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The role of SHIP2 in suppressing inflammatory signaling induced by LPS in immortalized murine macrophage… Wang, Tianren 2016

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  The role of SHIP2 in suppressing inflammatory signaling induced by LPS in Immortalized Murine Macrophage Cell Line by Tianren Wang B. Sc. (Hon), University of Toronto, 2014    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Biochemistry and Molecular Biology)   The University of British Columbia (Vancouver) January, 2016    © Tianren Wang 2016   ii  Abstract  Inflammation is an important step in the body’s defense against pathogen infection. However, it must be tightly regulated and appropriately terminated to prevent pathological consequences. Interleukin-10 (IL10) is one of the body’s most important anti-inflammatory cytokine that can inhibit many molecular events necessary for promoting inflammation including production of pro-inflammatory cytokines such as Tumor Necrosis Factor α (TNFα). Our laboratory has recently shown that SH2-domain containing Inositol 5ʹ phosphatase (SHIP1) is involved in IL10 signaling in macrophages, and although the mechanism of how this occurs is not well studied, our laboratory have obtained data suggesting SHIP1 mediates IL10 signalling through its phosphatase activity or interaction with other signalling proteins. SHIP2 is the only other known homologue of SHIP1 with approximately 38% amino acid sequence identity, yet they possess several similar functions including mediating FcγIIB signaling and phagocytosis. Because of their similarities and SHIP1’s involvement in IL10 signaling, we sought to investigate whether SHIP2 is also involved in inhibiting inflammatory response in macrophage by knocking it out using CRISPR/Cas9-mediated genome editing. Overall, we were unable to determine whether SHIP2 plays a role in macrophage anti-inflammatory response due to the large variation in cell sensitivity to IL10 and we also observed that transduction of macrophages with CRISPR/Cas9 virus alters the cellular response to IL10 which confounded our investigation of SHIP2 function.      iii  Preface  Design of all experiments and analysis of all research data were completed under the supervision of Dr. Alice Mui.  All experiments were performed by the author, with assistance from Sylvia Cheung and Lisa Lee on CRISPR/Cas9 constructs design and cloning.  Biosafety Approval The author has completed Biological Safety Training Course conducted by Risk Management Services of UBC. Course completion date: October 14, 2014 Certificate ID: 2014 – XaTk5 The author’s research laboratory has been approved for Biosafety by UBC. Biosafety Project ID: B12 – 0010         iv  Table of Contents  Abstract ............................................................................................................................... ii Preface ............................................................................................................................... iii Table of Contents .............................................................................................................. iv List of Tables ..................................................................................................................... vi List of Figures .................................................................................................................. vii List of Illustrations .......................................................................................................... viii List of Abbreviations ......................................................................................................... ix 1 Introduction ..................................................................................................................... 1 1.1 Inflammation ....................................................................................................................... 1 1.2 Innate immunity .................................................................................................................. 2 1.2.1 Macrophages ................................................................................................................................. 3 1.3 TLR/LPS signaling pathway .............................................................................................. 4 1.4 Tumor Necrosis Factor-α .................................................................................................... 7 1.5 Interleukin-10 ...................................................................................................................... 8 1.5.1 Biology of IL10 ............................................................................................................................ 9 1.5.2 IL10 inhibition of TNFα production ............................................................................................11 1.6 PI3K pathway .................................................................................................................... 11 1.6.1 Function of PI3K in TLR4 pathway ............................................................................................12 1.7 Inositol phosphatases ........................................................................................................ 13 1.7.1 SHIP1 ...........................................................................................................................................14 1.7.2 SHIP2 ...........................................................................................................................................15 1.7.3 Non-catalytic and PI3K-independent functions of SHIP1 and SHIP2 .........................................16 1.7.4 Overlapping functions of SHIP1 and SHIP2 ...............................................................................17 1.8 CRISPR/Cas9 genome editing .......................................................................................... 17 1.9 Hypothesis .......................................................................................................................... 18 2 Method ........................................................................................................................... 20 2.1 Cell culture ......................................................................................................................... 20 2.2 Plasmids/primers ............................................................................................................... 20 2.3 Generation of cell lines ...................................................................................................... 21 2.4 Immunoblotting ................................................................................................................. 21 v  2.5 Antibodies ........................................................................................................................... 22 2.6 TNFα ELISA ...................................................................................................................... 22 3 Result ............................................................................................................................. 24 3.1 CRISPR/Cas9 constructs design and rationale ............................................................... 24 3.2 CRISPR/Cas9 reduces SHIP2 level in J2M cells ............................................................ 26 3.3 IL10 sensitivity of J2M cells lacking SHIP2 .................................................................... 27 4 Discussion...................................................................................................................... 30 5 Conclusion ..................................................................................................................... 34 References ........................................................................................................................ 35 Appendices ........................................................................................................................ 49 Appendix A - Amino acid sequence alignment of mouse SHIP1 and SHIP2 protein ........ 49 Appendix B - Nomenclature of the cell lines used in this study .......................................... 51 Appendix C - All IL10 IC50 values obtained for all cell lines ............................................... 52           vi  List of Tables  Table 1. Biological effect of IL10 on different immune cells ....................................................... 10                  vii  List of Figures  Figure 1. Validation of CRISPR/Cas9 plasmid constructs by PCR .............................................. 25 Figure 2. Western Blot analysis of SHIP2 expression in cells transduced with CRISPR/Cas9 virus .. 27 Figure 3. IL10 sensitivity of ΔSHIP2 and ΔSHIP1/ΔSHIP2 J2M cells ........................................ 28                       viii  List of Illustrations  Illustration 1. TLR4 signaling pathway ........................................................................................... 7 Illustration 2. IL10 signaling pathway ........................................................................................... 11 Illustration 3. Domains of SHIP1 and SHIP2 ................................................................................ 13 Illustration 4. SHIP2 knockout strategy using CRISPR/Cas9 genome editing ............................. 24                      ix  List of Abbreviations AP1    Activator protein 1   Akt     Protein kinase B (PKB)  Arap3    Ankyrin repeat and PH domain 3   ARE    AU-rich element   Bcl3     B-cell lymphoma 3-encoded protein   BCAP    B-cell adaptor protein   BCR     B-cell receptor   cPPT     Central polypurine tract   CD     Cluster of differentiation   CRISPR    Clustered, regularly interspaced, short palindromic repeat   CRISPR/Cas9    CRISPR-associated protein-9 nuclease  crRNA     CRISPR-RNA   DAMPs   Danger Associated Molecular Patterns   DD    Death domain   DMEM    Dulbecco’s Modified Eagle Medium   DSB    Double stranded break   dsRNA    Double-stranded RNA  EphA2    Ephrin type A receptor 2   EFP    Elongation factor-1α promoter   FCS    Fetal calf serum   G-CSF    Granulocyte-colony stimulating factor    GM-CSF    Granulocyte-macrophage colony-stimulating factor   MHC    Histocompatability Complex   HDR     Homology directed repair   HGFR      Met/hepatocyte growth factor receptor  x   iNOS    inducible nitric oxide synthase   KSRP     KH-type splicing regulatory protein   IL-6     Interleukin-6  IL10     Interleukin-10   IL10R    Interleukin-10 Receptor  IMDM     Iscove’s modified Dulbecco’s medium   IRAK1    Interleukin-1 Receptor Associated Kinase   IRF3     Interferon regulatory factor 3  IRF5    Interferon regulatory factor 5   ITIM motif     Immunoreceptor tyrosine-based inhibition motif   JNK    c-Jun N-terminal Kinase   J2M WW cell    J2M Wild-type cell  J2M KW cells (ΔSHIP1)  J2M KW cells (ΔSHIP1)   J2M KK3 cells    J2M KK3 cells (ΔSHIP1, ΔSHIP2)  LPS    Lipopolysaccharide   LBP    LPS-binding protein   M-CSF    Macrophage colony stimulating factor   MAPKK6   MAP Kinase Kinase 6   MD2     Lymphocyte antigen 96  MyD88    Myeloid differentiation primary response gene 88   Mal     MyD88-adaptor like   NHEJ    Non-homologous end joining   NFκB    Nuclear factor κ-light-chain-enhancer of activated B cells   PAMP     Pathogen-associated molecule pattern   PDGFR    Platelet-derived growth factor receptor xi  PI3K    Phosphatidylinositol-3-kinase   PIP3     Phosphatidylinositol (3,4,5)-trisphosphate   p38    p38 mitogen-activated protein kinase   pTyr    Phosphorylated tyrosine   PTEN    Phosphatase and Tensin Homolog   PIP2    Phosphatidylinositol (4,5)-bisphosphate   PIP3    Phosphatidylinositol (3,4,5)-trisphosphate   PTB    Phospho-tyrosine binding   PH-R    Pleckstrin homology-related   PRR    Proline-rich region   RIP1    Receptor interacting protein 1   RRE    Rev response element   SARM    Sterile α- and armadillo-motif containing protein    SH2    Src Homology 2   SHIP1    SH2-domain containing Inositol 5ʹ phosphatase 1   SHIP2    SH2-domain containing inositol 5´phosphatase 2   STAT1    Signal transducer and activator of transcription 1  STAT3    Signal transducer and activator of transcription 3   STAT5    Signal transducer and activator of transcription 5  SAM    Sterile α motif   TAB2    TAK1-associated binding protein 2   TACE    Tumor necrosis factor α converting Enzyme   TBS     Tris buffered saline   TBS-T     Tris-buffered saline supplemented with 0.1% Tween 20   TAK1    TGF-β activated kinase 1   xii  TBK1 TRAF family member-associated NFκB activator (TANK)-binding kinase 1   TIR    Toll/IL1R   TLR    Toll-like receptors   TNFα    Tumor necrosis factor α   TracrRNA   transactivating CRISPR-RNA   TRAF    TNF receptor associated factor   TRIF TIR-domain containing adaptor protein inducing interferon-β   TRAM     TRIF related adaptor molecule   TRAF6    Tumor necrosis factor receptor-associated factor 6   TTP    Tristetraprolin   UIM    ubiquitin interacting motif  1  1 Introduction 1.1 Inflammation In a world that is replete with microorganisms and viruses, an immune system is crucial for vertebrate survival. Inflammation is the key step in activating the immune system to fight against these pathogens and is also the key control point in the body’s attempt to modulate the degree of immunological activity1. Inflammation is classically defined as the occurrence of redness, swelling, heat, pain1,2, and disturbance of function of the tissue affected by inflammation. Because of these damaging characteristics, inflammation poses a threat to the host’s health, and inappropriate regulation of inflammatory response can potentially lead to inflammatory diseases3–8.  Inflammation’s main function is the rapid elimination of pathogen and damaged cells and restoration of immune homeostasis. It is initiated and orchestrated by immune cells when they come into contact with signals of infection or tissue damage2,9–11. These signals can be in the form of cellular debris or components of pathogens, which upon recognition by immune cells, cause a behavioral change in the immune cells to combat the presented threat by activating or repressing specific intracellular signaling pathways that regulate antimicrobial functions such as engulfment of pathogens (phaghocytosis) and release of cytokines, which can recruit effector immune cells such as neutropils and monocytes12,13. The recruitment of some of these immune cells will further exacerbate the cytotoxic environment at the site of inflammation. 2  Cytokines can either promote or inhibit the inflammatory state of immune cells. For example, pro-inflammatory cytokines such as Tumor Necrosis Factor α (TNFα) and Interleukin-1 (IL-1) are some of the earliest cytokines induced by pathogen signals, and they act to further enhance their own production, promote the production of molecules that induce fever, vasodilation, and destruction of pathogens, and recruit immunocompetent cells to the site of infection14–17. Anti-inflammatory cytokines such as IL-10 and transforming growth factor-β repress the recruitment of immune cells to the site of infection or damage, inhibit the release of pro-inflammatory cytokines and reprogram the immune cells’ behavior to an anti-inflammatory state18–22. Therefore, cytokines are crucial regulators of inflammation. 1.2 Innate immunity The inflammatory response involves cells of innate immunity that perform anti-microbial functions immediately upon recognition of pathogens23. The major innate immune cells are phagocytes, antigen-presenting cells, granulocytes, and natural killer cells24. Phagocytes are capable of engulfing pathogens, and they include macrophages25, neutrophils26, and dendritic cells27. Macrophages and dendritic cells can also present antigen by redirecting the components of the phagocytosed pathogen to the cell surface together with Major Histocompatability Complex (MHC), which leads to the activation of adaptive immunity28,29. Granulocytes, as its name suggests, contain granules that pack cytotoxic proteins and reactive nitrogen and oxygen species that kill and inhibit growth of bacteria and fungi30–32. Neutrophils, basophils, eosinophils and mast cells are the most abundant granulocytes. Lastly, the natural killer cells secrete cytotoxic substances in the 3  presence of cells that have abnormally low surface expression of MHC, which are typically tumor cells or cells infected by virus33. 1.2.1 Macrophages Macrophage is involved in both the initiation and resolution of inflammation. During initiation, macrophages recognize molecules secreted by or expressed on the surface of pathogenic organisms called pathogen-associated molecule patterns (PAMP) using a family of immune receptors called Toll-like receptors. When these receptors are activated macrophages secret inflammatory cytokines to recruit and activate effector cells such as neutrophils and monocytes to eliminate pathogens2. Macrophages also reduce inflammation by phagocytosing apoptotic inflammatory cells34,35 and secreting anti-inflammatory cytokines to inhibit the inflammatory activity of other immune cells’. Because macrophage can be either pro- or anti-inflammatory depending on the type of stimuli it receives, nomenclatures for describing macrophage activation state were developed. Classically, macrophage has been divided into M1 and M2 activation states36, denoting that the macrophage is pro- and anti-inflammatory, respectively. But a nomenclature of macrophage only possessing two polar opposite activation states leads to a problem of over-grouping macrophages with different molecular signatures. As a result of this issue, a new nomenclature was recently proposed where the two broad activation states are replaced with a spectrum of macrophage phenotypes defined by the stimulus they receive37.  Over the years, many macrophage cell lines have been developed by immortalizing macrophages obtained directly from mouse38. One such cell line called J2M, derived by 4  transducing mice bone marrow stem cells with J2 recombinant v-raf/v-myc retrovirus while culturing in macrophage colony stimulating factor (M-CSF), has been used to look at the effect of anti-inflammatory cytokine on macrophage activity. In addition, another J2M cell line was generated from bone marrow stem cells that were extracted from mice lacking expression of SH2-domain containing inositol 5´phosphatase 1 (SHIP1), and this cell line has been used by our laboratory to study the function of SHIP1 in macrophages. 1.3 TLR/LPS signaling pathway Activating macrophages and recruiting them to the site of infection requires pathogen recognition9,10. Two activating signals that macrophages can detect are Pathogen Associated Molecular Patterns (PAMPs) and Danger Associated Molecular Patterns (DAMPs). PAMPs are molecules that are associated with pathogens such as Lipopolysaccharide (LPS) found on cell wall of bacteria and double-stranded RNA found in viruses, whereas DAMPs are debris or substances secreted by damaged cells such as the normally intracellular protein HMGB1. These molecules can bind to a family of receptors expressed on the surface of macrophages called Toll-like receptors (TLR) and trigger an inflammatory response. TLRs are type I transmembrane proteins, possessing Toll/IL1R (TIR) domain in its cytoplasmic side capable of interacting with other TIR domains39. TLRs can form dimer with other TLRs, which allows them to bind to a variety of ligands40. So far, thirteen TLRs have been identified in mouse, and ten of them are also found in human. TLR1, 2, and 6 recognize peptidoglycan, TLR3 recognizes double stranded DNA in viruses, TLR4 recognizes lipopolysaccharide (LPS), TLR5 recognizes flagellin, TLR7 and 8 recognize 5  single stranded DNA in RNA viruses, TLR9 recognizes unmethylated CpG sequence on DNA, and TLR11 recognizes flagellin and profilin9. LPS, the prototypical endotoxin and one of the most well-studied PAMPs, binds to TLR4 expressed on the surface of macrophages41. The interaction of LPS with TLR4 is initiated by LPS-binding protein (LBP)42,43, a serum lipid transferase that transfers the LPS on bacterial cell wall to a macrophage surface protein called CD1444,45. CD14 then facilitates the transfer of LPS to TLR4 with the help of MD246,47, which leads to the dimerzation of TLR4 and activation of TLR4’s downstream signaling cascade40 (Figure 1). The dimerized TIR-TIR interface on TLR4 acts as an adaptor for downstream signaling molecules48 including Myeloid differentiation primary response gene 88 (MyD88)49, MyD88-adaptor like (Mal)50, TIR-domain containing adaptor protein inducing interferon-β (TRIF)51, TRIF-related adaptor molecule (TRAM)52, and Sterile α- and armadillo-motif containing protein  (SARM)53. Mal and TRAM act as adaptors for MyD88 and TRIF, respectively, which allows TLR4 to facilitate a MyD88-dependent and a MyD88-independent (TRIF-dependent) signaling pathway. In the MyD88-dependent pathway, the recruitment of MyD88 to TLR4 receptor leads to further recruitment of downstream signaling molecules through its N-terminal death domain (DD), including Interleukin-1 Receptor Associated Kinase 1 and 4 (IRAK1 and IRAK4)54,55. When MyD88 and IRAK4 interact, IRAK4 becomes activated and phosphorylates IRAK1, which in turn activates Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6)55. TRAF6 is an ubiquitin E3 ligase that catalyzes the addition of polyubiquitin chain on itself and other proteins along with ubiquitin 6  conjugating enzyme Ubc13 and Ubc-like protein Uev1a56. Upon ubiquitination, TRAF6 recruits and activates TAK1-associated binding protein 2 (TAB2), an activator of TGF-β activated kinase 1 (TAK1)57–59. MyD88 also recruits the δ and γ isoforms60 of Phosphatidylinositol-3-kinase (PI3K) to the plasma membrane upon activation by LPS61–63 and positively regulates the production of TNFα.  Activation of TRAF6 leads to the up-regulation of pro-inflammatory cytokines via three transcription factors. One of the transcription factors is interferon regulatory factor 5 (IRF5) which translocates to the nucleus to regulate gene expression after it is activated by MyD88 and TRAF664. The other two transcription factors, Activator Protein 1 (AP1) and Nuclear Factor κ-light-chain-enhancer of Activated B Cells (NFκB), are regulated by TAK1. AP1 is activated by kinases downstream of TAK1 including MAP Kinase Kinase 6 (MAPKK6), c-Jun N-terminal Kinase (JNK) and p38 mitogen-activated protein kinase (p38)58. NFκB translocates to the nucleus to activate transcription after its inhibitor is targeted for proteasomal degradation by the activated TAK158,65. In the MyD88-independent pathway, TRAM recruits TRIF to the plasma membrane52,66,67, leading to the activation of Receptor Interacting Protein 1 (RIP1)68 that targets the inhibitor of NFκB for proteasomal degradation so that NFκB can translocate to the nucleus to activate transcription of pro-inflammatory genes in a similar fashion as MyD88-dependent pathway. TRIF can also regulate transcriptional activity of IRF3, the transcription factor of Interferon-γ (IFNγ), by activating TRAF family member-associated NFκB activator (TANK)-binding kinase 1 (TBK1) that phosphorylates IRF367,69. 7  1.4 Tumor Necrosis Factor-α Tumor necrosis factor α (TNFα) was initially identified as a cytotoxic molecule secreted by immune cells in response to endotoxin stimulation that had tumor-killing capabilities70. It was later found that TNFα is the driver of endotoxin stimulated septic shock71–73 and its presence in the body became a hallmark of inflammation74,75. TNFα sustains inflammation by binding to its cell surface receptor that enhances expression and activates pro-inflammatory signaling molecules such as inducible Nitric Oxide Synthase (iNOS)76,77 and NFκB78. Because it is a hallmark of inflammation, the production of TNFα in macrophages is a  Illustration 1. TLR4 signaling pathway 8  well studied phenomenon, with numerous regulatory events characterized. LPS stimulated TLR4 signaling pathway can activate transcription of TNFα by utilizing transcription factor NFκB79 and AP180. TNFα production can also be regulated post-transcriptionally by proteins that recognize and bind to the AU-rich element (ARE) in the 3ʹUTR of TNFα mRNA. LPS activates the TPL2/Erk signaling pathway that promotes the translocation of TNFα mRNA to the cytoplasm, a process that is enhanced when the ARE is removed81. There are also several stabilizing and destabilizing factors that control the level of TNFα mRNA by binding to its ARE, such as KSRP82, TTP83, and HuR84. TNFα translation is enhanced by LPS-stimulated p38 activation85, but TIA186 and hnRNP A187 are capable of inhibiting TNFα mRNA translation. Lastly, TNFα can be regulated at the level of its secretion by Tumor Necrosis Factor α Converting Enzyme (TACE)88,89. 1.5 Interleukin-10 IL10 was first discovered in 1989 as a factor secreted by Th2 cells to inhibit Th1 cytokine production18. Ever since then, studies have shown that Interleukin-10 (IL10) is the body’s most important anti-inflammatory cytokine that inhibits a broad spectrum of activated macrophage functions19, including production of cytokines such as TNF-α, IL-1β, IL-6, IL-8, G-CSF, GM-CSF90–92, and IL-1293. This powerful attribute of IL10 is further highlighted in studies showing that genetic insufficiencies in IL10 or its receptor leads to inflammation in mouse models94,95 and human diseases96–98. Current research is aiming to harness the anti-inflammatory effect of IL10 as a way of treating diseases of chronic inflammation99–105. However, early clinical trials of IL10 showed mixed results, with some trials showing improvement with IL10 treatment106,107, while some showing IL10 having little to no effect108,109. These inconsistent results were speculated to be caused by 9  the non-anti-inflammatory effects of IL10, the activation of B-cell, CD4+, CD8+, and natural killer cells, and also due to the dose of IL10 administered in the trials being too low to have its intended effect on the target site of treatment. To circumvent these problems, researchers are investigating alternative methods of delivering IL10 to the site of inflammation to enhance the specificity of the therapy and to reduce the dose of IL10 that is necessary to elicit a beneficial anti-inflammatory effect110–113. 1.5.1 Biology of IL10 Cytokines are small proteins between the sizes of 5 – 20 kDa that affect the intracellular signaling cascade of their target cells by binding to their cognate receptors. They have been known to influence a wide range of biological processes114, but they are especially important in regulating inflammation, as mentioned in a previous section. Interleukin is a category of cytokine that was first observed to be expressed by lymphocytes115, and today, it is known that the function of the immune system depends on a large part to the activities of the various different types of Interleukins. IL10 is an Interleukin made up of six helices that are folded in a barrel shape that becomes fully active when it homodimerzes via its C-terminus helix. IL10 is expressed by almost all leukocytes, but most important sources of IL10 in vivo appear to be T-helper cells and macrophages116–118. IL10 targets T-helper cells and macrophages in addition to other immune cell types such as mast cells, B-cells and natural killer cells (Table 1). In macrophages, IL10 inhibits the production of pro-inflammatory cytokines, enhances production of IL-1 receptor antagonist119, downregulate the expression of class II Major Histocompatibility Complex120, promotes the expression of anti-inflammatory 10  genes through its downstream signaling pathway121, and drive differentiation of monocytes into macrophages122. Cell Type  Biological Effect  Macrophage and monocyte  Inhibits secretion of TNF-α, IL-1β, IL-692 Inhibits secretion of TNF-α, IL-1α, IL-1β, IL-6 and GM-CSF induced by LPS and IFN-γ91 Inhibits secretion of TNF-α and hydrogen peroxide90 induced by LPS Enhances IL-1 receptor antagonist and soluble TNF-α receptor119 Downregulates expression of Major Histocompatibility Complex II molecules induced by IFN-γ120 Inhibits synthesis of IL-1293 Drives differentiation of monocyte into macrophage122 T – cells Inhibits synthesis of IL-2 and IFN-γ by Th1 and IL-4/5 by Th2123 Neutrophilic Granulocytes Inhibits production of TNF-α and IL-1β that are induced by LPS and phagocytosis of bacteria124 Inhibits production of prostaglandin E2125 Eosinophilic Granulocytes Inhibits production of TNF-α, GM-CSF, and IL-8126 Mast Cells Inhibits production of TNF-α and GM-CSF127 Inhibits expression of IgE receptor, Syk, Fyn, Akt, and Stat5128 B – cells Promotes survival129 and differentiation into plasma cell130 Natural Killer Cells Enhances cytotoxicity131 IL10 signals through IL10-receptor (IL10R), which is composed of two subunits, IL10R1 and IL10R2132–134 (Figure 2). Upon binding of IL10, the receptor subunits dimerize, leading to the recruitment of tyrosine kinases Jak1 and Tyk2 to the cytoplasmic side of IL10R1 and IL10R2, respectively135. The recruitment and activation of these kinases leads to the phosphorylation of two residues on IL10R1. Signal Transducer and Activator of Transcription 3 (STAT3) contains a Src Homology 2 (SH2) domain that recognizes and binds to the phosphorylated Tyrosine residue on IL10R1136. The recruited STAT3 becomes phosphorylated, which can then dimerize with  STAT1132, STAT3, or STAT5 molecule137. The dimerized STAT3 translocates to the nucleus to activate transcription of genes that promote anti-inflammatory response. Examples include Bcl3121, which inhibits the transcriptional activation function of LPS-induced NFκB by binding to its subunits138,139, and Etv3 and SBNO2140, which also inhibits NFκB transcriptional activity by acting as transcriptional co-repressors141–143. By an unknown mechanism, IL10 also Table 1. Biological effect of IL10 on different immune cells 11  inhibits activation of PI3K/Akt signaling pathway to enhance the activation of IκB Kinase activity144, leading to enhanced stability of NFκB inhibitor IκB. 1.5.2 IL10 inhibition of TNFα production One of IL10’s most well-known functions is the inhibition of TNFα production. At the level of transcription, IL10 inhibits the translocation of NFκB to the nucleus by inhibiting the MyD88-dependent TLR4 signaling pathway, which leads to a concomitant reduction in TNFα production145–147. IL10 also reduces TNFα mRNA stability in a tristetraprolin (TTP)-dependent manner by inhibiting its upstream inhibitor p38 MAPK83. p38 MAPK also promotes translation of TNFα, and thus IL10 inhibition of p38 pathway inhibits TNFα translation85,148. Lastly, IL10 can inhibit secretion of TNFα by enhancing the expression of TIMP388, an inhibitor of TACE. 1.6 PI3K pathway Phosphotinositide-3-kinase (PI3K) is a class of enzymes that catalyze the addition of phosphate to the 3′ hydroxyl group on the inositol ring of phosphatidylinositol that are enriched on the cytoplasmic surface of plasma membrane149. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), the product generated by PI3K, is an important second messenger Illustration 2. IL10 signaling pathway 12  signaling molecule that mediates a variety of cellular functions such as differentiation, growth, proliferation, and survival.  PI3K is divided into classes of IA, IB, II, and III. The class I enzymes, consisting of IA and IB, are heterodimers of a regulatory subunit and a catalytic subunit150. The catalytic subunits consists of p110α, p110β, p110δ, and p110γ isoforms. p110α and p110β are expressed ubiquitously in many tissues and organs, whereas p110δ and p110γ are primarily expressed in leukocytes. The regulatory subunits include p85α, p55α, p50α, p85β, and p55γ, p85α being the most expressed. p85α and p85β possess SH2 domains that allow them to interact with phosphorylated tyrosine (pTyr). Upon external stimulus, receptor tyrosine kinases become phosphorylated in their tyrosine residues, which lead to the recruitment of p85 subunits to the plasma membrane. Because p85 possesses an “inter-SH2” domain that interacts constitutively with the p110 subunit, recruitment of p85 also leads to the recruitment of p110 to the plasma membrane. 1.6.1 Function of PI3K in TLR4 pathway It is well known that PI3K becomes activated from stimulation of TLR4 by LPS. However, the molecular mechanism underlying this activation is not well studied. Several reports suggest that PI3K is recruited to the TLR4 signaling complex by the physical interaction between p85 subunit of PI3K with either MyD8861,62 or B-cell adaptor protein (BCAP)151–153, another TLR4 adaptor protein. Also, whether PI3K activates or inhibits TLR4-mediated LPS activation of macrophages has been controversial, with some labs reporting that PI3K activates this pathway154–166 and other labs reporting that PI3K inhibits this pathway151,152,167–174. However, most of these studies investigated the function of PI3K in this pathway using pan-PI3K inhibitors without taking into account 13  the specific function played by individual PI3K isoforms. Indeed, part of the controversy can be addressed using isoform specific inhibitors of PI3K, and one paper has shown that p110δ and p110γ are the major isoforms involved in upregulating TNFα production in macrophages stimulated with LPS60. 1.7 Inositol phosphatases Dysregulation of PI3K is commonly found in a variety of diseases, such as cancer175, inflammatory disease176, and Alzheimer disease177, therefore regulating the activity of PI3K is crucial for proper vertebrae survival. PI3K activity is antagonized by inositol phosphatases, enzymes that remove phosphatase from PIP molecules. The most well studied inositol phosphatase is Phosphatase and Tensin Homolog (PTEN), which converts Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) into Phosphatidylinositol (4,5)-bisphosphate (PIP2)178, and is an important tumor suppressor found mutated in many cancer types179. The SH2-domain containing inositol 5´phosphatase 1 and 2 (SHIP1 and SHIP2) converts PIP3 into Phosphatidylinositol (3,4)-bisphosphate (PIP2)180,181. PTEN and SHIP2 are expressed in a variety of tissues, whereas SHIP1 is expressed predominantly in hematopoietic cells. Because of SHIP1’s specific expression in this cell type, compounds that alter SHIP1 activity have been shown to treat hematological disorders involving PIP metabolism182,183.  Illustration 3. Domains of SHIP1 and SHIP2 14  Unlike PTEN, the role of SHIP1 and SHIP2 in tumorigenesis has remained controversial. It was originally thought that they are tumor suppressors that inhibit the cell survival and proliferation pathway activated by PI3K/Akt signaling182,184–186 However, growing body of evidence is showing that Phosphatidylinositol (3,4)-bisphosphate correlates with Akt activation187 and a slew of studies have shown that SHIP2 play proto-oncogenic role in tumor initiation and progression188–190. Currently it is thought that a specific amount of PI(3, 4, 5)P3 and PI(3, 4)P2 is necessary to promote malignant state191, and variation in their cellular levels between studies can potentially contribute to the variation of SHIP1 and 2’s observed role in tumorigenesis. 1.7.1 SHIP1 SHIP1 was originally discovered as a 145 kDa protein that interacted with Shc and Grb upon activation of cytokine receptors192. SHIP1 contains six important features: SH2 domain, Pleckstrin Homology-Related (PH-R) domain, phosphatase domain, C2 domain, and proline-rich region (PRR) containing two NPXY motifs. The SH2 domain is involved in the recruitment of SHIP1 to FcγRII during B-cell cell fate determination193,194. PH-R domain is important for the recruitment of SHIP1 to the plasma membrane to mediate phagocytosis195. C2 domain is involved in binding of SHIP1’s product, PIP2, and it is also the allosteric binding site responsible for enhancing SHIP1’s activity182,183. SHIP1 is capable of binding several SH3 domain-containing proteins such as Shc and Grb2 via its PRR196,197. When NPXY motifs are phosphorylated, SHIP1 can bind to proteins containing Phospho-tyrosine binding (PTB) domain, such as Shc, Dok1, and Dok2198. The phosphorylation of SHIP1 is triggered by the activation of various receptors such as B-cell receptor (BCR) and T-cell receptor (TCR)199,200. 15  SHIP1 is a negative regulator of immune activity, parts of it due to SHIP1’s ability to inhibit pathogen-stimulated PI3K signaling pathways. Consistent with this view, many studies have shown that knocking out SHIP1 results in increased phosphorylation of PI3K’s downstream target such as Akt even in unstimulated conditions. Macrophages lacking SHIP1 also produce more pro-inflammatory cytokines than wild-type macrophages and lose endotoxin tolerance201. Furthermore, our laboratory showed that SHIP1 is necessary for IL10 inhibition of p38 MAPK substrate Mnk1 (MAPK-signal integrating kinase)148 and miR-155 maturation202, both of which upregulate production of TNFα.  1.7.2 SHIP2 SHIP2 was discovered after SHIP1, and although they have the same catalytic function, SHIP2 performs slightly different biological functions compared to SHIP1, owing to their difference in expression pattern and structure. SHIP1 and SHIP2 share 38% of the amino acid sequence (refer to Appendix A for sequence alignment of SHIP1 and SHIP2), and unlike SHIP1, SHIP2 lacks the PH-R domain, but it possesses at the C-terminus an ubiquitin interacting motif (UIM) and a sterile α motif (SAM). No notable functions have been attributed to SHIP2’s UIM, but the SAM is known for interacting with SAM in Ephrin Type A Receptor 2 (EphA2)193 and Arf-GAP With Rho-GAP Domain, Ankyrin Repeat and PH Domain 3 (Arap3)203 (refer to Figure 4 for structures of SHIP1 and SHIP2) . SHIP2 inhibits the insulin signaling pathway by competitively inhibiting the association of Shc to Grb2 which prevents the phosphorylation of MAPK p42/44 and Akt204. It is interesting to note that SHIP1 possesses these exact same functions in the context of 16  immune cells198,201,205,206. In vivo studies of SHIP2 knockout mice revealed two potential biological functions of SHIP2 in host metabolism. One study showed that SHIP2 necessary for retaining a certain level of glucose in the bloodstream, and mice lacking SHIP2 dies to hypoglycemia207. In another study, SHIP2 was shown to be necessary for maintaining a lean body in mice when fed with high-fat diet and that lack of SHIP2 do not affect blood glucose level as seen in the first study208. Despite the difference in physiological phenotype observed in the two studies, they both showed that SHIP2 is a negative regulator of insulin signaling. Study of SHIP2 in the context of immune cells is limited to its function in FcγR-mediated signaling. SHIP2 is tyrosine phosphorylated upon FcγRIIa clustering and this leads to inhibition of NFκB activity209. SHIP2 also inhibits FcγR-mediated phagocytosis by disrupting the level of PIP3 and inhibiting recruitment of Akt to the phagocytic cup210.  1.7.3 Non-catalytic and PI3K-independent functions of SHIP1 and SHIP2 Numerous reports have shown that SHIP1 and SHIP2 do not function exclusively by antagonizing PI3K. SHIP1 and SHIP2 both contain SH2 domain, phosphorylatable tyrosines, and proline rich regions that can interact with other proteins. This property of SHIP comes into play when they become tyrosine phosphorylated by receptor tyrosine kinases and interact with Shc204,206,211,212, leading to inhibition of Grb2-mediated Ras/Erk signaling pathway. Erk1 can prevent the translocation of TNFα mRNA to the cytoplasm81 and also inhibit the production of IL-6 and IL-1β205, and thus protein-protein interaction of SHIP proteins may inhibit production of pro-inflammatory cytokines. In support of this, studies that reconstituted cell lines with catalytic or non-catalytic mutants of SHIP proteins showed that the two regions outside of the phosphatase domain are necessary for 17  SHIP’s function in FcγR196, TLR463, IR213, PDGFR213, and HGFR214 signaling in a manner that is independent of their catalytic activity. Furthermore, studies that identified interaction partners of SHIP1 and SHIP2 have shown that they frequently come into contact with proteins associated with cytoskeleton211, which suggests that SHIP proteins do not function exclusively at the region of the cytoplasm rich in PIP. 1.7.4 Overlapping functions of SHIP1 and SHIP2 Despite the difference in their amino acid sequence, SHIP1 and SHIP2 possess similar domain structures, which allow them to play many overlapping functions in the immune system. The SH2 domain in each protein is necessary for its recruitment to FcγRIIB212,215 and they have similar binding affinities for the ITIM motif on FcγRIIB216. SHIP1 and SHIP2’s proline rich regions are capable of binding Grb2 in association with phosphorylated FcγRIIB217 and they are both involved in FcγRIIB-mediated phagocytosis210,218. Because of their similarities, it is possible that SHIP2 may perform a similar function as SHIP1 in IL10 signaling. 1.8 CRISPR/Cas9 genome editing Clustered, regularly interspaced, short palindromic repeat (CRISPR) is a segment of DNA found in prokaryotic genome whose function is to defend against invading virus and plasmid219,220. The sequence contains repeats of a short base sequence, where between each repeat is segment of DNA obtained from previous exposure to foreign genetic material. CRISPR is closely associated with Cas, nuclease that utilizes the sequences within CRISPR for targeted cleavage of previously exposed foreign DNA. 18  CRISPR/Cas9 system has been engineered to perform genome editing. The components in this system are the CRISPR-RNA (crRNA) that contains the complementary sequence of the DNA that is targeted, transactivating CRISPR-RNA (tracrRNA) that helps the maturation of crRNA and basepairs with crRNA, and the Cas9 endonuclease. The crRNA:tracrRNA duplex is incorporated into the nuclease to act as the guide sequence.  CRISPR/Cas9 system of genome editing has been used extensively to study the function of genes in cell lines221 and many model systems, such as zebrafish222, yeast223, and mice224. This is accomplished by generating double stranded break (DSB) in a genomic sequence to induce gene deletion. DSB can be repaired by joining the two broken ends of the DNA by non-homologous end joining (NHEJ) or by copying existing homologous DNA by homology directed repair (HDR). During NHEJ, a segment of the 5ʹ DNA strand at each of the two broken ends of the DNA is excised, leaving a 3ʹ overhang. The overhangs seek for sequence complementarities between each other, and once they form basepairs, and gaps between 3ʹ and 5ʹ ends of each strand of DNA are filled in with DNA polymerase. This will result in insertion or deletion of DNA sequence at the site of DSB. This change can potentially introduce frameshift mutations that completely disrupt the amino acid sequence of the protein encoded by the targeted gene, effectively nullifying its function. 1.9 Hypothesis The overall objective of this study is to study the role of SHIP2 in IL10 mediated anti-inflammation. This study will primarily be done in immortalized mouse bone-marrow derived macrophage cell line J2M. We will knockout the SHIP2 gene in J2M cells derived from wild-type and SHIP1-knockout mice using CRISPR/Cas9 technology. We 19  predict that SHIP2 plays a similar role as SHIP1 in mediating IL10 anti-inflammation by inhibiting the the production of pro-inflammatory cytokines. If SHIP2 promote anti-inflammation by IL10 signaling like SHIP1, then drug developers may consider creating compounds that activate SHIP2 as another alternative for treating inflammatory diseases, since this was already done for SHIP1182,183,225.                20  2 Method 2.1 Cell culture J2M cells and J2M cells knocked out in SHIP1 were kindly provided by Dr. Gerald Krystal (British Columbia Cancer Agency, Vancouver, British Columbia, Canada) and maintained in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, Nepean, ON), supplemented with 10% (v/v) Fetal Calf Serum (FCS, Fisher Scientific, Ottawa, ON), 10 μM β-mercaptoethanol (Sigma Aldrich, Oakville ON), 150 μM monothioglyerol, and 1 mM L-glutamine (BD Scientific, Mississauga, ON). HEK293T cells were maintained in High Glucose Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Nepean, ON) supplemented with 9% (v/v) Fetal Calf Serum. All cells were cultured at 37oC, 5% CO2, 95% humidity. 2.2 Plasmids/primers The pLentiCRISPR vector expressing Cas9 was kindly provided by Dr. Keith Humphries (British Columbia Cancer Agency, Vancouver, British Columbia, Canada), and it was digested with BsmBI and inserted with oligonucleotides (Life Technologies, Pleasanton, California, USA) containing coding sequence for sgRNA targeting SHIP2 exon 4, 9, 14, and 19. sgRNA oligonucleotides were designed using CRISPR Design Tool (Zhang laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA). The following sgRNA primers were used. For sgRNA targeting exon 4, CACCGAGGGTCTGGAAGCGACGCAC and AAACGTGCGTCGCTTCCAGACCC TC. For sgRNA targeting exon 9, CACCGGGCCAAGACCATCCCCGTGC and AAACGCACGGGGATGGTCTTGGCCC. For sgRNA targeting exon 14, CACCG TCACACTGGACGTACTGACG and AAACCGTCAGTACGTCCAGTGTGAC. For 21  sgRNA targeting exon 19, CACCGAAAGATGAAGTCCTTGCGCT and AAACAGC GCAAGGACTTCATCTTTC. 2.3 Generation of cell lines         HEK293T cells were plated per well in a 6-well format, and allowed to adhere to the plate overnight. The following day, cells in each well were transfected with 62 µg of polyethylemine pre-incubated in 320 µL of Opti-MEM® I Reduced Serum Medium (Life Technologies, Pleasanton, California, USA) for 15 minutes, which was further pre-incubated with 0.32 µg of Vesicular Stomatitis Virus Envelope Protein expression vector, 0.96 µg of R8.9 plasmid, and 1.29 µg of pLentiCRISPR vector plasmid for 15 minutes. Cells were transfected for four hours in 37oC, 5% CO2, and 95% humidity, and incubated for 48 hours.         parental J2M cells and       J2M ΔSHIP1 cells were plated per well in a 6-well format prior to transduction. Viral supernatant obtained from the transfected HEK293T cells was supplemented with 8 µg/mL of protamine sulfate (Sigma-Aldrich Canada Co., Oakville, Ontario, Canada) and used to incubate J2M cells for 16 hours. 48 hours later, clones were selected by supplementing culture media with 6 µg/mL puromycin. 2.4 Immunoblotting  Cells lysates were obtained from lysing cells plated in 6-well dish format with plating density of       cells/well that were allowed to grow and adhere to plate overnight. Cells were lysed using 300 µL of 2×Laemmli’s buffer, sonicated at 80% power output for 10 seconds using Microson Ultrasonic Cell Disruptor (Heat Systems Ultrasonics Inc., Farmingdale, New York, USA), and boiled for 5 minutes prior to loading onto 7.5% gel which was ran at constant 100V for 90 minutes. Resolved proteins were transferred onto 22  Immobilon polyvinylidene difluoride membrane (Millipore, Etobicoke ON, Canada) using a wet transfer apparatus, blocked with 3% BSA in Tris buffered saline (TBS) for 45 minutes at room temperature, and probed with primary antibodies overnight at room temperature. Membranes were washed three times with Tris-buffered saline supplemented with 0.1% Tween (TBS-T) for 5 minutes per wash and incubated with Alexa-Fluor 680® secondary antibodies (Life Technology, Burlington, ON) diluted 1:10,000 in TBS-T for 60 minutes. Membranes were then washed three times with TBS-T for 5 minutes per wash and imaged using a Li-Cor Odyssey Infrared Imager (LI-COR biosceince, Lincoln NB, USA). Densitometry analysis was performed using the Image Studio software (LI-COR bioscience, Lincoln NB, USA) by measuring the integrated signal of each protein band and normalized to the integrated signal of an appropriate endogenous control protein band. 2.5 Antibodies Primary antibodies used in the experiments include α-SHIP1 (P1C1) purchased from Santa Cruz (Dallas, Texas), α-SHIP2-2 kindly offered by Dr. Steven Pelech (Kinexus, Vancouver, British Columbia, Canada), and α-Actin purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The secondary antibodies, including Alexa Fluor®680 Goat Anti-Rabbit IgG, and Alexa Fluor®680 Goat Anti-Mouse IgG, were all purchased from Life Technology (Burlington, ON, Canada) unless otherwise stated. 2.6 TNFα ELISA       J2M cells were plated per well and allowed to the plate overnight. Cells were stimulated with 1 ng/mL LPS + IL-10 (ranged from 0 to 20 ng/mL IL-10) in a total volume of 150 μL. 50 μL of supernatant was removed from each well for TNFα 23  concentration analysis. TNFα protein concentration was assayed by enzyme-linked immunosorbant assay (ELISA) using BD OptEIA™ Mouse TNF ELISA Set II kit (BD Scientific, Mississauga, ON). Assay plate (Sigma Aldrich, Oakville ON) was precoated with 50 uL of capture antibody (α-TNFα antibody, diluted 1:250 in 0.05 M carbonate/bicarbonate buffer, pH 9.6) overnight at 4°C. The following day, plate was blocked with assay diluent (10% FCS in PBS) for at least one hour at 23°C. The blocking solution was removed by washing three times using the wash buffer (0.05% Tween in PBS). 50 μL of the supernatant was loaded on 96 well plate, and incubated at 23°C for two hours or overnight. Then the supernatant was removed and the plate was then incubated with the 50 μL of detection antibody (biotinylated α-TNFα antibody, diluted 1:250 in assay diluent) for 1 hour at 23°C. The detection antibody solution was removed by washing three times using the wash buffer. 50 μL of streptavidin-HRP solution (diluted 1:250 in assay diluent) was then added to the plate and was incubated for 30 minutes at 23°C, followed by removal of the solution by washing seven times using the wash buffer. The assay was developed by adding 50 μL of the 3,3’, 5,5’ tetramethyl benzidine solution (TMB, 0.005% TMB, 0.006% H2O2 in 0.01M Acetate Buffer and 0.05% Sodium Nitroferricyanide) and the reaction was stopped by adding 50 μL of 2 N HCl. The plate was then read by the Epoch® Microplate Spectrophotometer at an absorbance of 450 nm.  24  3 Result 3.1 CRISPR/Cas9 constructs design and rationale In order to investigate the function of SHIP2, we generated cell lines lacking SHIP2 using the CRISPR/Cas9 system of gene knockout226. Prior to this project, one study reported the knockout of SHIP2 gene expression by deleting the region of SHIP2 locus containing exon 19 – 29. In order to mimic this SHIP2-knockout effect in the J2M cell                 A B C A: Plasmid map containing the lentiviral expression cassette of CRISPR/Cas9. B: Lentiviral expression cassette for Streptococcus pyogenes Cas9 and sgRNA in the lentiCRISPR v1. Psi packaging signal (psi+), rev response element (RRE), U6 promoter (U6), central polypurine tract (cPPT), FLAG octapeptide tag (FLAG), 2A self-cleaving peptide (P2A), puromycin selection marker (puro), posttranscriptional regulatory element (WPRE), and elongation factor-1α promoter (EFP). C: Diagram of SHIP2 gene, orange boxes represent exons, black line represent introns, and red arrows indicate the exons targeted by the various sgRNA.  sgRNA-mSHIP2-Exon4 sgRNA-mSHIP2-Exon9   sgRNA-mSHIP2-Exon14   sgRNA-mSHIP2-Exon19   psi+ RRE U6 sgRNA EFS cPPT FLAG NLS SpCas9 P2A 5ʹ LTR 3ʹ LTR chRNA NLS WPRE Puro Exon 4 Exon 9 Exon 14 Exon 19 Illustration 4. SHIP2 knockout strategy using CRISPR/Cas9 genome editing 25  line, CRISPR/Cas9 constructs were designed to induce DSB in mouse SHIP2 exon 4, exon 9, exon 14, or exon 19 (Figure 5). Mechanistically, inducing DSB in these exons is more likely to disrupt expression of SHIP2 gene than any other combinations of exons. Targeting exons 19 – 29 for cleavage has the possibility of retaining an intact exon 19. Spreading the targeted exons across exons 1 – 19 rather than making multiple constructs targeting the same exon avoids the scenario of the chosen exon being resistant to nuclease cleavage. After we constructed the plasmid that will be used to generate the lentiviral vectors, the presence of the inserted sgRNA sequence in the plasmids was verified using polymerase chain reaction (PCR). We chose to perform four different reactions, each reaction 100 bp100 bp 200 bp200 bp 300 bp300 bp 400 bp400  500 bp500 bp 600 bp600 bp 800 bp800 bp 1000 bp1000 bp Uninserted Vector Exon 9 Exon 14 No DNA Template  DNA Template Reverse Primer Exon 9 Exon 14 Exon 19 Exon 4 Exon 9 Exon 14 Exon 19 Exon 4 Exon 4 pLentiCRISPR Exon 4 pLentiCRISPR Exon 4 All reactions used the same forward primer that binds to a region of the plasmid 130 bp upstream of the sgRNA insert sequence. Exon 4, 9, 14, and 19 reverse primers have the same sequence as the sgRNA that corresponds to each CRISPR/Cas9 construct. pLentiCRISPR reverse primer binds to a region 100 bp downstream of the sgRNA insert sequence. Exon 4, 9, 14 and 19 DNA templates are the CRISPR/Cas9 plasmids that contain sgRNA sequence complementary to regions within these exons. Uninserted vector is the parental pLentiCRISPR plasmid that was digested with BsmBI and re-ligated without any inserts. Figure 1. Validation of CRISPR/Cas9 plasmid constructs by PCR 26  duplicated. Each pair of duplicate reactions used plasmid DNA that were purified from two different bacterial colonies transformed with the same plasmid. Each plasmid DNA was amplified using a forward primer that bound to a constant region of the plasmid DNA that was 130 bp away from the sgRNA sequence and a reverse primer with the sequence of sgRNA or the sequence of a constant region 100 bp upstream of the sgRNA sequence. As shown in figure 6, all reactions that utilized the correct pairing of DNA template and reverse primer generated bands that migrated slightly higher than 100 bp, which corresponds to the expected size of the product. We believe that the unexpected bands that appeared in lanes 9 and 10 are caused by unintended interaction between the reverse primer and the DNA templates. 3.2 CRISPR/Cas9 reduces SHIP2 level in J2M cells After generation of four CRISPR/Cas9 constructs targeting different exons of SHIP2, we packaged the CRISPR/Cas9 expression cassette into a lentiviral vector and transduced a retrovirally immortalized cell line generated from mouse bone marrow derived macrophage called J2M that either expresses or do not express SHIP1. After selection with puromycin the expression of SHIP2 protein in these cells were determined by analyzing cell lysates by Western Blot (Figure 7). In wild-type J2M cells, the level of SHIP2 band was knocked down when transduced with CRISPR/Cas9 constructs targeting exon 14 of SHIP2. In J2M ΔSHIP1 cells, the level of SHIP2 band was knocked down when transduced with CRISPR/Cas9 constructs targeting exon 19 of SHIP2. For the purpose of convenience, we will refer to parental J2M cells transduced with the pLentiCRISPR virus as J2M WW (no targeting virus), J2M WK1 (exon 4), WK2 (exon 9), WK3 (exon 14), and WK4 (exon 19). Simiarly, ΔSHIP1  J2M cells transduced with the A B 27  pLentiCRISPR virus will be referred as J2M KW (none targeting virus), J2M KK1 (exon 4), KK2 (exon 9), KK3 (exon 14), and KK4 (exon 19) (Refer to Appendix B for summary of nomenclature).  3.3 IL10 sensitivity of J2M cells lacking SHIP2 In response to LPS, macrophage activates an intracellular signaling cascade that leads to the production of pro-inflammatory cytokines such as TNFα and Interleukin-6 (IL6). IL10 is a well studied anti-inflammatory cytokine that is known to inhibit the production of TNFα at many regulatory steps83,85,88,145–148,227. SHIP1 is also important for inhibiting the pro-inflammatory response of macrophages, and it does this partly by inhibiting the production of TNFα60,63,201,205,228,229. Our laboratory has found that in macrophages, IL10 utilizes SHIP1 to inhibit p38148 and miR-155-mediated expression of TNFα202. Furthermore, we have found through knocking out SHIP1 in mouse primary macrophages that IL10/SHIP1 specifically inhibits the burst of TNFα production at one hour after stimulation with LPS (Cheung ST et al, unpublished).  Because evidence in the literature A. J2M cells and J2M cells lacking SHIP1 were transduced with CRISPR/Cas9 vector containing either no sgRNA (NT) or sgRNA complementary to part of SHIP2’s exon 4, 9, 14, and 19. Membranes were probed with α-SHIP1, α-SHIP2-2, and α-Actin antibodies. B. The average SHIP2 band signal from three replicates of the experiment (N = 3). Dunette’s multiple comparison test was used on the sets of Parental J2M cells and ΔSHIP1 J2M cells, with SHIP2 signal generated by J2M WW and KW acting as the controls for each set, respectively. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.   Fo  ΔSHIP1 CRISPR/Cas9 Parental J2M 4 WT CRISPR/Cas9 sgRNA Exon Target 9 14 19 NT Actin SHIP2 Actin SHIP1 sgRNA Exon Target 4 9 14 19 NT A B Figure 2. Western Blot analysis of SHIP2 expression in cells transduced with CRISPR/Cas9 virus 28  suggests that SHIP2 may possess overlapping functions as SHIP1 and our laboratory often observe that removing SHIP1 expression from macrophages does not completely abrogate IL10’s ability to inhibit TNFα production at one hour, we hypothesized that SHIP2 may play a dual role with SHIP1 in inhibiting TNFα production.  In order to test this hypothesis, we tested how well the J2M cells lacking SHIP2 were able to respond to IL10. The production of TNFα in all J2M cells expressing SHIP1 was inhibited by IL10 by 50% or more (Figure 8A, B and C). The cells transduced with the CRISPR/Cas9 virus produced more TNFα compared to the parental J2M cells, and this difference was not due to enhanced basal activation as all of the cells assayed produced less than 100 pg/mL of TNFα if not stimulated. The maximum inhibition of TNFα Figure 3. IL10 sensitivity of ΔSHIP2 and ΔSHIP1/ΔSHIP2 J2M cells TNFα production assay was performed on each cell line at least three times. J2M WT transduced with control CRISPR/Cas9 virus (J2M WW), J2M WT transduced with CRISPR/Cas9 virus expressing SHIP2 exon 14 sgRNA and knocked out in SHIP2 (J2M WK3), J2M ΔSHIP1 transduced with control CRISPR/Cas9 virus (J2M KW), and J2M ΔSHIP1 transduced with CRISPR/Cas9 virus expressing SHIP2 exon 19 sgRNA and knocked out in SHIP2 (J2M KK4). Each graph is the representative IL10 inhibition profiles for its corresponding cell line. Cells were plated at a density of 30000/well in 96-well format and incubated in 37oC at 5% CO2 for 20 hours. Cells were stimulated with media containing LPS and purified mouse IL10. LPS concentration was 10 ng/mL. TNFα level was analyzed using TNFα ELISA kit after 1 hour of stimulation. Cell line genotype, average IL10 IC50 and N indicated on each graph. A B C D E F IC50 = 5.5 ± 6.6 ng/ml N = 4 IC50 = 2.4 ± 1.2 ng/ml N = 6 IC50 = 4.1 ± 3.7 ng/ml N = 6 IC50 = 1.6 ± 1.8 ng/ml N = 3 IC50 = 2.4 ± 2.9 ng/ml N = 4 IC50 = 0.9 ng/ml N = 3 ΔSHIP2 ΔSHIP1 ΔSHIP1 ΔSHIP1 ΔSHIP2  29  production by IL10 in the parental J2M ΔSHIP1 was about 50%, but it was less than 30% in cells transduced with the CRISPR/Cas9 virus that lack SHIP1 (Figure 8D, E and F).  In order to determine whether J2M WW and J2M WK3 had differing sensitivity to IL10, we calculated the average IC50 of IL10 inhibition on these cell lines using GraphPad Prism 6 (refer to Appendix C for complete summary of IC50 values obtained from all repetitions of this experiment). The average IL10 IC50 of these cell lines ranged from 1.5 – 3 ng/ml and their standard deviations were widely dispersed. Therefore, the experimental technique and the variability of the cell line prevent us from distinguishing IL10 sensitivity between cells that express and lack SHIP2.            30  4 Discussion  IL10 is known for inhibiting the production of TNFα when macrophages are stimulated by Pathogen-associated molecular patterns such as LPS. Several groups have previously shown that SHIP1 is important for downregulating macrophage inflammatory response, and we were the first group to show that SHIP1 plays a significant role in the anti-inflammatory effect of IL10148,202. SHIP2 is the only known homologue of SHIP1, and in the immune system they are both recruited to FcγRIIB to inhibit B-cell proliferation212,215–217 and phagocytosis210,218. Because of their similarities, we hypothesized that SHIP1 and SHIP2 have overlapping functions in IL10 signaling such that they compensate for each other. In order to test this hypothesis, we generated CRISPR/Cas9-mediated SHIP2 knockout macrophage cell lines in a wild-type and SHIP1 knockout background. By Western Blot, we verified that CRISPR/Cas9 construct that targeted exon 14 of SHIP2 was able to knockout its expression level in wild-type cells. In cells lacking SHIP1, CRISPR/Cas9 construct that targeted exon 19 was able to knockout SHIP2 expression.  Because it is well known that IL10 can inhibit the production of TNFα at many regulatory steps83,85,88,145–148,227, a simple method for assaying IL10 responsiveness is to determine the IL10-inhibition sensitivity of TNFα production. To test whether SHIP2 plays a role in IL10 signaling, we stimulated cells lacking SHIP2 with LPS to induce the production of TNFα and simultaneously treated the cells with varying concentrations of IL10. We have observed the parental J2M cells that were transduced with CRISPR/Cas9 virus produced more TNFα than the parental J2M cells. We also observed the parental 31  ΔSHIP1 cells (J2M KW) that were transduced with CRISPR/Cas9 virus had weaker IL10 inhibition compared to the untransduced ΔSHIP1 cells (J2M ΔSHIP1).  This observation is likely due to two significant problems with the usage of CRISPR/Cas9 gene editing in macrophage cell lines. One is that macrophage expresses TLR3 that recognizes double-stranded RNA (dsRNA)230. Upon recognition of dsRNA, TLR3 activates adaptor protein TRIF which in turn activates TRAF667. As mentioned in section 1.3, TRAF6 regulates the activity of several signaling proteins involved in the production of TNFα which are also utilized by the LPS/TLR4 signaling cascade58,65. The CRISPR/Cas9 expression cassette was packaged into a lentiviral vector in the form of dsRNA, so the vector may have triggered the signaling events downstream of TLR3 and altered the macrophage physiology. The second issue with CRISPR/Cas9 gene editing is Cas9 nuclease’s off-target effect. After the cells were transduced with CRISPR/Cas9 virus, they were enriched by negative selection, a process that took approximately thirty days. By the end of the enrichment, Cas9 nuclease have likely caused significant amount of DNA damage to the genome, leading to the activation of DNA repair machinery that may have affected cellular physiology. However, this long-term mutagenesis may be avoided using more modern CRISPR/Cas9 products that significantly reduce the amount of time the Cas9 protein is active, such as transfecting cells with Cas9 proteins that could be degraded overtime rather than a CRISPR/Cas9 expression cassette that constantly expresses Cas9231. We observed in one experiment that IL10 IC50 of J2M WW (wild-type) was half of that of J2M WK3 (ΔSHIP2), which suggested that wild-type J2M cells are slightly more sensitive to IL10 than compared to cells lacking SHIP2. We were unable to replicate this 32  finding, and in fact we were unable to determine whether SHIP2 plays a role in macrophage anti-inflammatory response due to the large variation in the IL10 IC50 between experiments. However, our laboratory was able to consistently observe that cells lacking SHIP1 had an IL10 IC50 four-fold higher than that of wild-type cells (unpublished data), therefore further work must be done to determine the origin of this variation.  Although in a static cell culture setting the difference in IL10 response between wild-type and ΔSHIP1 J2M cells may not be completely obvious, in an experimental system that more mimics the physiological condition of macrophages, the difference may be much clearer. Our laboratory have previously performed experiments using primary macrophage cells obtained directly from mouse and stimulated cells with LPS and IL10 in a continuous flow cell system, where the cells are placed in an environment analogous to that of the bloodstream by allowing the cell culture media to flow past the cell rather than keeping it in a static environment (unpublished data). In that experiment, the production of TNFα peaked at about 75 minutes after the first exposure of the cells to the stimulation media containing LPS. This peak is inhibited by approximately 90% if the stimulation media also contained IL10 in addition to LPS. The primary macrophage cells that lack SHIP1 responded to LPS in the same fashion as the wild-type cells, but IL10 no longer inhibited the production of TNFα at the 75-minute peak. Although this study was unable to detect a difference between wild-type and ΔSHIP2 J2M cells, future efforts should aim to perform the same experiments done in this study but with finer control of cell behavior and experimental technique. If knocking out SHIP2 does cause a shift in macrophage’s IL10 IC50, there is the possibility that primary macrophages that lack 33  SHIP2 will completely lose their sensitivity to IL10 when they are subjected to the flow cell environment. As of writing of this thesis, the complete mechanism of how SHIP1 is involved in IL10 signaling has remained elusive and no groups have published data suggesting a relationship between IL10 and SHIP2 signaling. One potential route of investigation that can shed light on whether SHIP2 is involved in macrophage inflammatory response is by determining the signaling proteins that are regulated by SHIP1 and SHIP2. There are multiple signaling targets downstream of TLR4 that become phosphorylated after induction by LPS, including p38, IκBα, Akt, and Erk1/2, and our laboratory have previously shown that at least the p38 branch of signaling is regulated by SHIP1148. To investigate the possibility of SHIP2 involvement in TLR4 signaling, we would stimulate wild-type, ΔSHIP1, and ΔSHIP2 J2M cells with LPS and test the effect of IL10 on the phosphorylation of TLR4 signaling proteins. In wild-type cells, we expect IL10 to inhibit the phoshorylation of all signaling proteins mentioned above, and IL10 will not be able to inhibit the phosphorylation of p38 in cells lacking SHIP1. The protein whose IL10 inhibition is abolished in the cells lacking SHIP2 is likely regulated by SHIP2.      34  5 Conclusion  SHIP2 is primarily known to be involved in insulin signaling, phagocytosis, and negative signaling under FcγRIIB. 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Biotechnol. 208, 44–53 (2015).             49  Appendices Appendix A - Amino acid sequence alignment of mouse SHIP1 and SHIP2 protein   SHIP1      -------------MPAMVPGWNHGNITRSKAEELLSRAGKDGSFLVRASESIPRAYALCV SHIP2      MASVCGTPSPGGALGSPAPAWYHRDLSRAAAEELLARAGRDGSFLVRDSESVAGAFALCV                         : : .*.* * :::*: *****:***:******* ***:  *:****  SHIP1      LFRNCVYTYRILPNEDDKFTVQASEGVPMRFFTKLDQLIDFYKKENMGLVTHLQYPVPLE SHIP2      LYQKHVHTYRILPDGEDFLAVQTSQGVPVRRFQTLGELIGLYAQPNQGLVCALLLPVEGE            *::: *:******: :* ::**:*:***:* * .* :** :* : * ***  *  **  *  SHIP1      EEDAIDEAEEDTESVMSPPELPPRNIP----MSAGPSEAKDLPLATENPRAPEVTRLSLS SHIP2      REPDPPDDRDASDVEDEKPPLPPRSGSTSISAPVGPSSPLPTPE---TPTTPA-------            .*    : .: ::   . * ****.        .***.    *    .* :*          SHIP1      ETLFQRLQSMDTSGLPEEHLKAIQDYLSTQLLLDSDFLKTGSSNLPHLKKLMSLLCKELH SHIP2      -----------AESTPNGLSTVSHEYLKGSYGLDLEAVRGGASNLPHLTRTLVTSCRRLH                       :.. *:   .. ::**. .  ** : :: *:******.: :   *:.**  SHIP1      GEVIRTLPSLESLQRLFDQQLSPGLRPRPQ-----VPGEASPITMVAKLSQLTSLLSSIE SHIP2      SEVDKVLSGLEILSKVFDQQSSPMVTRLLQQQSLPQTGEQELESLVLKLSVLKDFLSGIQ            .** :.* .** *.::**** ** :    *       ** .  ::* *** *..:**.*:  SHIP1      DKVKSLLHEGSEST---------NRRSLIPPVTFEVKS-------ESLGIPQKMHLKVDV SHIP2      KKALKALQDMSSTAPPAPLQPSIRKAKTIPVQAFEVKLDVTLGDLTKIGKSQKFTLSVDV            .*. . *:: *.::         .: . **  :****         .:*  **: *.***  SHIP1      ESGKLIVKKSKDGSED--KFYSHKKILQLIKSQKFLNKLVILVETEKEKILRKEYVFADS SHIP2      EGGRLVLLRRQRDSQEDWTTFTHDRIRQLIKSQRVQNKLGVVFEKEKDRTQRKDFIFVSA            *.*:*:: : :  *::  . ::*.:* ******:. *** ::.*.**::  **:::*..:  SHIP1      KKREGFCQLLQQMKNKHSEQPEPDMITIFIGTWNMGNAPPPKKITSWFLSKGQGKTRDDS SHIP2      RKREAFCQLLQLMKNRHSKQDEPDMISVFIGTWNMGSVPPPKNVTSWFTSKGLGKALDEV            :***.****** ***:**:* *****::********..****::**** *** **: *:   SHIP1      ADYIPHDIYVIGTQEDPLGEKEWLELLRHSLQEVTSMTFKTVAIHTLWNIRIVVLAKPEH SHIP2      TVTIPHDIYVFGTQENSVGDREWLDLLRGGLKELTDLDYRPIAMQSLWNIKVAVLVKPEH            :  *******:****: :*::***:*** .*:*:*.: :: :*:::****::.**.****  SHIP1      ENRISHICTDNVKTGIANTLGNKGAVGVSFMFNGTSLGFVNSHLTSGSEKKLRRNQNYMN SHIP2      ENRISHVSTSSVKTGIANTLGNKGAVGVSFMFNGTSFGFVNCHLTSGNEKTTRRNQNYLD            ******:.*..*************************:****.*****.**. ******::  SHIP1      ILRFLALGDKKLSPFNITHRFTHLFWLGDLNYRVELPTWEAEAIIQKIKQQQYSDLLAHD SHIP2      ILRLLSLGDRQLSAFDISLRFTHLFWFGDLNYRLDMDI---QEILNYISRREFEPLLRVD            ***:*:***::** *:*: *******:******:::     : *:: *.::::. **  *  SHIP1      QLLLERKDQKVFLHFEEEEITFAPTYRFERLTRDKYAYTKQKATGMKYNLPSWCDRVLWK SHIP2      QLNLEREKHKVFLRFSEEEISFPPTYRYERGSRDTYAWHKQKPTGVRTNVPSWCDRILWK            ** ***:.:****:*.****:* ****:** :**.**: *** **:: *:******:***  SHIP1      SYPLVHVVCQSYGSTSDIMTSDHSPVFATFEAGVTSQFVSKNGPGTVDSQGQIEFLACYA SHIP2      SYPETHIICNSYGCTDDIVTSDHSPVFGTFEVGVTSQFISKKGLSKTSDQAYIEFESIEA            *** .*::*:***.*.**:********.***.******:**:* .....*. *** :  *  SHIP1      TLKTKSQTKFYLEFHSSCLESFVKSQEGENEEGSEGEL--VVRFGETLPKLKPIISDPEY SHIP2      IVKTASRTKFFIEFYSTCLEEYKKSFENDAQSSDNINFLKVQWSSRQLPTLKPILADIEY             :** *:***::**:*:***.: ** * : :...: ::  *   .. **.****::* **  SHIP1      LLDQHILISIKSSDSDESYGEGCIALRLETTEAQHPIYTPLTHHGEMTGHFRGEIKLQTS SHIP2      LQDQHLLLTVKSMDGYESYGECVVALKSMIGSTAQQFLTFLSHRGEETGNIRGSMKVRVP            * ***:*:::** *. *****  :**:    .: : : * *:*:** **.:**.:*::.   SHIP1      QG--KMREKLYDFVKTERDESSGMKCLKNLTSHD---PMRQWEPSGRVPACGVSSLNEMI SHIP2      TERLGTRERLYEWISIDKDDTGAKSKVPSVSRGSQEHRSGSRKPASTETSCPLSKLFEEP                  **:**:::. ::*::.. . : .::  .      . :*:.   :* :*.* *    50  SHIP1      NPNYIGMGPFGQPLHGKSTLSPDQQLTAWSYDQLPKDSSLGPGRGEGPPTPP----SQPP SHIP2      --------------------------------------EKPPPTG-RPPAPPRAVPREEP                                                  .  *  *  **:**     : *  SHIP1      LSPKKFSSSTANRGPCPRVQEARPGDLGKVEALLQEDLLLTKPEMFENPLYGSVSSFPKL SHIP2      LNPRLKSEGTSEQEG-----VAA----------------PPPKNSFNNPAYYVLEGVPHQ            *.*:  *..*:::        *                     : *:** *  :...*:   SHIP1      VPRK-----------------------------------E------QESPK---MLRKEP SHIP2      LLPLEPPSLARAPLPPATKNKVAITVPAPQLGRHRTPRVGEGSSSDEDSGGTLPPPDFPP            :                                             ::*          *  SHIP1      PPCPDPGISSPSIVLPKAQEV-ES-VKGTSKQAPVPVLGPTPRIRSFT-CSSSAEGRMTS SHIP2      PPLPDSAIFLPPNLDPLSMPVVRGRSGGEARGPPPPKAHPRPPLPPGTSPASTFLGEVAS            ** ** .*  *  : * :  * ..   * ::  * *   * * :   *  :*:  *.::*  SHIP1      GDKSQGKPK-----ASASSQAPVPVKRPVKPSRSEMSQQTTPIPAPRPPLPVKSPAVL-- SHIP2      GDDRSCSVLQMAKTLSEVDYAPGPGRSALLPNPL-------ELQPPRGPSDYGRPLSFPP            **. . .        *  . ** * :  : *.          :  ** *     *  :    SHIP1      -QLQHSKGRDYRDNTELPHHG------------------KHRQEEGLLGRTAMQ------ SHIP2      PRIRESIQEDLAEEAPCPQGGRASGLGEAGMGAWLRAIGLERYEEGLVHNGWDDLEFLSD             :::.*  .*  :::  *: *                   .* ****: .   :        SHIP1      ---------------------------- SHIP2      ITEEDLEEAGVQDPAHKRLLLDTLQLSK                51   Appendix B - Nomenclature of the cell lines used in this study  Cell Line Full Name SHIP2 Targeting Site Genotype Cell Line Abbreviation   SHIP1 locus SHIP2 locus  J2M WT N/A WT WT J2M WT J2M WT CRISRP/Cas9 - control None WT WT J2M WW J2M CRISRP/Cas9 - exon 4 Exon 4 WT targeted J2M WK1 J2M CRISRP/Cas9 - exon 9 Exon 9 WT targeted J2M WK2 J2M CRISRP/Cas9 -exon 14 Exon 14 WT targeted J2M WK3 J2M CRISRP/Cas9 - exon 19 Exon 19 WT targeted J2M WK4 J2M KO N/A KO WT J2M KO J2M KO CRISRP/Cas9 - control None KO WT J2M KW J2M KO CRISRP/Cas9 - exon 4 Exon 4 KO targeted J2M KK1 J2M KO CRISRP/Cas9 - exon 9 Exon 9 KO targeted J2M KK2 J2M KO CRISRP/Cas9 - exon 14 Exon 14 KO targeted J2M KK3 J2M KO CRISRP/Cas9 - exon 19 Exon 19 KO targeted J2M KK4                 52  Appendix C - All IL10 IC50 values obtained for all cell lines Note that the graphical data of figure 8 was obtained from FW124.                  J2M KW Exp. ID IC50 (ng/ml) R2 95% C.I. FW115 6.5 0.55 (Very Wide) FW119 2.3 0.86 1 – 5.3 FW122 0.1 0.32 (Very Wide) FW124 0.5 0.71 0.3 – 0.9 Average IC50 = 2.4 ± 2.9 ng/ml, N = 4  J2M WK3 Exp. ID IC50 (ng/ml) R2 95% C.I. FW115 6.7 0.9 3.7 – 12 FW118 3.1 0.89 2.5 – 3.9 FW119 2.3 0.86 1 – 5.4 FW122 10.2 0.68 4.4 – 23.8 FW123 1.3 0.87 0.8 – 2.1 FW124 0.8 0.79 0.3 – 2.6 Average IC50 = 4.1 ± 3.7 ng/ml, N = 6  J2M KK4 Exp. ID IC50 (ng/ml) R2 95% C.I. FW119 No Inhibition N/A N/A FW122 No Inhibition N/A N/A FW124 0.9 0.70 0.5 – 1.7 Average IC50 = 0.9 ng/ml, N = 3  J2M ΔSHIP1 Exp. ID IC50 (ng/ml) R2 95% C.I. FW115 3.7 0.9 2.3 – 5.9 FW119 0.1 0.56 (Very Wide) FW124 0.9 0.69 0.3 – 2.8 Average IC50 = 1.6 ± 1.8 ng/ml, N = 3  J2M WW Exp. ID IC50 (ng/ml) R2 95% C.I. FW115 3.6 0.95 2.6 – 5.1 FW117 4.4 0.93 Very Wide FW118 1.2 0.93 0.7 – 2.1 FW119 4 0.85 2.5 – 6.4 FW120 1.5 0.94 0.9 – 2.5 FW122 2.2 0.85 0.9 – 5.7 FW123 1.8 0.95 1.2 – 2.6 FW124 1.4 0.9 0.9 – 2.4 Average IC50 = 2.5 ± 1.2 ng/ml, N = 8  J2M WT Exp. ID IC50 (ng/ml) R2 95% C.I. FW115 15 0.78 1.3 – 179 FW117 1.2 0.59 Very Wide FW119 1.6 0.69 0.6 – 3.9 FW120 4.1 0.47 Very Wide FW123 1.5 0.79 0.2 – 14.2 FW124 2.1 0.89 1.3 – 3.4 Average IC50 = 4.9  ± 5.8 ng/ml, N = 6  

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