2006). 533. Knowling, M., et al. A phase II study of perifosine (D-21226) in patients with previously untreated metastatic or locally advanced soft tissue sarcoma: A National Cancer Institute of Canada Clinical Trials Group trial. Invest New Drugs 24, 435-439 (2006). 534. Posadas, E.M., et al. A phase II study of perifosine in androgen independent prostate cancer. Cancer Biol Ther 4, 1133-1137 (2005). 535. Powis, G., Ihle, N. & Kirkpatrick, D.L. Practicalities of drugging the phosphatidylinositol-3-kinase/Akt cell survival signaling pathway. Clin Cancer Res 12, 2964-2966 (2006). 536. Van Ummersen, L., et al. A phase I trial of perifosine (NSC 639966) on a loading dose/maintenance dose schedule in patients with advanced cancer. Clin Cancer Res 10, 7450-7456 (2004). 537. Warino, L. & Libecco, J. Cutaneous effects of sirolimus in renal transplant recipients. J Drugs Dermatol 5, 273-274 (2006). 538. Iijima, M. & Devreotes, P. Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109, 599-610 (2002). 539. Downes, C.P., et al. Acute regulation of the tumour suppressor phosphatase, PTEN, by anionic lipids and reactive oxygen species. Biochem Soc Trans 32, 338- 342 (2004). 540. Gericke, A., Munson, M. & Ross, A.H. Regulation of the PTEN phosphatase. Gene 374, 1-9 (2006). 541. Lo, T.C., et al. Inactivation of SHIP1 in T-cell acute lymphoblastic leukemia due to mutation and extensive alternative splicing. Leuk Res 33, 1562-1566 (2009). 542. Hollander, M.C., Blumenthal, G.M. & Dennis, P.A. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer 11, 289- 301 (2011). 543. Li, J., et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943-1947 (1997). 544. Laporte, J., et al. A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. Nat Genet 13, 175- 182 (1996). 545. Aderem, A. & Underhill, D.M. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17, 593-623 (1999). 546. Crowley, M.T., et al. A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages. J Exp Med 186, 1027-1039 (1997). 547. Fitzer-Attas, C.J., et al. Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn. J Exp Med 191, 669-682 (2000).  201 548. Cox, D., Tseng, C.C., Bjekic, G. & Greenberg, S. A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J Biol Chem 274, 1240- 1247 (1999). 549. Caron, E. & Hall, A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 282, 1717-1721 (1998). 550. Hall, A.B., et al. Requirements for Vav guanine nucleotide exchange factors and Rho GTPases in FcgammaR- and complement-mediated phagocytosis. Immunity 24, 305-316 (2006). 551. Massol, P., Montcourrier, P., Guillemot, J.C. & Chavrier, P. Fc receptor-mediated phagocytosis requires CDC42 and Rac1. EMBO J 17, 6219-6229 (1998). 552. Dewitt, S., Tian, W. & Hallett, M.B. Localised PtdIns(3,4,5)P3 or PtdIns(3,4)P2 at the phagocytic cup is required for both phagosome closure and Ca2+ signalling in HL60 neutrophils. J Cell Sci 119, 443-451 (2006). 553. Dormann, D., Weijer, G., Dowler, S. & Weijer, C.J. In vivo analysis of 3- phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis. J Cell Sci 117, 6497-6509 (2004). 554. Marshall, J.G., et al. Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis. J Cell Biol 153, 1369-1380 (2001). 555. Ai, J., et al. The inositol phosphatase SHIP-2 down-regulates FcgammaR- mediated phagocytosis in murine macrophages independently of SHIP-1. Blood 107, 813-820 (2006). 556. Hoppe, A.D. & Swanson, J.A. Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol Biol Cell 15, 3509-3519 (2004). 557. Ravetch, J.V. & Clynes, R.A. Divergent roles for Fc receptors and complement in vivo. Annu Rev Immunol 16, 421-432 (1998). 558. Larkin, M.A., et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007). 559. Ong, C.J., et al. Small-molecule agonists of SHIP1 inhibit the phosphoinositide 3- kinase pathway in hematopoietic cells. Blood 110, 1942-1949 (2007). 560. Marley, J., Lu, M. & Bracken, C. A method for efficient isotopic labeling of recombinant proteins. Journal of Biomolecular NMR 20, 71-75 (2001). 561. Shojania, S. & O'Neil, J.D. HIV-1 Tat is a natively unfolded protein: the solution conformation and dynamics of reduced HIV-1 Tat-(1-72) by NMR spectroscopy. Journal of Biological Chemistry 281, 8347-8356 (2006). 562. Neidhardt, F.C., Bloch, P.L. & Smith, D.F. Culture medium for enterobacteria. J. Bacteriol. 119, 736-747 (1974). 563. Kay, L.E., Keifer, P. & Saarinen, T. Pure Absorption Gradient Enhanced Heteronuclear Single Quantum Correlation Spectroscopy with Improved Sensitivity. Journal of the American Chemical Society 114, 10663-10665 (1992). 564. Wittekind, M. & Mueller, L. HNCACB, a High-Sensitivity 3D NMR Experiment to Correlate Amide-Proton and Nitrogen Resonances with the Alpha- and Beta- Carbon Resonances in Proteins. Journal of Magnetic Resonance Series B 101, 201-205 (1993).  202 565. Grzesiek, S. & Bax, A. Correlating Backbone Amide and Side-Chain Resonances in Larger Proteins by Multiple Relayed Triple Resonance NMR. Journal of the American Chemical Society 114, 6291-6293 (1992). 566. Ikura, M., Kay, L.E. & Bax, A. A Novel-Approach for Sequential Assignment of H-1, C-13, and N-15 Spectra of Larger Proteins - Heteronuclear Triple-Resonance 3-Dimensional Nmr-Spectroscopy - Application to Calmodulin. Biochemistry 29, 4659-4667 (1990). 567. Yamazaki, T., et al. An HNCA Pulse Scheme for the Backbone Assignment of 15N,13C,2H-Labeled Proteins - Application to a 37-kDa Trp Repressor DNA Complex. Journal of the American Chemical Society 116, 6464-6465 (1994). 568. Delaglio, F., et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. Journal of Biomolecular NMR 6, 277-293 (1995). 569. Godard, T. & Kneller, D.G. SPARKY 3.115. (, University of California, San Francisco, 2008).  Accessed March 3rd 2008. 570. Cavanagh, J., Fairbrother, W.J., Palmer, A.G. & Skelton, N.J. Protein NMR Spectroscopy: Principles and Practice, (Academic Press, San Diego, 1996). 571. Wishart, D.S., et al. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. Journal of Biomolecular NMR 6, 135-140 (1995). 572. Blasi, E., Radzioch, D., Merletti, L. & Varesio, L. Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus. Cancer Biochem Biophys 10, 303-317 (1989). 573. Ng, D.H., Harder, K.W., Clark-Lewis, I., Jirik, F. & Johnson, P. Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells. J Immunol Methods 179, 177-185 (1995). 574. Marsh, J.A., Singh, V.K., Jia, Z. & Forman-Kay, J.D. Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma- synuclein: implications for fibrillation. Protein Sci 15, 2795-2804 (2006). 575. Zhang, H., Leung, A., and D. Wishart. "The THRIFTY web server, version 1.0". University of Alberta, Edmonton(2005). Accessed February 13th 2009. 576. Lemmon, M.A. Pleckstrin homology domains: not just for phosphoinositides. Biochem Soc Trans 32, 707-711 (2004). 577. Lemmon, M.A. Pleckstrin homology (PH) domains and phosphoinositides. Biochem Soc Symp, 81-93 (2007). 578. Li, X., Romero, P., Rani, M., Dunker, A.K. & Obradovic, Z. Predicting Protein Disorder for N-, C-, and Internal Regions. Genome Inform Ser Workshop Genome Inform 10, 30-40 (1999). 579. Romero, Obradovic & Dunker, K. Sequence Data Analysis for Long Disordered Regions Prediction in the Calcineurin Family. Genome Inform Ser Workshop Genome Inform 8, 110-124 (1997). 580. Romero, P., et al. Sequence complexity of disordered protein. Proteins 42, 38-48 (2001). 581. Guermeur, Y., et al. Combining protein secondary structure prediction models with ensemble methods of optimal complexity. Neurocomputing 56, 305-327 (2004).  203 582. Garnier, J., Gibrat, J.F. & Robson, B. GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266, 540-553 (1996). 583. Kay, L., Keifer, P. & Saarinen, T. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. Journal of the American Chemical Society 114, 10663-10665 (1992). 584. Neidhardt, F.C., Bloch, P.L. & Smith, D.F. Culture medium for enterobacteria. J Bacteriol 119, 736-747 (1974). 585. Geourjon, C. & Deleage, G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11, 681-684 (1995). 586. Cole, C., Barber, J.D. & Barton, G.J. The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36, W197-201 (2008). 587. Pollastri, G. & McLysaght, A. Porter: a new, accurate server for protein secondary structure prediction. Bioinformatics 21, 1719-1720 (2005). 588. Ouali, M. & King, R.D. Cascaded multiple classifiers for secondary structure prediction. Protein Sci 9, 1162-1176 (2000). 589. Pollastri, G., Przybylski, D., Rost, B. & Baldi, P. Improving the prediction of protein secondary structure in three and eight classes using recurrent neural networks and profiles. Proteins 47, 228-235 (2002). 590. Farrow, N.A., et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984- 6003 (1994). 591. Godard, T., Kneller, D.G. SPARKY 3.115. in University of California, San Francisco (2008). 592. Dowler, S., et al. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem J 351, 19-31 (2000). 593. Keightley, D.D. & Cressie, N.A. The Woolf plot is more reliable than the Scatchard plot in analysing data from hormone receptor assays. J Steroid Biochem 13, 1317-1323 (1980). 594. Vieira, O.V., et al. Distinct roles of class I and class III phosphatidylinositol 3- kinases in phagosome formation and maturation. J Cell Biol 155, 19-25 (2001). 595. Wientjes, F.B. & Segal, A.W. PX domain takes shape. Curr Opin Hematol 10, 2- 7 (2003). 596. Kiener, P.A., et al. Cross-linking of Fc gamma receptor I (Fc gamma RI) and receptor II (Fc gamma RII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J Biol Chem 268, 24442-24448 (1993). 597. Nimmerjahn, F. & Ravetch, J.V. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol 8, 34-47 (2008). 598. Larsen, E.C., et al. A role for PKC-epsilon in Fc gammaR-mediated phagocytosis by RAW 264.7 cells. J Cell Biol 159, 939-944 (2002). 599. Cozier, G.E., Carlton, J., Bouyoucef, D. & Cullen, P.J. Membrane targeting by pleckstrin homology domains. Curr Top Microbiol Immunol 282, 49-88 (2004).  204 600. Edlich, C., Stier, G., Simon, B., Sattler, M. & Muhle-Goll, C. Structure and phosphatidylinositol-(3,4)-bisphosphate binding of the C-terminal PH domain of human pleckstrin. Structure 13, 277-286 (2005). 601. Yogo, K., et al. Src homology 2 (SH2)-containing 5'-inositol phosphatase localizes to podosomes, and the SH2 domain is implicated in the attenuation of bone resorption in osteoclasts. Endocrinology 147, 3307-3317 (2006). 602. Wada, T., et al. Role of the Src homology 2 (SH2) domain and C-terminus tyrosine phosphorylation sites of SH2-containing inositol phosphatase (SHIP) in the regulation of insulin-induced mitogenesis. Endocrinology 140, 4585-4594 (1999). 603. Damen, J.E., Ware, M.D., Kalesnikoff, J., Hughes, M.R. & Krystal, G. SHIP's C- terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation. Blood 97, 1343-1351 (2001). 604. Aman, M.J., et al. Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling. Mol Cell Biol 20, 3576-3589 (2000). 605. Zhang, Y., Hoppe, A.D. & Swanson, J.A. Coordination of Fc receptor signaling regulates cellular commitment to phagocytosis. Proc Natl Acad Sci U S A 107, 19332-19337. 606. Jaumouille, V. & Grinstein, S. Receptor mobility, the cytoskeleton, and particle binding during phagocytosis. Curr Opin Cell Biol 23, 22-29. 607. Golebiewska, U., et al. Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages. Mol Biol Cell 22, 3498-3507 (2011). 608. Mehta, P., et al. LyGDI, a Novel SHIP-Interacting Protein, Is a Negative Regulator of FcgammaR-Mediated Phagocytosis. PLoS One 6, e21175. 609. Harris, S.J., et al. Evidence that the lipid phosphatase SHIP-1 regulates T lymphocyte morphology and motility. J Immunol 186, 4936-4945. 610. Tsukamoto, K., et al. Critical roles of the p110 beta subtype of phosphoinositide 3-kinase in lipopolysaccharide-induced Akt activation and negative regulation of nitrite production in RAW 264.7 cells. J Immunol 180, 2054-2061 (2008). 611. Elkon, R., Linhart, C., Halperin, Y., Shiloh, Y. & Shamir, R. Functional genomic delineation of TLR-induced transcriptional networks. BMC Genomics 8, 394 (2007). 612. Wells, C.A., et al. Genetic control of the innate immune response. BMC Immunol 4, 5 (2003). 613. Alam, M.M. & O'Neill, L.A. MicroRNAs and the resolution phase of inflammation in macrophages. Eur J Immunol 41, 2482-2485. 614. Gracias, D.T. & Katsikis, P.D. MicroRNAs: key components of immune regulation. Adv Exp Med Biol 780, 15-26. 615. Li, T., et al. MicroRNAs modulate the noncanonical transcription factor NF- kappaB pathway by regulating expression of the kinase IKKalpha during macrophage differentiation. Nat Immunol 11, 799-805. 616. El Gazzar, M. & McCall, C.E. MicroRNAs distinguish translational from transcriptional silencing during endotoxin tolerance. J Biol Chem 285, 20940- 20951.  205 617. Luers, A.J., Loudig, O.D. & Berman, J.W. MicroRNAs are expressed and processed by human primary macrophages. Cell Immunol 263, 1-8. 618. Lu, L.F. & Liston, A. MicroRNA in the immune system, microRNA as an immune system. Immunology 127, 291-298 (2009). 619. Bamias, G. & Cominelli, F. Immunopathogenesis of inflammatory bowel disease: current concepts. Curr Opin Gastroenterol 23, 365-369 (2007). 620. Balla, T. Inositol-lipid binding motifs: signal integrators through protein-lipid and protein-protein interactions. J Cell Sci 118, 2093-2104 (2005). 621. Yao, L., et al. Pleckstrin homology domains interact with filamentous actin. J Biol Chem 274, 19752-19761 (1999). 622. Brugnoli, F., Bavelloni, A., Benedusi, M., Capitani, S. & Bertagnolo, V. PLC- beta2 activity on actin-associated polyphosphoinositides promotes migration of differentiating tumoral myeloid precursors. Cell Signal 19, 1701-1712 (2007).     206       APPENDICES                   207              Figure A.1 Cloning strategy for generation of inducible siRNA lentiviral vectors.  Double stranded siRNA designed using Invitrogen’s BLOCK-iT design software was annealed into linearized pcDNA 6.2-GW/EmGFP-miR vector via complementary overhanging nucleotides to create pcDNA 6.2-GW/EmGFP-miR-siRNA.  Using Invitrogen’s Gateway system, a BP recombination reaction was then performed between pcDNA 6.2-GW/EmGFP-miR-siRNA and pDONR-221 to generate the pENTR-221- siRNA vector.  A LR recombination reaction between the generated pENTR-221-siRNA vector and pTRIPZ-Dest vector was then performed to create the pTRIPZ-siRNA vector used for lentiviral production. attB = attB recombination site. EmGFP = GFP gene.  miR = microRNA-like sequence. attP = attP recombination site.  attL = attL recombination site. attR = attR recombination site. TRE = tetracycline response element.  Figure was provided courtesy of Gary Golds.  208      Target Forward Primer Reverse Primer Primary Response Genes ccl2 TTAAAAACCTGGATCGGAACCAA GCATTAGCTTCAGATTTACGGGT ccl3 TTCTCTGTACCATGACACTCTGC CGTGGAATCTTCCGGCTGTAG cxcl2 CCAACCACCAGGCTACAGG GCGTCACACTCAAGCTCTG egr1 TCGGCTCCTTTCCTCACTCA CTCATAGGGTTGTTCGCTCGG icam1 GTGATGCTCAGGTATCCATCCA CACAGTTCTCAAAGCACAGCG il23 ATGCTGGATTGCAGAGCAGTA ACGGGGCACATTATTTTTAGTCT junb TCACGACGACTCTTACGCAG CCTTGAGACCCCGATAGGGA marksl1 CAATGGAGACTTAACCCCCAAG GGCCACTCAATTTGAAAGGCT nfkbia TGAAGGACGAGGAGTACGAGC TTCGTGGATGATTGCCAAGTG nfkbiz GCTCCGACTCCTCCGATTTC GAGTTCTTCACGCGAACACC nr4a1 TTGAGTTCGGCAAGCCTACC GTGTACCCGTCCATGAAGGTG peli1 GCCCCAGTAAAATATGGCGAA CCCCATTTGCCTTAGGTCTTT pim1 CTGGAGTCGCAGTACCAGG CAGTTCTCCCCAATCGGAAATC sod2 CAGACCTGCCTTACGACTATGG CTCGGTGGCGTTGAGATTGTT tnfa TCTTCTCATTCCTGCTTGTGG GGTCTGGGCCATAGAACTGA tnfsf9 CGGCGCTCCTCAGAGATAC ATCCCGAACATTAACCGCAGG  Normalization Control Genes gapdh AATGTGTCCGTCGTGGATCT GCTTCACCACCTTCTTGATGT  Colitis Model Inflammatory Genes ccl2 Same as above il17 CTCCAGAAGGCCCTCAGACTAC GGGTCTTCATTGCGGTGG  Promoter Specific Sequences tnfa CCGCTTCCTCCACATGAGA TCATTCAACCCTCGGAAAACTT actin TGACGGGGTCACCCACACTGTGCCCATCT CTAGAAGCATTTGCGGTGGACGATGGAGGGG gapdh AGTGCCAGCCTCGTCCCGTAGACAAAATG AAGTGGGCCCCGGCCTTCTCCAT  Table A.1: Primer sequences for real-time quantitative PCR using SYBR green detection.      209 C ontrol LPS LPS +  IL-10 0 1000 2000 3000 ** ** P I-3 ,4 ,5 -P 3 ( C P M ) C ontrol LPS LPS +  IL-10 0 1000 2000 3000 ** * A     -      5    10   20   30    -     5    10   20   30 p-Tyrosine SHIP1 Wildtype IL-10R 446/496 Mutant SHIP1 p-4 46/ 496 446 /49 6B C P I-3 ,4 -P 2 ( C P M )           Figure B.1 IL-10 activates SHIP1 (A) Immunoblot analysis of J774.1 macrophage cells retrovirally transduced to express wildype hIL-10R or mutant hIL-10R-TyrFF stimulated with hIL-10 (50 ng/ml) in the presence of 10 µg/ml IBI.2 rat anti-mIL-10R blocking antibody for the indicated times, probed with anti-Phospho Tyrosine antibody (4G10) and protein SHIP1 (Loading Control).  (B) Immunoblot analysis of J774.1 cell lysates precipitated with a phosphorylated synthetic hIL-10R peptide (p-446/496) or an unphosphorylated hIL-10R peptide (446/496) and probed for SHIP1. HPLC Inositol phospholipid analysis of orthophosphate labeled J16 BMDMs treated with control buffer, LPS (50 ng/ml) or co- treated with LPS + IL-10 (100ng/ml) for 15 minutes.  Data represent mean CPMs ± s.d. (n=3).   *p<0.05, **p<0.01 (One-way ANOVA).  Figure provided courtesy of Dr. Ali Ghanipour.      210                     Figure C.1 Continuous Flow Apparatus. Continuous-flow apparatus facilitates constant stimulation and removal of cell supernatants to determine kinetic profiles of cytokine production over time.  A=Syringe Pump set to dispense at a constant rate of 150 µl stimulation media/min injects stimulation media through “Inlet Line”.  B=Continuous-flow chamber plate.  Cells plated in a 24-well tissue culture plate are fitted with rubber stoppers adapted with 18 gauge needles.  Stimulation media volume within each well is maintained at a volume of 500 µl. Pressure generated by stimulation media injection forces the simultaneous removal of cell supernatants through the “Outlet line”.  C=Fraction collector set to advance every 5 minutes.  Component A and B are maintained in a 37°C incubator.  211 0 50 100 150 200 250 0 250 500 750 LPS LPS + IL-10 T ime (min) 0 50 100 150 200 250 0 250 500 750 T ime (min) [T NF _ ] ( [p g/ m l) 0 50 100 150 200 250 0 250 500 750 LPS LPS + IL-10 T ime (min) 0 50 100 150 200 250 0 250 500 750 T ime (min) [T NF _ ] ( [p g/ m l) [T NF _ ] ( [p g/ m l) [T NF _ ] ( [p g/ m l) A B SHIP1 WT SHIP1 KO SHIP1 WT SHIP1 KO                    Figure D.1 Replicate experiments demonstrating IL-10 enhancement of TNFα production during a specific phase of the first peak of production in the absence of SHIP1. (A) TNFα ELISA of fractions collected from SHIP1+/+ and SHIP1-/- PMΦs stimulated with LPS (1 ng/ml) with or without IL-10 (100 pg/ml) under continuous-flow conditions. (B) Replicate experiment as (A) but only fractions collected at 75 through 150 minutes were analyzed by TNFα ELISA.     212   S H IP 1 E nz ym e A ct iv ity (p m ol es  p ho sp ha te /m in ) 0.0 2.0 4.0 6.0 No  d ru g Pe lor ol AQ X- 01 6A 0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0 12.5 SHIP1 SHIP2 AQX-016A (+M) P ho sp ha ta se  A ct iv ity (p m ol es  p ho sp ha te /m in ) 5 mg/ml                          AQX-016A TN F_  (%  o f m ax im um ) 0 25 50 75 100 SHIP1+/+ +/+ -/--/- 1 mg/ml 0 500 1000 1500 2000 2500 PI -3 ,4 ,5 - P 3  ( CP M  ) + + + 0 1000 2000 3000 4000 5000 PI -3 ,4 - P 2 ( C PM  ) - -           -           + -           -            -           + ++ + LPS AQX-016A LY294002 - - -           -                        - -           -            - + + LPS AQX-016A LY294002 C A B D                    Figure E.1 Pelorol and AQX-016A enhance SHIP1 phosphatase activity. (A) Purified Pelorol and AQX-016A were tested at 2 µM for their ability to enhance recombinant SHIP1 enzyme activity.  (B) HPLC Inositol phospholipid analysis of orthophosphate labeled J16 BMDMs treated with vehicle, AQX-016A (15 µM) or LY294002 (25 µM) 30 minutes prior to stimulation with LPS (50 ng/ml).  (C) The effect of AQX-016A on SHIP1 (n) and SHIP2 (Δ) enzyme activity was compared in in vitro enzyme assays.  (D) (A) SHIP+/+ and SHIP -/- macrophages were pre-treated with AQX- 016A or vehicle for 30 minutes prior to stimulation with 10 ng/mL of LPS at 37°C for 2 h and TNFα production determination by ELISA.  Absolute TNFα levels for SHIP+/+ and SHIP-/- cells were 623 +/- 30 and 812 +/- 20 pg/ml, respectively. Data are expressed as mean +/ SEM and are representative of three independent experiments.  Figure provided courtesy of Dr. Ali Ghanipour.  213           Figure F.1 AQX-MN100 specifically enhances SHIP1 phosphatase activity and has minimal off-target effects Compound profiling activity was undertaken using 100 protein kinase and phosphatase targets by SignalChem (Richmond, BC) against compound AQX-MN100 (2 µM final concentration).  Protein kinase assays were performed in the presence of 50 µM ATP at 30°C for 15 min. Protein phosphatase activites were determined using pNPP as substrate and were also performed at 37°C for 15 min.  The activity of the enzymes in the presence of AQX-MN100 was compared to that in the vehicle control and expressed as a % change in activity relative to that observed in the vehicle control.  Changes in activity of <25% were not considered significant.  Enzymes affected by AQX-MN100 are plotted in an expanded graph in B.      214               Figure G.1 Phosphatidyl inositol lipid binding ability of single mutant SHIP1 PH- R domains. (A) Initial screen of SHIP1 PH-R domain point mutant constructs made using a standard PCR-based site directed mutagenesis method.  Mutant constructs were expressed in BL21 (DE3) competent cells and purified as previously described. Recombinant domains at a concentration of 625 nM in 6 ml of blocking buffer were tested in PLO assays for their ability to bind (A) PI-4,5-P2, (B) PIP3 or (C) PI-3,4-P2 spotted onto PVDF membrane in amounts ranging from 0 to 50 pmols  (n=1).  215                     Figure H.1 Double reciprocal plots of phosphatidyl inositol lipid binding ability of mutant SHIP1 PH-R domains (A) Recombinant K370A or K397A single mutant domains or (B) KAKA SHIP1 PH-R domain were expressed in BL21 (DE3) competent cells and purified as previously described.  Recombinant domains at a concentration of 625 nM in 6 ml of blocking buffer were tested in PLO assays for their ability to bind PI-4,5-P2, PIP3, or PI-3,4-P2 spotted onto PVDF membrane in amounts ranging from 0 to 50 pmols.  Blots were probed with primary and secondary antibodies, and quantified as in Materials and Methods.  The reciprocal of the mean intensities were plotted against the reciprocal of the amount of lipid spotted.  KD values were calculated from the slope of the lines 593 as determined by linear regression (GraphPad Prism, San Diego, CA).


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items