Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

SHIP deficiency is associated with the Crohn’s disease susceptibility variant in ATG16L1 and leads to… Ngoh, Eyler Ndumeya 2015

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

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
24-ubc_2016_february_ngoh_eyler.pdf [ 3.81MB ]
Metadata
JSON: 24-1.0216551.json
JSON-LD: 24-1.0216551-ld.json
RDF/XML (Pretty): 24-1.0216551-rdf.xml
RDF/JSON: 24-1.0216551-rdf.json
Turtle: 24-1.0216551-turtle.txt
N-Triples: 24-1.0216551-rdf-ntriples.txt
Original Record: 24-1.0216551-source.json
Full Text
24-1.0216551-fulltext.txt
Citation
24-1.0216551.ris

Full Text

SHIP deficiency is associated with the Crohn’s disease susceptibility variant in ATG16L1 and leads to increased interleukin-1beta transcription and intestinal autoinflammation  by  Eyler Ndumeya Ngoh    M.Sc. Linkoping University, 2010 B.Sc. (Honours), University of Buea, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   November 2015  © Eyler Ndumeya Ngoh, 2015 ii  Abstract Crohn`s disease (CD) is a polygenic immune-mediated disease of the gastrointestinal tract characterized by chronic inflammation. The SH2-domain-containing inositol polyphosphate 5΄phosphatase (SHIP) is a hematopoietic-specific negative regulator of inflammatory cytokine production and plays an important role in regulating immune homeostasis. Using the SHIP deficient mouse model of intestinal inflammation, we found that IL-1β is increased in SHIP-/- mouse macrophages due to increased class I PI3K p110α activity. Macrophage depletion or treatment with an IL-1 receptor antagonist reduced development of intestinal inflammation in SHIP-/- mice.  To interrogate if SHIP is dysregulated in people with ileal CD, we demonstrate that subjects with ileal CD have reduced SHIP mRNA expression and enzymatic activity at sites of inflammation and in PBMCs, compared to control subjects. A single nucleotide polymorphism (SNP) in the gene encoding ATG16L1 (T300A) causes an autophagy defect and is associated with increased IL-1β production and susceptibility to CD. In all tissues from our patient cohort and in PBMCs from a second healthy control cohort, subjects, who were homozygous for the CD-associated ATG16L1 T300A encoding gene variant, had reduced SHIP mRNA expression and enzymatic activity, which correlated with increased IL-1β production. In addition, starvation-induced autophagy increased SHIP protein levels, which were reduced in the presence of the ATG16L1 CD-associated risk allele.  Examining the effects of additional autophagy and CD-related gene variants, which may affect SHIP mRNA expression and enzymatic activity, on IL-1β production in PBMCs from a cohort of healthy control subjects, we found that the NOD2 rs2066844 and the XBP-1 iii  rs35873774 gene variants were associated with increased IL-1β production in response to specific PAMPs. Collectively, these data identify SHIP up-regulation as a novel mechanism by which autophagy regulates IL-1β production and intestinal autoinflammation. Our findings also identify a subgroup of CD patients that could be amenable to treatment with therapy that targets IL-1β.    iv  Preface Animal studies were reviewed and approved by the University of British Columbia according to guidelines provided by the Canadian Council on Animal Care, protocol numbers A09-0027 and A09-0032. Collection of human samples used in the studies was approved by the University of British Columbia Research Ethics Boards, protocol numbers H09-01826, H04-0534, and H14-00622. Chapter 1. Figure 1.1 was modified and reproduced with permission of nature publishing Group: Xavier RJ & Podolsky DK. Unraveling the pathogenesis of inflammatory bowel disease. Nature 2007: 448(7152). Figure 1.2 was reproduced with permission of ELSEVIER LTD: Zaki MH, Lamkanfi M & Kanneganti TD. The NLRP3 inflammasome: contributions to intestinal homeostasis. Trends in Immunology 2011; 32(4). Figure 1.6 was reproduced with permission of Nature Publishing Group: Murthy A et al. A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3. Nature 2014; 506(7489). Chapter 2. A version of this material has been published as Eyler N. Ngoh, Shelley B. Weisser, Lisa K. Kozicky, Roger Jen, Hayley K. Brugger, Dominika Nackiewicz, Nico van Rooijen, Kevan Jacobson, Jan A. Ehses, Stuart E. Turvey, and Laura M. Sly. Activity of SHIP, which prevents expression of IL-1β is reduced in patients with Crohn`s disease in the journal, Gastroenterology. Eyler N. Ngoh conducted all the experimental work, and data analysis described herein, with the exception of the following contributions from additional authors: Shelley B. Weisser and Lisa K. Kozicky acted as scorers for quantification of crypt-villus length, immune cell infiltration and macrophage number. Roger Jen helped with YVAD-FLICA and F4/80 staining. Hayley K. Brugger helped with isolation of peripheral blood mononuclear cells from peripheral blood, and quantitative image analysis of immune v  cell infiltration for the human samples. Dr. Laura M. Sly measured SHIP activity in the human samples.  Chapter 3. A version of this material has been published as Eyler N. Ngoh, Hayley K. Brugger, Mahdis Monajemi, Susan C. Menzies, Aaron F. Hirschfeld, Kate L. Del Bel, Kevan Jacobson, Pascal M. Lavoie, Stuart E. Turvey, and Laura M. Sly. The Crohn’s diseases-associated polymorphism in ATG16L1 (rs2241880) reduces SHIP gene expression and activity in human subjects in Genes & Immunity, 2015. Eyler N. Ngoh conducted all the experimental work, and data analysis described herein, with the exception of the following contributions from additional authors: Hayley K. Brugger helped with isolation of peripheral blood mononuclear cells from peripheral blood, and measurement of SHIP mRNA expression in some of the samples. Mahdis Monajemi measured ATG16L1 mRNA in Figure 3.4A. Susan C. Menzies helped with isolation of peripheral blood mononuclear cells from peripheral blood for some of the experiments. Aaron F. Hirschfeld and Kate L. Del Bel genotyped the ATG16L1 single nucleotide polymorphism. Dr. Laura M. Sly measured SHIP activity and performed the Western blots seen in Figures 3.5A and B.  Chapter 4. Contains preliminary data for a future publication. Eyler N. Ngoh conducted all the experimental work and data analysis described herein, with the exception of the following contributions from additional authors: Aaron F. Hirschfeld and Kate L. Del Bel helped with genotyping for the single nucleotide polymorphisms examined.      vi  Table of Contents  Abstract ................................................................................................................................ ii Preface ................................................................................................................................. iv Table of Contents ................................................................................................................ vi List of Tables ....................................................................................................................... xi List of Figures .................................................................................................................... xii List of Abbreviations ......................................................................................................... xv Acknowledgements ......................................................................................................... xxiii Dedication ......................................................................................................................... xxv Chapter 1: Introduction ...................................................................................................... 1 1.1 Inflammatory bowel disease .................................................................................. 1 1.2 Clinical presentation and diagnosis ....................................................................... 3 1.3 Etiology and pathogenesis ..................................................................................... 4 1.3.1 The role of genetics in Crohn’s disease ............................................................. 5 1.3.2 Environmental factors in Crohn’s disease ......................................................... 7 1.3.3 The microbiome in Crohn’s disease .................................................................. 9 1.3.4 The epithelial barrier in Crohn’s disease ........................................................... 9 1.3.5 The immune response in Crohn’s disease ....................................................... 11 1.3.5.1 Innate immune response .......................................................................... 12 1.3.5.2 Adaptive immune response...................................................................... 13 1.3.6 Therapeutic options ......................................................................................... 17 1.4 The Interleukin 1 family of cytokines ................................................................. 18 vii  1.4.1 Interleukin 1β (IL-1β) ...................................................................................... 21 1.4.2 Inflammasome activation and IL-1β production ............................................. 22 1.4.3 IL-1β-mediated diseases (auto-inflammatory diseases) .................................. 26 1.4.3.1 Cryopyrin-associated periodic syndrome (CAPS)................................... 26 1.4.3.2 TNF-receptor-associated periodic syndrome (TRAPS)........................... 27 1.4.3.3 Familial Mediterranean Fever (FMF) ...................................................... 27 1.4.3.4 Adult onset Still’s disease (AOSD) ......................................................... 28 1.4.3.5 Other autoinflammatory diseases ............................................................ 28 1.4.4 IL-1β in intestinal inflammation ...................................................................... 29 1.5 Autophagy............................................................................................................ 30 1.5.1 Description and overview ................................................................................ 30 1.5.2 Autophagy related 16-like 1 (ATG16L1) ........................................................ 32 1.5.3 Autophagy protein 5 (ATG5) .......................................................................... 34 1.5.4 Immunity-related GTPase family M (IRGM).................................................. 34 1.5.5 Nucleotide-binding oligomerization domain containing 2 (NOD2) ................ 35 1.5.6 Endoplasmic reticulum stress and autophagy .................................................. 36 1.5.6.1 X-box binding protein 1 (XBP1) ............................................................. 36 1.5.6.2 Orosomucoid 1-like 3 (ORMDL3) .......................................................... 37 1.6 Src homology 2 domain-containing inositol polyphosphate 5´-phosphatase ...... 37 1.6.1 Description and function.................................................................................. 37 1.6.2 SHIP enzymatic activity .................................................................................. 38 1.6.3 The SHIP deficient mouse ............................................................................... 40 1.6.4 The SHIP-/- mouse model of Crohn’s disease-like intestinal inflammation ... 41 viii  1.6.5 The role of SHIP in inflammation ................................................................... 42 1.7 Thesis hypothesis and objectives ......................................................................... 43 1.7.1 Summary of rationale ...................................................................................... 43 1.7.2 Hypothesis and objectives ............................................................................... 44 1.7.3 Significance ..................................................................................................... 45 Chapter 2: SHIP deficiency leads to increased IL-1β transcription in macrophages and intestinal autoinflammation in mice ................................................................................ 46 2.1 Introduction and rationale .................................................................................... 46 2.2 Materials and methods ......................................................................................... 47 2.3 Results ................................................................................................................. 53 2.3.1 Ileal macrophages from SHIP-/- mice produce high levels of IL-1 and   IL-18…. ....................................................................................................................... 53 2.3.2 In vivo differentiated SHIP-/- macrophages produce high levels of IL-1 and  IL-18…. ....................................................................................................................... 56 2.3.3 Class I PI3Kp110drives transcription of IL1B ............................................. 61 2.3.4 Macrophage depletion reduces intestinal inflammation in SHIP-/- mice  ....... 64 2.3.5 Anakinra treatment reduces intestinal inflammation in SHIP-/- mice............. 67 2.3.6 SHIP activity is lower in subjects with Crohn’s disease. ................................ 69 2.4 Discussion ............................................................................................................ 72 Chapter 3: The Crohn`s disease-associated polymorphism in ATG16L1 reduces SHIP gene expression and activity in human subjects ............................................................. 77 3.1 Introduction and rationale .................................................................................... 77 3.2 Materials and methods ......................................................................................... 80 ix  3.3 Results ................................................................................................................. 86 3.3.1 SHIP mRNA expression is reduced in subjects with ileal Crohn’s disease .... 86 3.3.2 Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with low SHIP mRNA expression and activity ........................................................... 88 3.3.3 SHIP mRNA expression and activity are lower in PBMCs from healthy control subjects, who are homozygous for the ATG16L1 T300A-encoding gene variant ...... 90 3.3.4 SHIP and ATG16L1 are not transcriptionally co-regulated ............................ 91 3.3.5 Starvation-induced autophagy up-regulates SHIP protein, which is dependent on ATG16L1 or autophagy, and the risk allele genotype ................................................. 94 3.3.6 Homozygosity for the Crohn’s disease-associated ATG16L1 gene variant is associated with high IL-1β production and IL1B transcription ................................... 97 3.3.7 Reduced SHIP mRNA expression is associated with high IL-1β production and IL1B transcription ...................................................................................................... 100 3.4 Discussion .......................................................................................................... 102 Chapter 4: Autophagy-related CD genetic variants modulate IL-1β production upon selective action of PRRs................................................................................................... 106 4.1 Introduction and rationale .................................................................................. 106 4.2 Materials and methods ....................................................................................... 108 4.3 Results ............................................................................................................... 111 4.3.1 Determination of PRR ligand concentrations to be used for stimulations..... 111 4.3.2 Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased IL-1β production in response to TLR4 and TLR5 ligands ................ 112 x  4.3.3 ATG5 rs12201458 gene variant is associated with an anti-inflammatory response to TLR4 ligand ........................................................................................................... 115 4.3.4 The NOD2 rs2066844 gene variant is associated with increased IL-1β production in response to TLR4 ligand ..................................................................... 118 4.3.5 The IRGM rs7714584 gene variant does not affect IL-1β production in response to TLRs and NOD2 ligands ....................................................................................... 120 4.3.6 The XBP-1 rs35873774 gene variant is associated with increased IL-1β production in response to PRR ligands ...................................................................... 122 4.4 Discussion .......................................................................................................... 125 Chapter 5: Concluding remarks and future directions ............................................... 130 5.1 Concluding remarks ........................................................................................... 130 5.2 Future directions ................................................................................................ 136 5.3 Thesis model ...................................................................................................... 138  References ........................................................................................................................ 139  xi  List of Tables  Table 4.1   Crohn's disease-associated single nucleotide Polymorphisms ........................ 109 Table 4.2   Subject numbers (n) per genotype ................................................................... 109 Table 4.3   TLR & NOD2 ligands and concentrations used for stimulations .................... 111  xii  List of Figures  Figure 1.1   Etiology of Crohn’s disease (CD) ...................................................................... 5 Figure 1.2   The epithelial barrier separates the gut lumen from the lamina propria. ........ 11 Figure 1.3   NLRP3 inflammasome activation and IL-1β production ................................. 22 Figure 1.4   The NLRP3 inflammasome .............................................................................. 23 Figure 1.5   Autophagy. ....................................................................................................... 31 Figure 1.6   The ATG16L1 T300A-encoding gene variant predisposes people to CD ....... 33 Figure 1.7   SHIP negatively regulates the immune response ............................................. 39 Figure 2.1   IL-1β production is increased in ileal sections from SHIP-/- mice compared to SHIP+/+ ............................................................................................................................... 55 Figure 2.2   SHIP-/- ileal macrophages produce more IL-1β and IL-18 than wild type ileal macrophages ........................................................................................................................ 56 Figure 2.3   In vivo differentiated SHIP-/- macrophages produce high levels of IL-1β and   IL-18  ................................................................................................................................... 59 Figure 2.4   IL-1β and IL-18 production are dependent on the NLRP3 inflammasome and caspase-1 activity ................................................................................................................. 60 Figure 2.5   IL-1β production is dependent on class I PI3K p110α activity ........................ 63 Figure 2.6   SHIP deficiency increases transcription of IL-1β ............................................ 64 Figure 2.7   Macrophage depletion reduces development of intestinal inflammation in   SHIP-/- mice ........................................................................................................................ 66 Figure 2.8   Macrophage depletion reduces IL-1β and IL-6 levels in the ileum of SHIP-/- mice...................................................................................................................................... 67 xiii  Figure 2.9   Anakinra reduces development of intestinal inflammation in SHIP-/- mice ... 68 Figure 2.10   Anakinra reduces IL-1β levels in the ileum of SHIP-/- mice ......................... 69 Figure 2.11   SHIP activity is lower in ileal biopsies from subjects with CD compared to control subjects .................................................................................................................... 71 Figure 2.12   SHIP activity is lower in peripheral blood mononuclear cells (PBMCs) from subjects with CD compared to control subjects and is inversely proportional to IL-1β production by PBMCs ......................................................................................................... 72  Figure 3.1   Subjects with ileal CD have lower SHIP mRNA expression compared to control subjects ................................................................................................................................ 88 Figure 3.2   Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with low SHIP mRNA expression and activity ................................................................... 90 Figure 3.3   SHIP mRNA expression and activity are lower in PBMCs from healthy control subjects, who are homozygous for the ATG16L1 T300A-encoding gene variant .............. 91 Figure 3.4   ATG16L1 mRNA expression does not vary linearly with SHIP mRNA expression ............................................................................................................................ 93 Figure 3.5   Autophagy up-regulates SHIP protein and SHIP up-regulation is dependent on ATG16L1 expression and/or autophagy .............................................................................. 96 Figure 3.6   SHIP up-regulation is dependent on the ATG16L1 genotype .......................... 97 Figure 3.7   Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased (LPS+ATP)-induced IL-1β production ....................................................... 99 Figure 3.8   Reduced SHIP mRNA expression is associated with increased LPS-induced    IL-1β production ................................................................................................................ 101 Figure 4.1   TLRs/NOD2 dose response assays ................................................................ 112 xiv  Figure 4.2   Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased IL-1β production in response to TLR4/5 ligands+ATP ............................ 114 Figure 4.3   The ATG5 rs510432 gene variant does not affect IL-1β production in response to TLR and NOD2 ligands ..................................................................................................... 116 Figure 4.4   The ATG5 rs12201458 gene variant is associated with increased in IL-10 production in response to TLR4 ligand ............................................................................. 117 Figure 4.5   The NOD2 rs2066844 gene variant is associated with increased IL-1β production in response to TLR4 ligand................................................................................................ 119 Figure 4.6   The IRGM rs7714584 gene variant does not affect IL-1β production in response to TLR and NOD2 ligands ................................................................................................. 121 Figure 4.7   The XBP1 rs35873774 gene variant is associated with increased IL-1β production in response to TLR and NOD2 ligands ........................................................... 124 Figure 5.1   Autophagy regulates IL-1β production and intestinal auto-inflammation via SHIP up-regulation ............................................................................................................ 138   xv  List of Abbreviations  ACTB                         Gene encoding β-actin  AIEC   Adherent and invasive Escherichia coli AIM2                 Absent in melanoma 2 Akt                     A serine/threonine protein kinase also known as protein kinase B                                      (PKB) ANOVA           Analysis of variance AOSD                Adult onset Still’s disease Arg                     Arginine Arg I                   Arginase I AS605240          A class I PI3K p110γ selective inhibitor ASC                   Apoptosis-associated speck-like protein containing a CARD ATG16L1          Autophagy related 16-like 1 ATG5                Autophagy protein 5 ATP                   Adenosine triphosphate BMMs               Bone marrow macrophages Ca2+                  Calcium CAPS    Cryopyrin-associated periodic syndrome CARD    Caspase recruitment domain CCL2    Chemokine (C-C motif) ligand 2 CD    Crohn’s disease CD4                   Cluster of differentiation 4 xvi  CGD                  Chronic granulomatous disease CINCA              Chronic infantile neurological syndrome CLRs                 C-type lectin receptors CT                     Computer tomography CXCL8             Chemokine (C-X-C motif) ligand 8 DAMPs            Danger associated molecular patterns DAPI                4΄ 6-diamino-2-phenylindole DCs                  Dendritic cells DMSO               Dimethyl sulphoxide DNA                 Deoxyribonucleic acid DSS                  Dextran sodium sulphate EDTA               Ethylenediaminetetraacetic acid ELISA              Enzyme-linked immunosorbent assay ER                    Endoplasmic reticulum FBS                  Fetal bovine serum FCAS               Familial cold autoinflammatory syndrome FCS                  Fetal calf serum FLICA              Fluorescent labeled inhibitor of caspases FMF                 Familial Mediterranean Fever FSL                  Pam2CGDPKHPKSF (TLR6 ligand) GAPDH           Glyceraldehyde 3-phosphate dehydrogenase Gln                   Glutamine GM-CSF          Granulocyte-monocyte colony-stimulating factor xvii  GWAS             Genome wide association studies H&E                 Hematoxylin and eosin  H2O2                 Hydrogen peroxide HBSS               Hanks balanced salt solution HKLM             Heat-killed Listeria monocytogenes hr                      Hour IBD                  Inflammatory bowel disease IECs                 Intestinal epithelial cells IFI16                Gene-encoding interferon gamma-inducible protein 16 (human) IFNγ                 Interferon gamma IgG      Immunoglobulin G IL-1Ra              Interleukin 1 receptor antagonist IL-1α                Interleukin 1 alpha IL-1β                Interleukin 1 beta IL-23                Interleukin 23 IL-23R             Interleukin 23 receptor IMDM              Iscove’s modified Dulbecco’s medium INPP5D              Gene encoding the SH2-domain-containing inositol polyphosphate-5΄- phosphatase (SHIP) protein (human) IRAK               Interleukin 1 receptor-associated kinase IRF3                 Interferon regulatory factor 3 IRGM               Immunity-related GTPase family M protein  JAK2                Janus kinase 2 xviii  K+                     Potassium KO                    Knockout  LC3                   Microtubule-associated protein light chain 3 LP                      Lamina propria LPS                   Lipopolysaccharide LRR                  Leucine rich repeat LY29                LY294002, a pan-PI3K inhibitor LY30                LY303511, a non-inhibiting structural analogue of LY294002 MAMPs            Microbial associated molecular patterns MAPK              Mitogen activated protein kinase MCSF               Macrophage colony-stimulating factor MDP                 Muramyl dipeptide MEFV               Gene-encoding Mediterranean fever  mg                     Milligram MHC                 Major Histocompatibility complex min                    Minute miR-155            microRNA-155 ml                      Milliliter mM                    Millimolar MMPs                Matrix metalloproteinase mRNA               messenger RNA mTOR                mammalian target of rapamycin MUC19              Mucin-19 xix  MWS                  Muckle-Wells syndrome MyD88               Myeloid differentiation primary response gene 88  NACHT                      A family of NTPase domains that mediate ATP-dependent                                           oligomerization of NLRPs      NFκB                 Nuclear factor kappa B NH4+Cl-              Ammonium chloride NK cells              Natural killer cells NLRP3                Nod-like receptor family, pyrin domain containing 3 NLRs                   Nod-like receptors NO                       Nitric oxide NOD2                  Nucleotide-binding oligomerization domain-containing protein 2 NSAIDs               Nonsteroidal anti-inflammatory drugs ORMDL3            Orosomucoid 1-like 3 Pam3CSK4         Pam3CysSerLys4, which is a TLR1/2 ligand PAMPs               Pathogen associated molecular patterns PBMCs               Peripheral blood mononuclear cells PBS                     Phosphate-buffered saline PI(3,4,5)P3          Phosphatidylinositol 3,4,5-trisphosphate PI3K                   Phosphoinositide 3-kinase PIK-90/PI-103    Class I PI3K p110α inhibitors PMA                   Phorbol 12-myristate 13-acetate PolyI:C LMW     Low molecular weight Polyinosinic-polycytidylic acid, which is a TLR3 ligand xx  PRRs                   Pattern recognition receptors PTGER4             Prostaglandin E receptor 4 PTPRC               Protein tyrosine phosphatase, receptor type, C, which is the gene name for CD45 antigen PVDF                 Polyvinylidene fluoride PYD                   Pyrin domain PYHIN               Pyrin and HIN protein domain qRT-PCR           Quantitative real-time polymerase chain reaction RLRs                  RIG-like receptors RNA                   Ribonucleic acid RNAi                  RNA interference RORt                  Retinoic acid-related orphan receptor        ROS                    Reactive oxygen species Rplp0                  Gene encoding the ribosomal protein, large, P0 RPMI                  Roswell Park Memorial Institute medium SDS-PAGE         Sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM                    Standard error of the mean SHIP                    src homology 2 domain containing inositol polyphosphate 5’-                                       phosphatase                                         siRNA                 Small interfering RNA SMAD7                 Gene encoding the Mothers against decapentaplegic homolog 7 protein (A TGFβ signalling antagonist) SNPs                   Single nucleotide polymorphisms xxi  SOCS                  Suppressor of cytokine signalling  SOJIA                 Systemic onset juvenile idiopathic arthritis ssRNA                single stranded RNA STAT3               Signal transducer and activator of transcription 3 SW18                 A Class I PI3K p110γδ selective inhibitor SW30                 A Class I PI3K p110δ selective inhibitor TCR                    T-cell receptor TGF-β                 Transforming growth factor beta    TGX-221             A Class I PI3K p110β selective inhibitor TH                        T helper cell TL1A                   TNF-like ligand 1A TLRs                   Toll-like receptors TNFα                   Tumor necrosis factor alpha  TRAPS                TNF receptor-associated periodic syndrome Tregs                    regulatory T cells TREM2               Triggering receptor expressed on myeloid cells 2 UC                       Ulcerative colitis UK                      United Kingdom UPR                    Unfolded protein response US FDA              United States Food and Drug Administration UTR                    Untranslated region Wm                     Wortmannin XBP1                  X-box binding protein 1 xxii  YVAD                A selective irreversible inhibitor of caspase-1  γδ T cells           gamma delta T cells xxiii  Acknowledgements This work was funded by research grants from the Canadian Institutes of Health Research (CIHR). I am also grateful for salary support provided by the Child & Family Research Institute (CFRI) Immunity in Health & Disease Graduate Studentship, CIHR Transplantation Training Program, and the Mitacs-Accelerate Graduate Research Award.  I would like to thank my supervisor, Dr. Laura Sly, for providing continuous support and guidance throughout my doctoral training; for providing me with the tools to become a better scientist; for teaching me effective scientific and communication skills; for editing my written work anytime I knocked on your door; and for your generosity and kindness. I will forever be grateful that you accepted me as a PhD student, and provided me with all the opportunities I have had in your laboratory.   I would also like to thank my supervisory committee: Dr. Megan Levings, Dr. Kevan Jacobson, and Dr. Pascal Lavoie. You have been extremely supportive and have provided excellent suggestions that have helped me throughout my doctoral training. Thank you to Dr. Stuart Turvey for invaluable contributions to the direction of this work. Our collaboration with your laboratory, helped improve the quality of this work. I also want to thank the following staff at CFRI, who have assisted with my research: Lisa Xu (Flow cytometry), Baoping Song (Histology core), and German Fernandez (Animal Care Facility). I am also grateful to the Vallance laboratory at CFRI for helping me with microscopy. I would also like to thank all past and present members of the Sly laboratory and my friends at CFRI for their help, support, and the wonderful memories that I will take with me. This includes Dr. Shelley Weisser, Dr. Keith McLarren, Hayley Brugger, Roger Jen, Susan Menzies, Lisa Kozicky, Mahdis Monajemi, Young Lo, Jean-Philippe Sauvé, Bonnie Cheung, xxiv  Ashish Sharma, Ashish Marwaha, Brian Chung, Jonathan Han, Kevin Tsai, Ganive Bhinder, Kathleen Wee, Dominika Nackiewicz, Brian Tenant, Jacques Courtade, and Jens Vent-Schmidt. I am also grateful for the contributions of all the students that I mentored including: Linnette Ocariza, Lindsay Richter, and Rania Khelifi.  I would like to thank Alex Persson (Sweden) for the part you played in my coming to Vancouver. You are such a special friend.   Personally, I would also like to thank all my friends and family for supporting me throughout this journey.  Special thanks to Muriel, Gladys, Yvette, Barbara, and Jeffroy for continuous encouragement, support, and motivation. I deeply appreciate all your support.        xxv  Dedication  To my sweetheart, Felicia Choung Anyambong You stood by me throughout this journey, providing me with words of encouragement, support, and endless love.  Thank you for coming into my life.  I love you.  To my mother, Jannet Abiliwuh Ngoh  You supported me throughout my years of education, both morally and financially. You have always encouraged me and stood by me from the beginning.  This success is yours too. I love you Mami.    1  Chapter 1: Introduction  1.1 Inflammatory bowel disease Inflammatory bowel disease (IBD) is a chronic, relapsing, idiopathic inflammatory disorder of the gastrointestinal tract that manifests in two main forms: Crohn’s disease (CD) and ulcerative colitis (UC). IBD can affect individuals of any age, with peak incidence reported amongst individuals in the third decade of life.1 Patients usually suffer from recurring episodes of abdominal pain, diarrhea, rectal bleeding, and nutritional deficiencies.2 No difference has been seen in the incidence rate of IBD between men and women, but other factors, such as race and ethnicity have been shown to play a significant role.  UC is typically confined to the colon and affects the rectum in 95% of patients.1 Some UC patients also develop inflammation in the distal ileum, which is thought to result from backwash of cecal content.3 Inflammation in UC is restricted to the mucosal and submucosal layers.2 It is characterized by edema, which is swelling resulting from retention of excess fluid; ulceration, and rectal bleeding.1 Histological analysis of tissue sections from UC patients reveals the presence of immune cell infiltrates and crypt abscesses, reduced goblet cell number, and alteration of crypt architecture.1  On the other hand, CD can affect any part of the gastrointestinal tract from the mouth to the perianal region.1  Inflammation is usually discontinuous with patches of inflammation separated by areas of healthy tissue, and can involve the entire intestinal wall (transmural). The most common site of intestinal inflammation in CD is the distal part of the ileum.4 Histological analysis of tissue sections from CD patients reveals transmural inflammation with the presence of immune cell aggregates known as granulomas.1 Other key features 2  usually present in the affected areas of the gastrointestinal tract of CD patients include: fistulas, which are channels connecting the intestine to other sites; and strictures, which result in narrowing of the intestine and may require surgical intervention if the intestine becomes blocked.5 The incidence rate of IBD is highest in northern Europe, the United Kingdom (UK), and North America.6,7 It has been revealed that, Jewish populations have a threefold higher incidence of IBD compared to non-Jewish populations.8 According to estimates from 2012, Canada has the highest incidence of IBD in the world, with an estimated 233,000 people having the disease.9,10 Amongst these people, 129,000 were diagnosed with CD and 104,000 were diagnosed with UC.9,10 The incidence rate of IBD among children less than 10 years old is rising in Canada. It is estimated that 5900 children less than 18 years of age have IBD, making Canada one of the countries with the highest incidence of pediatric IBD in the world.11 The high incidence of IBD places a huge burden on the affected families and the Canadian healthcare system. The consequences of IBD on the Canadian economy cannot be over emphasized. In 2012, it was estimated that both direct and indirect costs of IBD in Canada, amounted to about 2.8 billion dollars. Direct costs include hospitalizations, surgical, and other procedures, such as colonoscopy, medications, laboratory tests, and services provided by physicians and other healthcare professionals.10 The chronic nature of IBD and the financial strain also causes psychological stress and affects the quality of life of patients and their families.12 People with IBD have also been reported to have poor sleep.13 IBD is also associated with social stigma, which can be reduced by increasing awareness of the disease amongst the population.10 Because of the health, psychological, social, and economic consequences of IBD, organizations, such as the Canadian Institutes of Health Research and Crohn’s and Colitis Canada are committed to 3  funding research aimed at improving the lives of Canadians living with IBD. A key research priority is to identify and validate new therapeutic strategies to treat the disease thereby reducing the burden of IBD on patients, their families, and the Canadian health care system.  In keeping with these goals, my doctoral work focusses on characterizing mechanisms and exploring novel interventions to treat intestinal inflammation in Crohn’s disease.  1.2 Clinical presentation and diagnosis Patients with UC present with abdominal pain, cramping, and diarrhea containing blood mixed with mucus.1 CD patients, on the other hand, may experience pain in the right lower quadrant of the abdomen and disease complications may result in swelling, thickening of the bowel wall, and blockage of the intestine.1 Patients also suffer from anorexia, diarrhea, and weight loss.1 In children, IBD can result in delayed growth and delayed sexual maturation.14 Diagnosis of IBD usually requires a review of the patient’s history and a combination of different tests and procedures to exclude the pathological effects caused by the presence of enteric pathogens, such as Clostridium difficile, Salmonella typhimurium, Shigella flexneri, Mycobacterium tuberculosis, or Escherichia coli.1,15 Review of patient’s history include assessment for symptoms such as diarrhoea with blood and mucus, abdominal pain and cramping, fever, weight loss, and perianal disease, if CD is suspected.15 Also, the patient’s recent use of medications such as antibiotics and NSAIDs and the history of IBD in the family are important considerations during diagnosis of IBD. Various blood examinations, such as complete blood count; erythrocyte sedimentation rate and C-reactive protein (which negatively correlate with inflammation and disease severity); and liver function tests are also performed to help exclude other causes and confirm diagnosis.15 If a 4  patient presents without a history of blood in stool, colonoscopy and endoscopic examination may be performed with or without occult blood, with biopsies taken for histological analysis.1,15 Sigmoidoscopy (used to see inside the sigmoid colon and rectum) or colonoscopy are used for examination of ulcers, bleeding, inflammation, and to determine the presence of multiple biopsies of the colon and terminal ileum.15 Patients may also have X-rays, abdominal ultrasounds, CT scans (computer tomography scans), MRI (magnetic resonance imaging) or small bowel imaging, which is usually done for CD patients to determine the extend and severity of disease, the involvement of the terminal ileum and the rest of the small bowel, and also to look for extraintestinal complications.1,15 In children in which upper GI disease (with symptoms such as nausea and vomiting) is more common, upper GI endoscopy may be perfomed.15    1.3 Etiology and pathogenesis  Although the etiology of IBD remains unknown, current thinking is that IBD occurs in genetically susceptible individuals due to an inappropriate initiation or perpetuation of an immune response to intestinal flora (Figure 1.1).16,17 CD is characterized by relapsing and remitting inflammation, and it is estimated that approximately 90% of CD patients will experience relapse at some point, while between 38-71% of CD patients will require surgery within 10 years of diagnosis for their disease.18 Moreover, studies indicate that about 10% of CD patients are refractory to current anti-TNFα therapy and up to 30% of CD patients are predicted to become refractory to these therapies.19,20 Identification of disease phenotypes and unique 5  molecular signatures of disease are essential to develop urgently needed novel strategies to effectively target intestinal inflammation in all patients with CD.    Figure 1.1   Etiology of Crohn’s disease (CD) The etiology of CD lies at the intersection of genetic, environmental, and immunologic factors. It is believed that, CD results from complex interactions between the intestinal microenvironment, including the microbiome and the environment external to the host, and the immune response in genetically susceptible individuals. GWAS have identified 140 single nucleotide polymorphisms associated with Crohn’s disease, including those in ATG16L1 and NOD2. Current therapeutic intervention relies on non-specific suppression of the immune response. Modified and reproduced with permission of Nature Publishing Group: Xavier R.J. & Podolsky D.K, Nature 2007.4  1.3.1 The role of genetics in Crohn’s disease The first studies aimed at understanding the role of genetics in the onset and pathogenesis of CD was familial aggregation studies and twin studies, which revealed a steady and consistently increased prevalence of CD amongst relatives.21  Studies in Europe revealed a very high CD concordance rate in monozygotic twins. In Sweden, the CD concordance rate in monozygotic twins was 50% whereas it was only 3.8% for dizygotic twins.22 In Denmark, the CD concordance rate was 50% in monozygotic twins and 0% for dizygotic twins.23 In the UK, the CD concordance rate was reported to be 33.3% in monozygotic twins and 9.9% for dizygotic twins.24 Amongst CD patients, 6  between 2-14% have a family history of disease.25-31 However, very little is now known of the effects of a positive family history on the severity and pathogenesis of CD. After the familial aggregation and twin studies, came the era of genome-wide association studies (GWAS), which focused on identifying single nucleotide polymorphisms (SNPs) and candidate genes that may underlie disease susceptibility.32-42  GWAS have strengthened our understanding of the role that genetics play in the pathogenesis of CD. These studies have been facilitated by the completion of the Human Genome Project (2003) and the International HapMap Project (2005). The Human Genome Project completely mapped out the human genome and identified about 20,500 genes. The International HapMap project determined genetic variations in the human genome and generated a set of tools for researchers to utilize to investigate the effects of specific gene variants in disease.43 Both projects have successfully generated a set of computerized databases containing genetic variants or single nucleotide polymorphisms (SNPs), some of which contribute to common diseases.44 Linkage studies, which are used to identify inherited genetic markers associated with diseases in a large family, were the first studies used to identify a genetic locus on chromosome 16 strongly associated with CD, 44 which was later confirmed by GWAS as a SNP in the nucleotide-binding oligomerization domain-containing protein 2 (NOD2).32,33  A recent meta-analysis of GWAS identified 163 SNPs associated with susceptibility to IBD, of which 140 are susceptibility loci for CD.16,42 The SNPs associated with disease often converge on specific biological processes, such as autophagy, innate and adaptive immune responses, suggesting that CD may comprise distinct pathological subsets of disease. GWAS have identified genes associated with increased susceptibility to CD including PTGER4 7  (encoding the prostaglandin E receptor 4),45 and MUC19 (encoding mucin 19),39 both of which are associated with epithelial barrier function; and genes associated with the interleukin 23 (IL-23) signalling pathway, such as IL23R, STAT3 (signal transducer and activator of transcription 3), and JAK2 (Janus kinase 2).34,46,47 GWAS analyses have also highlighted an unexpected but central role for autophagy in intestinal inflammation.16 Autophagy-related SNPs that predispose people to develop CD include those in genes encoding NOD2, which is required for induction of autophagy in response to intracellular bacterial infection;48,49 XBP1 and ORMDL3, which are required for the unfolded response;49-51 and autophagy genes ATG5,16,52 IRGM,38,53 and ATG16L1;35,36 all of which have been implicated in autophagy-related defects in CD. Identification of these polymorphisms in key innate and adaptive immune genes associated with CD susceptibility provides more evidence that crosstalk between genetic, environmental, and immunological factors play a crucial role in the development of CD.  1.3.2 Environmental factors in Crohn’s disease The prevalence of CD has steadily increased in the past 50-60 years and part of this could be attributed to the fact that different populations have migrated from areas that had very low incidence to areas with higher incidence, such as North America and Europe.54 This migration pattern coupled with the different genetic backgrounds and dietary changes of the populations, have demonstrated a role for environmental factors in the pathogenesis of CD.30 Several environmental factors have been associated with increased risk for CD including smoking, antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), oral contraceptives, diet, vitamin D, and stress. Cigarette smoking increases the risk of developing CD by two-8  fold.55,56 Studies have shown that, smoking reduces T helper cell proliferation and alters the ratio of regulatory T cells to T helper cells in the gut.57 Interestingly, non-smokers are at an increased risk of developing UC.55 Repeated use of antibiotics has also been associated with increased risk of developing CD in pediatric patients.58 Antibiotics may alter the microbiota and thus may predispose people to the development of CD.58 Oral contraceptives also increase the risk of developing CD, with studies revealing that the risk of CD is two-fold higher in women on oral contraceptives than control subjects.59 NSAIDs are also associated with increased risk of developing IBD. This has been modeled using IL-10 KO mice, which develop spontaneous colonic inflammation when treated with NSAIDs.60 Studies have also associated vitamin D deficiency with IBD. Patients diagnosed with IBD have been reported to be deficient or insufficient in vitamin D compared to healthy controls.61 Stress is another environmental factor that has been associated with increased risk of developing IBD.62,63 Both chronic and acute stress result in changes in the inflammatory response and may play a role in the development of intestinal inflammation during IBD. Chronic stress results in increased cortisol levels, which are associated with immunosuppression64 and may lead to reduced intestinal inflammation. In addition, depression has been associated with reduced immune cell numbers (macrophages, NK cells) in the blood.65 In middle aged to elderly patients, chronic psychological stress leads to subclinical increases in inflammation,66 suggesting that, chronic stress could also enhance intestinal inflammation. Acute stress, on the other hand, leads to increased adrenaline and pro-inflammatory cytokines such as IL-6, TNFα, and IFNγ, which may enhance intestinal permeability and inflammation.67   9  1.3.3 The microbiome in Crohn’s disease The gut lumen in humans is composed of a plethora of different bacteria with approximately 1012 microorganisms that are in close proximity to the intestinal epithelial barrier.68 Migration results in alteration of the gut microbiota due to: changes in the environment external to the host, dietary changes, changes in behaviour, and adaptation to new life styles, all of which could explain the increased incidence of CD in new populations, which have relocated.69 In CD patients, dysbiosis in luminal bacteria, characterized by less diversity in the microbiota is very common.70,71 CD is also associated with adherent and invasive E. coli (AIEC), with close to 22% of CD patients having AIEC in the lumen compared to 6.2% of healthy subjects.72 Viral infections have also been implicated in CD pathogenesis and have been shown to alter the gut microflora. Evidence for this has been seen in mice harboring the homologue of the ATG16L1 CD susceptibility gene found in humans.73 Upon infection with norovirus, these mice show abnormal Paneth cell structure and granules similar to that observed in CD patients, who are homozygous for the ATG16L1 gene variant.73 Indeed, the CD risk alleles in NOD2 and ATG16L1 have also been associated with changes in the composition of intestinal microbiota.74 These studies suggest an important role for intestinal microbiota in the pathogenesis of CD.   1.3.4  The epithelial barrier in Crohn’s disease Intestinal immune homeostasis is maintained by coordinated action of the intestinal epithelial cells and the innate and adaptive cells and responses. The intestinal epithelial barrier is a single layer of cells made up of four main types of epithelial cells: enterocytes 10  or colonocytes, goblets cells, Paneth cells, and enteroendocrine cells; separating the gut lumen from the lamina propria (LP).75 It serves as a dynamic physical barrier preventing entry of microbes and other luminal antigens into the LP and also allows the passage of nutrients and water from the gut lumen into circulation.75,76 Tight junctions seal the space between adjacent epithelial cells preventing entry of luminal bacteria into the LP.77 Paneth cells and goblet cells secret mucin and antimicrobial peptides to form the protective mucus layer that limits contact between commensal bacteria and epithelial cells. It is believed that during CD, the epithelial barrier is defective, resulting in increased epithelial permeability.78 In addition, MUC1 (a cell surface glycoprotein) mRNA expression is reduced in the inflamed ileal mucosa of CD subjects compared to the healthy mucosa.79  Moreover, patients homozygous for the ATG16L1 risk variant have abnormal Paneth cells, which may affect secretion of antibacterial peptides, such as defensins.73,80 Furthermore, in patients with IBD, a dysregulated immune response to normal enteric microflora leads to increased mucosal secretion of IFNγ and TNFα, which have been associated with defective epithelial barrier permeability.81  In all, maintaining an intact intestinal epithelial barrier with fully functional Paneth and goblet cells, together with effective immunoregulatory mechanisms are crucial to prevent the onset of intestinal inflammation and CD (Figure 1.2).  11   Figure 1.2   The epithelial barrier separates the gut lumen from the lamina propria. The intestinal epithelial barrier prevents LP immune cells from interacting with commensal microbes in the gut lumen. (Left) In a healthy individual, there exists a state of immune tolerance that allows commensal bacteria to live alongside immune cells in the gut. Epithelial cells, DCs, and Paneth cells sample the gut lumen for microbes. DCs present microbial antigens to T cells, which in turn initiate a regulatory response to maintain immune homeostasis; epithelial cells release IL-18 that stimulates growth and proliferation of epithelial stem cells to repair damaged tissue; Paneth cells secrete anti-microbial host defense proteins to maintain homeostasis; while type 3 innate lymphoid cells (ILC3), which form the majority of ILCs in a healthy intestine, secrete IL-22, mucus, and antimicrobial peptides that mediate tissue repair and maintain tolerance. (Right) In a susceptible host, the intestinal epithelial barrier may be compromised allowing luminal bacteria and antigens to enter the sterile LP where they encounter DCs and macrophages. These cells sense the presence of these microbes using their PRRs, and initiate an inflammatory response with production of pro-inflammatory cytokines, such as IL-1β, IL-18, IL-12, IL-6, TNFα, and IFNγ, resulting in inflammation. Activated immune cells also produce chemokines, such as IL-8, CCL2, which attract more immune cells to the site of inflammation, where they encounter these microbes and amplify the inflammatory response. IL-12 and IL-18 produced by macrophages and DCs stimulate type 1 innate lymphoid cells (ILC1), which are the majority of ILCs present during an inflammatory state, to produce TNFα, and IFNγ, which promote chronic inflammation. Modified and reproduced with permission of ELSEVIER LTD: Zaki, M.H. et al, Trends in Immunology 2011.82    1.3.5  The immune response in Crohn’s disease The immune system is made up of two arms: the innate immune system and the adaptive immune system. Both play critical roles in mucosal homeostasis and immune 12  dysregulation in CD. Below, I describe the roles the innate and adaptive immune systems play in the onset and pathogenesis of CD.  1.3.5.1 Innate immune response  The innate immune system is the first line of defense against invading microbes and is composed of cells including: epithelial cells, and leukocytes, such as monocytes, neutrophils, basophils, and eosinophils; macrophages, dendritic cells (DCs), and Natural Killer (NK) cells. Innate immune cells recognize pathogen-derived molecules in the intestinal microenvironment. Intraepithelial DCs sample the gut lumen for the presence of non-pathogenic microbes resulting in a regulatory response that provides tolerance.82 Innate immune cells contain extracellular and endosomal pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs),83 and C-type lectin receptors (CLRs)84 that monitor the extracellular and subcellular compartments for signs of infection or damage.85 In humans, there are 10 TLRs, TLR1-10 (13 in mice), each recognizing specific microbial associated molecular patterns (MAMPs). Intestinal epithelial cells (IECs) on the other hand express TLRs basolaterally to limit interaction with MAMPs in the gut lumen.86 NLRs and RLRs are intracellular PRRs that sense factors released during perturbations in tissue homeostasis known as damage-associated molecular patterns (DAMPs).83 In healthy individuals, there is a steady-state induction of protective factors by TLRs when they come in contact with commensal bacteria, which results in immune tolerance.87  Studies in mice have revealed that alteration in the proteins required for immune defense responses could lead to intestinal inflammation.88 TLR4 expression in intestinal epithelial cells (IECs) and both 13  TLR2 and TLR4 expression in intestinal macrophages and DCs are increased in CD patients compared to control subjects.89,90 Innate lymphoid cells (ILCs) are cells, which do not express the T-cell receptor (TCR) and serve as a source of cytokines, especially IL-23, at mucosal surfaces.91 Patients with CD produce more IL-2392 and express more of the transcription factor, retinoic acid-related orphan receptor (RORt)93,94 in the LP compared to control patients. Thus the innate immune response is crucial to prevent the development of IBD since it serves as the first line of defense against microbes after they cross the intestinal epithelial barrier. Defects in this arm of the immune response may result in increased production of inflammatory mediators that drive or contribute to intestinal inflammation in CD.  1.3.5.2 Adaptive immune response  The adaptive immune system is made up of T and B lymphocytes and acts as the second line of defense to invading pathogens. It is highly specific, recognizes ‘self’ from ‘non-self’ antigens, and generates appropriate immune responses. It also confers long lasting immunological memory. CD4+ T cells are important effector cells of the adaptive immune response that are important in defense against pathogenic microbes and excessive entry into the gut lumen of commensal bacteria. They can be grouped into different classes including: helper T cells (Th1, Th2, or Th17), or regulatory T cells (Tregs). Th1 cells are induced by IL-12 and IL-18 and produce high levels of interferon-γ (IFNγ) and TNFα. They protect against intracellular bacterial infection. Th2 cells produce high levels of IL-4, IL-5, IL-9, and IL-13, and protect against parasitic helminthes infections.95 Th17 cells on the other hand, are 14  induced by IL-6 in the presence of transforming growth factor beta (TGFβ) in mice,96 or both IL-6 and IL-1β in humans,97 to produce high levels of IL-17A, IL-17F, IL-21, and IL-22.98 Th17 cells are important for defense against extracellular pathogens via recruitment of neutrophils and macrophages to the site of infection.99 IL-23 promotes the expansion of Th17 cell responses.98 An imbalance or overreaction of CD4+ T cells relative to Tregs is believed to cause intestinal inflammation.100-103 CD is widely believed to be a Th1/Th17 mediated disease with increased secretion of IFNγ, TNFα, IL-17A, and IL-2 reported in T cells from CD patients compared to those from control subjects.104,105 It has been shown that, IFNγ and TNFα levels are increased in the inflamed mucosa of CD patients,106,107 while their LP cells produce high levels of IL-12.108 In UC patients, there is increased production of IL-4 and IL-13,105 suggesting that Th1 and Th2 cytokines play an important role in the pathogenesis of CD and UC, respectively. IL-17 producing Th17 cells are also increased in the inflamed mucosa of IBD patients and are regulated by IL-23.109,110 LP macrophages from CD patients produce high levels of IL-23, which drives Th1 and Th17 responses.111,112 In addition, SNPs in the IL-23R gene have been associated with IBD.34 In fact, the IL-23R SNP, Arg381Gln (arginine381glutamine), has been reported to confer a 2-3 fold protection against development of pediatric CD.34,113 These studies suggest that both IL-17 and IL-23 play an important role in the pathogenesis of IBD and consequently, targeting the IL-17/IL-23 pathway may serve as a therapeutic option in IBD. Monoclonal antibodies directed against the p40 subunit of IL-12/23 (ABT-874) and IL-23 (ustekinumab) have been tested as treatments for CD patients, but have failed in phase two clinical trials114; the human anti-IL-17A monoclonal antibody (secukinumab) induced modest clinical response in patients, who 15  were not responsive to anti-TNFα therapy.115 Indeed, secukinumab has been shown to reduce moderate to severe CD in patients with the TNF-like ligand 1A (TL1A) gene variant.115 The TL1A gene encodes a cytokine that belongs to the TNF ligand family, and drives pathogenic T cells in inflammatory processes116  IL-21 is another cytokine that is increased in intestinal tissues from IBD patients and enhances IFNγ production and Th1 signalling.117 Moreover, IL-22 expression is increased in the gut of CD patients compared to control patients and drives epithelial cell migration and pro-inflammatory gene expression.118  In mice, IL-17 has also been shown to play an important role in different models of intestinal inflammation. IL-17F enhances dextran sodium sulfate (DSS)-induced colitis, whereas IL-17A protects mice against DSS-induced colitis.119 Natural killer (NK) cells produce IL-22, which protects mice from intestinal inflammation by promoting production of mucus and defensins by intestinal epithelial cells.120,121 In all, Th17 cells enhance intestinal inflammation by promoting the recruitment of inflammatory cells, such as neutrophils, and production of inflammatory mediators, such as cytokines, chemokines, and matrix metalloproteinases (MMPs), which cause tissue damage.122-124   Tregs are adaptive immune cells that are crucial for the maintenance of mucosal immune homeostasis. They exert their action by producing IL-10 and TGF-β, suppressing the proliferation of naïve T helper cells and aberrant immune responses to commensal bacteria and microbial antigens.125,126 In patients with active IBD, Treg numbers are significantly reduced in peripheral blood compared to control subjects.127,128 In contrast, Treg activity is reduced in the intestinal mucosa of IBD patients despite increased Treg numbers.129,130 Smad7 (inhibitory molecule) is up-regulated in the inflamed mucosa of IBD 16  patients and blocks Treg function by regulating TGFβ signalling.131 Blocking Smad7 activity restores TGFβ signalling and Treg function.132 In mice, studies have shown that, knockdown of Smad7 using a Smad7 antisense oligonucleotide (GED0301) blocked inflammatory cytokine production and reduced experimental colitis.132 Furthermore, Phase I clinical trial studies have shown that patients with active steroid-dependent/resistant CD treated with GED0301 had reduced inflammatory cytokine-expressing CCR9+ T cells in their blood.133 Recently, Phase II clinical trials with an oral Smad7 antisense oligonucleotide (Mongersen) caused clinical remission in patients with moderate to severe CD with rates of between 55 and 65% and remission was maintained for up to 3 months,134 suggesting that targeting Smad7 could be an important therapeutic strategy for IBD. Taken together, IBD may develop due to defects in Treg activity resulting from increased Smad7 in the intestinal mucosa. Reduced Treg activity may lead to reduced anti-inflammatory cytokine production and uncontrolled Th1, Th2, and/or Th17 inflammatory cytokine production that promotes tissue damage and inflammation.  To summarize, the onset and pathogenesis of CD is such that, in a healthy individual, there exists a state of immune tolerance that allows commensal bacteria to live alongside the immune cells in the gut. Intraepithelial DCs sample the gut lumen for microbes and present microbial antigens to T cells, which in turn initiate a regulatory response to maintain immune tolerance. In a genetically susceptible host, the intestinal epithelial barrier may be compromised allowing luminal bacteria and antigens to enter the sterile LP where they encounter resident immune cells (DCs and macrophages). These cells sense the presence of these microbes using their PRRs, and initiate an inflammatory response with production of chemokines, such as IL-8, CCL2 (chemokine ligand 2), 17  CXCL8 (chemokine ligand 8), and pro-inflammatory cytokines, such as IL-6, TNFα, and IL-1β, resulting in inflammation.   1.3.6 Therapeutic options There is currently no cure for IBD. Generally, medical management of IBD requires long-term treatment based on a combination of drugs designed to relieve patients of the symptoms, provide long-term remission, and diminish the risks of complications. Treatment takes into account the severity, location, and phenotype of disease, as well as disease complications and individual tolerance to medical intervention.15 Futhermore, the patient’s past disease course and the duration and number of relapses in a calendar year are also considered during IBD management. In addition, management of pediatric IBD takes into account the age and pubertal status of the child.15 Treatment begins with 5-aminosalicyclic acids (5-ASAs), progresses to steroids if symptoms persist, and finally to biological therapies.1,135   5-ASAs, such as sulfasalazine, mesalamine, olsalazine, and balsalazide, are used for treating mild to moderate UC and can be used for mild ileocolonic and colonic CD.1,15 For moderate disease, corticosteroids, such as prednisone, which reduce inflammation, are used. Because of the side effects of corticosteroids, long-term use is avoided. Exclusive enteral nutrition is an effective and often preferred option, which involves exclusion of normal diet for a period of time and use of liquid nutritional products.1,136 CD Patients with complications  such as perianal disease, fistulas, bacterial over-growth in the settings of strictures, may be treated with antibiotics, such as ciprofloxacin and metronidazole.1,15 In patients with moderate to severe IBD, immunosuppressive drugs, such as azathioprine, 6-18  mercaptopurine or methotrexate that suppress the immune response can be used. A disadvantage of using immune-modulatory drugs such as methotrexate is that they are non-selective and reduce the patient’s ability to fight infections.1   Biological therapies are designed to target specific immune mediators of diseases, such as cytokines. Biological therapies have been used for IBD treatment and have produced mixed results. Some of these biological therapies include infliximab, adalimumab, and certolizumab, which are monoclonal antibodies (mAb) directed against the pro-inflammatory cytokine, TNFα.  These are effective at inducing and maintaining remission in patients and have revolutionized the treatment for CD and UC.137 Others, such as secukinumab (human anti-IL-17A monoclonal antibody), have produced mixed results and has been shown to reduce moderate to severe CD in patients with the TL1A gene variant.115 Ustekinumab, which is a human mAb that specifically binds the p40 subunit shared by IL-12 and IL-23, has failed to induce remission in patients with moderate to severe CD in phase two clinical trials.114 Despite that, some patients are refractory to biological therapy and for others, treatment loses its efficacy over time.138 Loss of efficacy may be caused by low drug levels or development of antibodies to the biological therapy, but also occurs in some patients with neither of these causes.138 This underscores the need for identification of disease subtypes and development of novel therapeutic strategies to treat disease.  1.4 The Interleukin 1 family of cytokines The interleukin 1 (IL-1) family of cytokines is composed of eleven members including seven ligands (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ); three receptor antagonists (IL-1Ra, IL-36Ra, and IL-38), and an anti-inflammatory cytokine (IL-37).139 IL-1β and IL-19  18 are processed through the same mechanism, involving activation of the inflammasome and generation of active caspase-1. IL-33 is produced by many cell types including fibroblast, mast cells, macrophages, DCs, osteoblasts, epithelial cells, and endothelial cells.140 It was initially thought to be produced by the same process as IL-1β and IL-18. However, recent studies have shown that caspase -1 cleavage is not required for production of biologically active IL-33.141 IL-33 is increased in bronchial epithelial cells of asthma patients compared to control subjects.142 IL-33 has been shown to promote goblet cell hyperplasia, airway hyper-responsiveness, eosinophilia, M2 macrophage polarization and accumulation of IL-4, IL-5, and IL-13 in the lungs.143-145 IL-36 is produced by innate immune cells, lymphocytes,146,147 and epithelial cells,148 and activates nuclear factor kappa B (NFκB) and mitogen-activated protein kinases (MAPKs).149 Some members of the IL-1 family play an important role in the development and differentiation of T helper cells. IL-1β, together with IL-6, and TGFβ, induce differentiation of Th17 cells.150 In the presence of IL-23, IL-1β induces development of RORγt+ IL-17A-producing cells.150 IL-18, directs development of IFNγ-producing Th1 cells,151 whereas IL-33 is known to induce Th2 responses.152 Both IL-1α and IL-33 mediate sterile inflammation since their precursors are constitutively active and are released when cells undergo necrosis.153,154 The IL-1α precursor is present in epithelial layers of the gastrointestinal tract, the kidney, liver, and the lungs.153,154 Furthermore, IL-36 activates release of IL-2 and enhances T-cell proliferation.147   The IL-1 family also includes anti-inflammatory cytokines that are critical for the regulation and control of the actions of the pro-inflammatory cytokines. These anti-inflammatory members include IL-37, IL-38, IL-1Ra, and IL-36Ra. Endogenous IL-37 is a 20  natural anti-inflammatory cytokine, and reduces LPS (lipopolysaccharide) and IL-1β-induced pro-inflammatory cytokine production by 2-3 folds in human blood monocytes.155,156 IL-37 production is stimulated by TGFβ,155 and exerts its suppressive functions by translocating to the nucleus, where it binds to Smad3 (a transcription factor of TGFβ) and inhibits transcription of pro-inflammatory genes.157 Recombinant IL-37 binds the IL-18Rα chain and transmits its inhibitory signals.155 IL-37 has also been shown to inhibit adaptive immune responses. DCs expressing IL-37 induce Tregs and are unable to activate effector T cell responses155 IL-38 on the other hand, binds IL-36R and acts as a partial receptor antagonist.158 Polymorphisms in the IL-38 gene have been associated with increased susceptibility to inflammatory diseases, such as psoriatic arthritis and ankylosing spondylitis.159 Furthermore, IL-38 inhibits IL-17A and IL-22 production by human memory T cells.158  The IL-1 receptor antagonist (IL-1Ra) is a naturally occurring cytokine that competes with IL-1β and IL-1α for IL-1R1 and prevents IL-1 signalling. The IL-1Ra gene is conserved in humans and mice with identical intron-exon organization.160 There has been no report of interspecies differences in the IL-1Ra gene between humans and mice. Anakinra is a synthetic IL-1Ra that has been shown to reduce disease symptoms in patients with autoinflammatory diseases, such as rheumatoid arthritis.161,162 Deficiency in IL-1Ra is associated with severe systemic and local inflammation in children.163 In mice, deficiency in IL-1Ra leads to increased susceptibility to carcinogens, psoriatic-like skin lesions, and spontaneous arthritis.164,165  The IL-36 receptor antagonist (IL-36Ra) competes with IL-36 for the IL-36R and prevents IL-36 signalling. IL-36Ra also prevents IL-36-induced IL-23, IL-17, and IL-22 21  production as well as development of psoriasiform dermatitis.148,166 Mutations in the IL-36Ra gene have been associated with psoriasis.167   1.4.1 Interleukin 1β (IL-1β) The inflammatory response is critical in host defense against infectious pathogens but must be tightly regulated to prevent excessive tissue damage during injury or infection. This is particularly evident for production of the pro-inflammatory cytokine IL-1β, which has been described as the “master mediator,”168,169 “initiator,”168 or “gatekeeper” of inflammation.170 Cells, such as epithelial cells, monocytes, macrophages, DCs, B lymphocytes, and NK cells can produce IL-1β to initiate and/or amplify innate immune responses.170  IL-1β and IL-18 are produced by the same two-step process (Figure 1.3) that is tightly regulated in immune cells. In the first step, known as the priming step, microbial associated molecular patterns (MAMPs), such as lipopolysaccharide (LPS) bind to pattern recognition receptors (e.g. TLR4) and initiate a signalling cascade, which leads to NFκB activation and translocation into the nucleus, where it drives transcription of the IL-1β precursor, pro-IL-1β,171 and up-regulates NLRP3 (NOD-like receptor family, pyrin domain containing 3) expression.172,173 A second signal, typically a danger associated molecular pattern (DAMP), stimulates assembly of the inflammasome.   22   Figure 1.3   NLRP3 inflammasome activation and IL-1β production IL-1β production is a two-step process. The first step involves activation of a PRR (such as a TLR) by a MAMP (e.g. LPS), resulting in NFκB activation, its translocation into the nucleus, and production of pro-IL-1β and pro-IL-18. Activation of PRR also results in increased NLRP3 transcription. In the second step, ATP (a DAMP) binds to the P2X7 receptor, resulting in potassium ion (K+) efflux. The NLRP3 inflammasome senses a drop in intracellular K+ levels, activating caspase-1, which cleaves pro-IL-1β and pro-IL-18 to mature IL-1β and IL-18 that are secreted out of the cell.     1.4.2 Inflammasome activation and IL-1β production The inflammasome is a multi-protein complex composed of a nod-like receptor protein (NLRP), an adaptor protein (ASC), and an effector protein. Structurally, NLRs are composed of a C-terminus leucine rich repeat (LRR) that acts as the sensor of intracellular pathogen associated molecular patterns (PAMPs) and DAMPs,174 a central nucleotide binding oligomerization domain (NOD or NACHT), which is important for oligomerization 23  and activation of the NLR,175 and an N-terminal domain, which is usually a PYD (pyrin domain) or a CARD (caspase recruitment domain)176 ( Figure 1.4).    Figure 1.4   The NLRP3 inflammasome The NLRP3 inflammasome is made up of three main components: NLRP3, which contains a LRR, NACHT, and a Pyrin domain (PYD); an adaptor protein (ASC), which contains a PYD and a caspase recruitment domain (CARD); and pro-caspase-1. In the presence of a DAMP, the NLRP3 inflammasome is assembled by interaction of the PYD of NLRP3 with the PYD of ASC, and the CARD of ASC with the CARD of pro-caspase-1.  Six inflammasomes containing a NLR sensor have been identified to date and include: NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 (also known as IPAF).85 Two other inflammasomes that contain a PYHIN (pyrin and HIN domain-containing protein) have also been described and include Absent in melanoma 2 (AIM2) and IFNγ-inducible protein 16 (IFI16)177. These inflammasomes are usually assembled in response to stimuli, such as: Muramyl dipeptide (MDP) and Bacillus anthracis lethal toxin (NLRP1b); microbiota (NLRP6); cytosolic flagellin or stimuli from type III or IV secretion system-24  containing bacteria, such as Salmonella sp serovar Typhimurium, P. aeruginosa, L. pneumophilia, and S. flexneri (NLRC4); cytoplasmic double-stranded deoxyribonucleic acid (DNA) viruses, Francisella tularensis, and Listeria monocytogenes (AIM2). It is still not known exactly how these inflammasomes sense the presence of DAMPs within the cell. NLRP3 is the best described of the inflammasomes and its activation is often used as a model to study the production of IL-1β by monocytes and macrophages. The assembly and activation of the NLRP3 inflammasome occurs via many different pathways; the first is via detection of increased levels of extracellular ATP (adenosine triphosphate), which is released from dying cells and acts as a danger signal. ATP binds to the P2X7 receptor, opening the potassium channel, and induces formation of pores in the cell membrane, allowing MAMPs or DAMPs to gain access into the cell and activate the inflammasome.178,179 Pore forming toxins, such as nigericin also create pores in the cell membrane that facilitate potassium efflux.180 The drop in intracellular potassium concentration triggers activation of the NLRP3 inflammasome.181 Particulate and non-particulate stimuli, such as uric acid crystals, silica, asbestos, aluminum salt, and amyloid-β also activate the NLRP3 inflammasome. These crystals and particles are thought to destabilize the phagosomes in which they are stored upon phagocytosis, causing lysosome damage and release of cathepsin B into the cytosol, which then activates NLRP3.182-184 A wide range of pathogens including Escherichia coli,185 Neisseria gonorrhoeae,186 Candida albicans,187  and Vibrio cholera188 have also been shown to activate NLRP3. The mechanism by which this occurs is not completely known but NLRP3 activation by C. albicans requires release of cathepsin B and V. cholera acts via release of cytotoxins. Mitochondrial reactive oxygen species (ROS) was initially thought to activate the NLRP3 inflammasome, but recent 25  studies have now shown that mitochondrial ROS actually enhances expression of pro-IL-1β and NLRP3 via increased NFκB transcription.172,189 It is not known if there is direct contact between the LRR and the different DAMPs that activate NLRP3 inflammasome, but considering their diversity, it seems unlikely.  Activation and assembly of the inflammasome results in cleavage of pro-caspase-1 to active caspase-1. Caspase-1 produced, cleaves pro-IL-1β/pro-IL-18 to their active forms, which are secreted out of the cell. Secreted IL-1β signals through the IL-1 receptor, driving transcription of IL-1β thus initiating positive feedback activation.190-192 Circulating blood monocytes in humans have constitutively active caspase-1193 and when monocytes are stimulated with PAMPs that activate PRR, they produce mature IL-1β for several hours.170 This process can be accelerated by addition of exogenous ATP that activates the NLRP3 inflammasome.194 Thus, the rate-limiting step in IL-1β production is the synthesis of pro-IL-1β from IL-1 mRNA.170   Extracellular serine proteases, such as proteinase-3 released by infiltrating neutrophils,195,196 elastase, granzyme A, and matrix metalloprotease 9,169 can also cleave pro-IL-1β to mature IL-1β. This is relevant physiologically in that neutrophils are recruited to sites of inflammation and they release these proteases when they become apoptotic, thereby enhancing IL-1β production.  After mature IL-1β is produced, it is released out of the cell, though the mechanism of its release is not completely understood. An increase in intracellular calcium levels and the presence of phospholipase C are necessary to facilitate the process.197 IL-1β release can occur through vesicular bodies that contain exosomes,198 exocytosis of secretory lysosomes,199,200 and shedding of plasma membrane micro-vesicles.198 Even though a lot has been done to characterize the molecular mechanisms that underlie inflammasome activation, 26  there are still avenues for future research especially in identifying new intrinsic cellular mechanisms that regulate expression levels of NLRP3 and other inflammasome components.  To summarize, production of IL-1β is a 2 step process: The first step is activation of a TLR/cytokine receptor (e.g. IL-1R1) by a PAMP/cytokine (e.g. IL-1) resulting in downstream activation of NFκB transcription, which in turn drives IL-1β/IL-18 transcription and pro-IL-1β/pro-IL-18 production. The second step is activation of the inflammasome by DAMPs, resulting in caspase-1 activation, which permits cleavage of the pro-forms of IL-1β/IL-18 to their active forms, which are then secreted out of the cell.     1.4.3 IL-1β-mediated diseases (auto-inflammatory diseases) Auto-inflammatory diseases are initiated by sterile inflammation caused by excessive innate immune responses to DAMPs.168 IL-1 mediates these diseases and IL-1 blockade or treatment with a decoy IL-1 receptor (IL-1RII) or monoclonal antibody to IL-1β prevents pathology. In contrast, autoimmune diseases occur when one develops an adaptive immune response to self-antigens, usually initiated by excessive innate immune responses to DAMPs and PAMPs.168 IFNγ and TNFα produced by Th1 cells or IL-17 produced by Th17 and γδ T cells mediate autoimmune diseases.201 IL-1β has been shown to mediate many human diseases, which are described in the following sections.    1.4.3.1 Cryopyrin-associated periodic syndrome (CAPS)  CAPS are a group of autoinflammatory diseases including FCAS (Familial cold auto-inflammatory syndrome), MWS (Muckle-Wells syndrome), and CINCA (Chronic infantile 27  neurological, cutaneous, and articular syndrome), which occur when individuals with a gain of function mutation in NLRP3 are exposed to cold.202 Patients usually have recurrent fever, increased acute phase proteins, and leukocyte infiltration.202 CAPS are uniquely mediated by IL-1β and hence patients respond to treatment with the synthetic IL-1Ra, Anakinra,203-205 the soluble IL-1 receptor, rilonacept,206 or the monoclonal antibody to IL-1β, canakinumab.207     1.4.3.2 TNF-receptor-associated periodic syndrome (TRAPS) This is an auto-inflammatory disease that results from a gain of function mutation in the coding region of the TNF receptor and is characterized by recurrent fever and systemic inflammation.208 People with this mutation have defects in the translocation of the TNF receptor to the cell membrane, resulting in accumulation of misfolded proteins, endoplasmic reticulum stress (ER), and hence inflammation.170 ER stress results in increased mitochondrial ROS production that has been associated with potassium efflux, NLRP3 activation, and IL-1β release.209 Patients with TRAPs respond to treatment with an IL-1Ra (Anakinra) suggesting a key role for IL-1 in disease pathogenesis.210,211  1.4.3.3 Familial Mediterranean Fever (FMF) FMF is a chronic inflammatory condition that results from a gain of function mutation in pyrin, a protein component of the inflammasome complex.212 The marentosin-encoding fever gene (MEFV), which encodes pyrin, is associated with FMF. Pyrin is a protein found in neutrophils, monocytes, eosinophils, and fibroblasts from the skin, peritoneum, and synovium.213,214 They regulate caspase-1 activation and IL-1β production through its 28  interaction with the adaptor protein, ASC.215 A mutation in the MEFV gene leads to cleavage of pyrin and results in increase caspase-1 activation and IL-1β production.215 Furthermore, caspase-1 has been shown to cleave pyrin, releasing the N-terminal fragment of this protein that is translocated to the nucleus and activates NFκB by interacting with its p65 subunit.216 FMF is characterized by systemic and local inflammation resulting from increased activation of the inflammasome and IL-1β production.170 Anakinra has been shown to improve clinical outcome in patients with FMF.170  1.4.3.4 Adult onset Still’s disease (AOSD) AOSD is a rheumatologic condition characterized by fever, sore throat, arthritis, splenomegaly, lymphadenopathy, increased neutrophil infiltration, and release of hepatic acute phase proteins.170 Reducing IL-1β activity with Anakinra reduces systemic and local manifestations of disease.217  1.4.3.5 Other autoinflammatory diseases Other diseases mediated by increased IL-1β include Systemic onset juvenile idiopathic arthritis (SOJIA),211,218 osteoarthritis,219 chronic granulomatous disease (CGD),220 Type 2 diabetes,221,222 and smoldering myeloma.223,224 In gouty arthritis, uric acid crystals activate synovial macrophages in the presence of fatty acids to synthesize IL-1β.225-230 In all of these diseases, there is increased release of DAMPs, which are known activators of the inflammasome. The resultant IL-1β produced feeds back to produce more IL-1β. Anakinra or a monoclonal Ab to IL-1β has been shown to reduce pathology. Blocking IL-1β signalling 29  reduces IL-1β production and feedback, recruitment of neutrophils, and release of proteases that cause tissue damage and DAMPs production, and consequently reduces auto-inflammation.   1.4.4 IL-1β in intestinal inflammation Despite being a central mediator in the inflammatory response, the role of IL-1β in intestinal inflammation has been confounded by conflicting reports. In humans, reduced expression of NLRP3 has been associated with increased susceptibility to CD.231 Gene variants around the regulatory region of NLRP3 (rs4353135 and rs10733113 SNPs) are associated with reduced NLRP3 gene expression and decreased IL-1β production suggesting that NLRP3 and IL-1β may play a protective role early on during CD.231 However, IBD patients have increased levels of IL-1 in their intestinal tissue, which correlates with the level of intestinal inflammation.232 It has also been reported that, there is an imbalance between IL-1 and IL-1ra in the intestinal mucosa of IBD patients.233 Moreover, elevated IL-18 levels have been reported in colonic inflammatory lesions in CD patients.234 During the later stages of disease, inflammasome activation and IL-1β production have been shown to alter epithelial tight junctions and increase intestinal permeability in CD subjects.235 Hence, IL-1 may play protective or pathogenic roles in IBD depending on the stage of the disease. In mice, studies have shown that NLRP3 activation and increased IL-18 production, protects mice from chemically-induced intestinal inflammation by stimulating differentiation of epithelial stem cells and repair of damaged intestinal epithelium.236-238 Consistent with that, deficiency in NLRP6 inflammasome239 or IL-1β,240 exacerbates DSS-induced colitis in mice. These findings support the hypothesis that, in the initial phase of intestinal 30  inflammation, IL-1β and IL-18 produced by inflammasome activation in non-hematopoietic cells play a protective role, promoting epithelial repair237 after injury and limiting bacterial translocation into the lamina propria. During the later stages of disease, IL-1β promotes intestinal pathology by enhancing neutrophil recruitment and IL-17 production by innate lymphoid cells (ILCs) and CD4+ Th17 cells in the gut.241 In IL-10 KO mice, inflammasome activation and IL-1β production promote development of spontaneous colitis.242 Moreover, IL-1β is increased in intestinal tissues and peritoneal macrophages during DSS-induced colitis243 and increased IL-1β relative to IL-1Ra has been shown to drive colitis in TLR5 KO mice.244   1.5 Autophagy 1.5.1 Description and overview Autophagy is a multistep, catabolic self-preservation process that controls clearance and re-use of intracellular components during homeostasis and provides amino acids required for cell survival under stress conditions including starvation, accumulation of unfolded proteins, or intracellular infection.245 During autophagy, cytoplasmic contents including damaged organelles, misfolded/unfolded proteins, or intracellular bacteria are engulfed in a double membrane vesicle called the autophagosome, and degraded by fusion with lysosomes.246 Autophagy is usually determined by measuring expression levels of the microtubule-associated protein light chain 3 (LC3-II). LC3 is ubiquitously expressed in mammalian tissues as LC3-I in the cytosol.247 During autophagy, cytosolic LC3-I is conjugated with phosphatidylethanolamine to form LC3-II, which is integrated into the autophagosome membrane, and usually degraded during fusion with lysosomes (Figure 31  1.5).247,248 The amount of LC3-II is indicative of the number of autophagosomes present in a cell.247     Figure 1.5   Autophagy.  Autophagy is a cell homeostatic mechanism for degradation of cytoplasmic constituents, such as intracellular bacteria, misfolded/unfolded proteins, or damaged organelles, in autophagosomes. A phagophore nucleates, expands, and engulfs cytoplasmic constituents to form an autopahgosome. Cytosolic LC3-I is conjugated with phosphatidylethanolamine to form LC3-II, which is integrated into the autophagosome membrane. The completely formed autophagosome fuses with lysosomes to form an autolysosome, where its contents are degraded by lysosomal enzymes and proteases. Ammonium chloride (NH4+Cl-) prevents autophagosome-lysosome fusion while Bafilomycin A1 prevents degradation of the autophagosome-lysosome complex (autolysosome).   Autophagy negatively regulates the inflammatory response by regulating production of pro-inflammatory IL-1β.  In mice, autophagy stops inflammasome activation (signal 2) by degrading cytosolic DAMPs that activate inflammasomes, mitochondrial DNA, ROS, or by degrading inflammasomes/caspase-1.48,249-251 Autophagy also targets and segregates pro-IL-1β in autophagosomes for degradation by lysosomes, hence, inhibition of autophagy leads to availability of more pro-IL-1β, and consequently increased IL-1β production.251 Finally, autophagy limits IL-1β signalling by promoting degradation of p62 (multi domain protein involved in activation of NFκB) by proteasomes and other lysosomal enzymes.252 SNPs in 32  genes encoding NOD2, IRGM, and ATG16L1 have been implicated in autophagy-related defects in CD. The association of these SNPs in autophagy with CD highlights a crucial role for autophagy in the pathogenesis of CD.     1.5.2 Autophagy related 16-like 1 (ATG16L1)  ATG16L1 is crucial for autophagy in that, it sets the platform for recruitment of the ATG12-ATG5 complex to the immature phagophore membrane, which in turn permits progression, and closure of the membrane to form the autophagosome.253 ATG16L1 is ubiquitously expressed. A non-synonymous SNP in the ATG16L1 gene has been associated with increased risk for developing CD and specifically ileal CD.35-37 The presence of the ATG16L1 gene variant renders ATG16L1 susceptible to cleavage by Caspase 3/7 released during cellular/metabolic stress, death receptor ligation, or bacterial infection, resulting in a reduction in full-length, functional ATG16L1 (Figure 1.6).254,255 The association between the ATG16L1 gene variant and CD has been replicated in more than 50 patient cohorts and the gene variant has been associated with defects in autophagy256 and increased inflammatory responses.257 The ATG16L1 CD-associated SNP, rs2241880, is common in the population, and about 33.2% of people from Western European descent are homozygous for the gene variant.258 Individuals homozygous for the ATG16L1 CD risk allele (GG) have an odds ratio (which is a measure of association of a disease between an exposed and a non-exposed group) of 2.38 (95% confidence interval 1.40-4.04),while heterozygotes (AG) have an odds ratio of 1.86 (95% confidence interval 1.09-3.24),35,259 suggesting that the ATG16L1 CD risk allele increases the risk of developing CD. The ATG16L1 gene variant is denoted as T300A and codes for a threonine (T) to alanine (A) amino acid substitution at position 300.36   33    Figure 1.6   The ATG16L1 T300A-encoding gene variant predisposes people to CD In the absence of the T300A-encoding variant, ATG16L1 is fully functional, and facilitates bacterial clearance during autophagy. The substitution of Alanine (A) for Threonine (T) at position 300 in the protein creates a cleavage site on ATG16L1 for caspase 3/7. ATG16L1 cleavage causes a defect in autophagy and xenophagy (bacterial clearance by autophagy) in the gut, and chronic inflammation. Reproduced with permission of Nature Publishing Group: Murthy A et al, Nature 2014.254,255  ATG16L1 deficiency in mice or homozygosity for the ATG16L1 CD susceptibility gene variant in humans causes defects in Paneth cell granule exocytosis,80 which may be important in susceptibility to disease in response to specific infections and/or environmental influences.73 Moreover, CD patients, who are homozygous for the ATG16L1 CD susceptibility SNP, are unable to adequately clear pathosymbionts and monocytes from subjects with the ATG16L1 T300A-encoding gene variant have impaired ability to clear invading pathogens.260 In humans, defects in autophagy and the ATG16L1 gene variant have also been associated with increased IL-1β production.254,255,261   34  1.5.3 Autophagy protein 5 (ATG5) ATG5 is an autophagy-related protein that forms a complex with ATG12 and ATG16L1 and provides a platform for the formation of the autophagosome.245 ATG5 plays a second role, regulating anti-viral responses. As part of the autophagy protein complex, ATG5 enhances autophagic clearance of viruses262 and antigen presentation, 263 whereas the ATG5-ATG12 complex promotes viral replication by negatively regulating type I IFN production.264-266 The ATG5 SNP, rs510432, enhances ATG5 promoter activity and, together with the ATG5 rs12201458 SNP, has been associated with childhood asthma.267 Besides, ATG5 mRNA expression is increased in nasal epithelial cells of asthma patients, 267 suggesting that the ATG5 SNPs are associated with increased expression of ATG5. Despite its central role in autophagy, it is not known if the ATG5 SNPs cause defects in autophagy.  1.5.4 Immunity-related GTPase family M (IRGM) The immunity-related GTPase family M (IRGM) localizes to the mitochondria and regulates the initial phase of anti-bacterial autophagy and cellular homeostasis.53,268-270 IRGM has been implicated in the regulation of AIEC, 271 Salmonella sp serova-typhimurium, and Mycobacterium tuberculosis infections37,53 by autophagy. Three synonymous SNPs, rs13361189 rs4958847, and rs7714584, within the human IRGM locus around chromosome 5q33.1 have been shown to alter expression levels of IRGM272 and disrupt autophagy.53,270 The IRGM SNPs are associated with increased risk of developing CD, with individuals carrying the risk alleles for the rs13361189, rs4958847, and rs7714584 SNPs having an OR of 1.38, 1.36, and 1.37 respectively, suggesting that the presence of these SNPs increase the risk for developing CD.38,272 In CD patients harboring the IRGM 35  CD-associated genetic variant, miR-196 expression is increased and this down regulates IRGM expression, resulting in decreased autophagy in the inflamed intestinal mucosa.273   1.5.5 Nucleotide-binding oligomerization domain containing 2 (NOD2) The nucleotide-binding oligomerization domain 2, NOD2 (also known as CARD15), is an intracellular PRR, expressed in macrophages, DCs, intestinal epithelial cells,274 and T cells.275 NOD2 is activated by a component of bacterial peptidoglycan, muramyl dipeptide (MDP),276,277 or by viral single stranded RNA (ssRNA),278 which causes activation of the NFκB pathway or the transcription factor, interferon regulatory factor 3 (IRF3), respectively. Polymorphisms in NOD2 were the first SNPs to be associated with CD.32 These CD-associated gene variants in NOD2 are located in the coding region of the gene and are localized in the leucine-rich-repeat (LRR) region of NOD2.32 The odds ratio for CD in the presence of these SNPs is 2-4 for heterozygotes and 20-40 for homozygotes.32,33,279,280 In the presence of the CD-associated NOD2 variants, NFκB activation is reduced in response to stimulation with MDP.281 Along these lines, TLR2-induced activation of NFκB is inhibited by the CD-associated NOD2 variants.282 Ileal CD patients with the NOD2 variants have been shown to express reduced levels of α-defensins in their Paneth cells.283 During bacterial infection, NOD2, which is localized within the cell, recruits ATG16L1 to the plasma membrane at the site of bacterial entry to initiate autophagic degradation of the bacteria.284 In dendritic cells, the degraded bacterial antigens are processed and presented through major histocompatibility complex (MHC) class II to activated CD4+ T cells.256 In all, the CD-associated NOD2 SNPs result in defective autophagic processing of intracellular bacteria, which is believed to account for their contribution to intestinal inflammation.  36  1.5.6 Endoplasmic reticulum stress and autophagy Accumulation of unfolded or misfolded proteins within the ER causes ER stress.285,286 In response to ER stress, cells initiate the unfolded protein response (UPR), a process that facilitates the folding, processing, export, and degradation of proteins emanating from the ER during stress conditions.287,288 Autophagy is also induced downstream of ER stress as an alternative pathway for degradation of misfolded proteins.289-291 Hence, it is evident that autophagy and ER stress may interact at various levels to regulate the cellular stress response. Two CD susceptibility SNPs in genes encoding proteins required for the UPR have been identified, X-box binding protein 1 (XBP1) and orosomucoid 1-like 3 (ORMDL3).49 It is not yet known if they cause defects in autophagy.  1.5.6.1 X-box binding protein 1 (XBP1) The X-box binding protein 1 (XBP1) is a transcription factor that regulates expression of genes crucial for the functioning of the ER, the immune system, and the UPR.292,293 TLR 2, 4, and 5 agonists activate XBP1, enhancing sustained production of inflammatory mediators IL-6 and TNFα.294 XBP1 deficiency in mice leads to spontaneous intestinal inflammation with impaired Paneth cell function, reduced goblet cell number, and increased ER stress, and increased pro-inflammatory responses to microbial pathogens.288 Non-synonymous SNPs within the XBP1 gene locus around chromosome 22q12.1 have been associated with increased risk for IBD.295-297 Carriers of the minor C allele of XBP1 rs35873774 SNP, which is strongly associated with both CD and UC, have an odds ratio of 0.74,298 suggesting that this SNP could play a protective role in IBD. 37  1.5.6.2 Orosomucoid 1-like 3 (ORMDL3) Orosomucoid 1-like 3 (ORMDL3) is expressed ubiquitously and is localized on the ER membrane where it mediates Ca2+ signalling and homeostasis, and regulates protein folding and the UPR.42,299 The ORMDL3 rs2872507 SNP located on chromosome 17q21 have been associated with increased susceptibility to asthma and CD.39,42 Individuals carrying the ORMDL3 CD-associated risk allele have an OR of 1.14 of developing CD.39,42 The ORMDL3 SNP disrupts Ca2+ signalling, which causes the accumulation of misfolded proteins, and subsequently ER stress that initiates the UPR.300,301 In children with the ORMDL3 SNP, IL-17 secretion is increased in peripheral blood mononuclear cells (PBMCs).302 All of these studies demonstrate that ORMDL3 is involved in many pathways in the ER that regulate inflammation.    1.6 Src homology 2 domain-containing inositol polyphosphate 5´-phosphatase 1.6.1 Description and function The src homology 2 domain-containing inositol polyphosphate 5΄- phosphatase (SHIP) is a hematopoietic-specific lipid phosphatase that negatively regulates class I phosphatidylinositol 3-kinase (PI3K) activity.  SHIP is also expressed in osteoblasts and mesenchymal stem cells.303,304 The human gene encoding the 145kDa SHIP protein (INPP5D) is located at chromosome 2q37.1.305 Two other SHIP isoforms exist, the 150kDa SHIP2 that is similar in structure and biochemical function to SHIP,306-308 and the 104kDa sSHIP, which lacks the SH2 domain. SHIP2 is ubiquitously expressed and is seen in high levels in human skeletal muscles, placenta and heart.309 sSHIP is restricted to murine hematopoietic and embryonic stem cells.306,310 38  PI3Ks are a family of enzymes that are critical in cellular processes including cell growth, differentiation, proliferation, and inflammation.311,312  These enzymes phosphorylate the 3´position of the inositol ring of phosphatidylinositol-4,5-bisphosphate PI(4,5)P2 to generate PI(3,4,5)P3, a critical second messenger. PI3Ks can be grouped into three main classes, class I, II, and III, based on their substrates, molecular structures, and regulation within the cell.311,313 Class I PI3Ks are heterodimeric enzymes: Class IA is composed of 1 of 5 regulatory subunits, p50α, p55α or p55γ, p85α, p85β, and 1 of 3 catalytic subunits, p110, p110, or p110; and Class IB is composed of 1 of 2 regulatory subunits, p87 or p101, and the catalytic subunit, p110.  p110 and p110 are ubiquitously expressed whereas p110 and p110are mainly restricted to hematopoietic cells.312  PI3Kp110 catalytic subunits have overlapping as well as unique functions downstream of specific receptor tyrosine kinases, growth factor, cytokine, and TLRs.314 Class II PI3K is membrane bound, usually activated by tyrosine kinases and integrins,315,316 and is involved in cell migration.317 Class III PI3K consists of a single catalytic subunit Vps34 and a regulatory subunit Vps15, and has been implicated in autophagy.318 Class II PI3K catalyzes the phosphorylation of PI and PIP to PI(3)P and PI(3,4)P 2,318 whereas class III PI3K only catalyzes the production of PI(3)P from PI.313  1.6.2 SHIP enzymatic activity  To exert its action, SHIP is translocated from the cytoplasm, where it resides, to the site of synthesis of PI(3,4,5)P3 at the inner leaflet of the cell membrane through association with adaptor and scaffold proteins and/or direct binding of its SH2 domain. SHIP antagonizes class I PI3K signalling by dephosphorylating the 5' position of the inositol ring 39  to form PI(3,4)P2 (Figure 1.5).305 PI(3,4,5)P3 recruits serine-threonine kinases, such as the serine/threonine protein kinase, Akt, and PDK1 to the plasma membrane,311 driving cellular processes, such as growth, proliferation, differentiation, and immune activation.319 SHIP therefore blocks recruitment of Akt and PDK1; and inhibits downstream cellular processes, such as cytokine production and inflammation (Figure 1.7).320    Figure 1.7   SHIP negatively regulates the immune response Ligation of receptor tyrosine kinases (RTKs), cytokine receptors (cytokine Rs), growth factor receptors (GFRs), G protein-coupled receptors (GPCRs), and TLRs activate Class I PI3K, which is comprised of a p110 catalytic and a p85 regulatory subunit. Class I PI3K phosphorylates PI(4,5)P2 to produce the second messenger PI(3,4,5)P3. SHIP dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2, and blocks cellular processes, such as growth, proliferation, differentiation, and immune activation. PTEN reverses the action of class I PI3K dephosphorylating PI(3,4,5)P3 to PI(4,5)P2.  In addition, SHIP has also been shown to promote activation of Akt via production of PI(3,4)P2,321,322 suggesting that SHIP can have either an inhibitory or enhancing role in cell signalling and inflammation. SHIP also regulates cell signalling events independent of its enzymatic activity, by binding to DNAX-activating protein of 12kD (DAP12), blocking 40  TREM2- (Triggering receptor expressed on myeloid cells 2) and DAP12-induced signalling in macrophages, and osteoclasts.323  SHIP can be regulated either at the level of transcription or post-transcriptionally.324 Transcriptionally, Activin and TGFβ up regulate SHIP mRNA expression in both human and mouse cells,325 while microRNA-155 (miR-155) negatively regulates SHIP expression  during physiological conditions through direct 3΄ UTR interaction.326 SHIP protein is up regulated by LPS/CpG-induced TGFβ via SMAD4,327 while SMAD7, which blocks TGFβ activity by competing locally with SMAD2/3 for TGFβ receptor,328 may reduce SHIP expression. On the other hand, miR-155-induced reduction in SHIP protein levels result in increased pro-inflammatory cytokine production.329 Post transcriptionally, IL-4 induces SHIP protein degradation in macrophages.330 Studies have also shown that, tyrosine phosphorylation of SHIP, reduces SHIP protein levels through polyubiquitination and proteasomal degradation.330   1.6.3 The SHIP deficient mouse The SHIP deficient (SHIP-/-) mouse was developed in 1998 by deleting the first exon of SHIP.331 These mice are relatively smaller in size than their wild type counterparts because they fail to thrive, they have asthmatic-like lungs, and reduced lifespan, and suffer from splenomegaly and myeloproliferative disorder (characterized by abnormal growth of blood cells; red blood cells, white blood cells, and platelets).331 Moreover, SHIP-/- mice have increased numbers of Gr-1+Mac-1+ myeloid suppressor cells332 and reduced B cell numbers333 compared to SHIP+/+ mice.  41  SHIP-/- macrophage responses to microbial pathogens vary, and depend on whether they are derived in vivo or in vitro. In vivo derived SHIP-/- peritoneal and alveolar macrophages are profoundly M2 skewed,334 and are hyper-responsive to cytokine, growth factor, and chemokine stimulation compared to SHIP+/+ macrophages.331,335 These macrophages constitutively express high levels of the M2 markers, Arginase I (Arg I) and Ym1.334 When stimulated with LPS, SHIP-/- peritoneal and alveolar macrophages secrete low levels of IL-6, TNFα, IL-12, and nitric oxide (NO), and high levels of the anti-inflammatory cytokines IL-10 and TGF-β,334 confirming their M2 properties. In contrast, in vitro derived SHIP-/- bone marrow macrophages (BMMs) produce high levels of TNFα, IL-6, IL-1β, and NO when stimulated with LPS.327 Studies have shown that, SHIP-/- BMMs derived in vitro in standard culture medium (MCSF), develop an M1 phenotype whereas, when these macrophages are derived in standard culture medium in the presence of mouse or human serum, IL-10, or TGFβ, or when derived in GM-CSF or IL-3, they develop an M2 phenotype.334 GM-CSF and IL-3 skew SHIP-/- macrophages to an M2 phenotype because they act on basophils, which use STAT5 to drive transcription of IL-4.336 IL-4 in turn acts through type I/II receptors on macrophages, leading to phosphorylation, dimerization, and translocation of STAT6 to the nucleus, where it drives transcription of STAT6 responsive genes, and skews macrophages to an M2 phenotype.336   1.6.4 The SHIP-/- mouse model of Crohn’s disease-like intestinal inflammation SHIP-/- mice develop spontaneous CD-like ileal inflammation beginning at the age of 4 weeks.337 Inflammation, which is present in all SHIP-/- mice from the age of 6 weeks onwards is restricted to the distal part of the ileum and is characterized by increased 42  Th2/Th17 cytokines, massive granulocyte infiltration, increased collagen deposition, and fibrosis, thickened muscularis, goblet cell hyperplasia, and the presence of immune cell aggregates that resemble poorly formed granulomas.337,338 There is a paucity of T cells in the inflamed mucosa of SHIP-/- mice, suggesting that T cells might not play an important role in onset of inflammation.338 Furthermore, both arginase-1 expression and activity are increased in the SHIP-/- mouse ilea compared to their wild type littermates.337  1.6.5 The role of SHIP in inflammation The role of SHIP in inflammation has been studied extensively in mice, with very limited studies done in humans. SHIP has been shown to play a critical role in regulating immune homeostasis. It acts in concert with other negative regulators of inflammation, such as the interleukin 1 receptor-associated kinase M (IRAK-M), Suppressor of cytokine signalling proteins 1 and 3 (SOCS1 and SOCS3), and the spliced variant of myeloid differentiation primary response gene (MyD88s), to promote endotoxin tolerance.327 SHIP has been shown to play a critical role in mast cell biology in that, it sets the threshold,339 and reduces adhesion, degranulation, and cytokine production by mast cells.340 Moreover, SHIP enhances neutrophil apoptosis,341 decreases B cell proliferation, chemotaxis, and activation.342-344 Furthermore, SHIP-/- B cells have reduced FcRγIIB inhibitory signals.342 SHIP has also been shown to be involved in NK cell physiology. Studies have shown that, SHIP inhibits FCγRIII signalling345 and promotes NK cell effector function by enhancing IFNγ production.346 In addition, SHIP promotes T cell survival and maintains innate immune balance at mucosal surfaces.347 SHIP deficient mice have reduced CD4+ and CD8+ T cell numbers in the small intestine.338 Moreover, myeloid cells from SHIP deficient mice are 43  hyper-proliferative,331 and when recruited to the intestinal mucosa secret numerous inflammatory mediators and proteolytic enzymes that promote tissue destruction and fibrosis.348  Regulation of SHIP expression levels and activity could therefore be an important strategy to limit chronic inflammation. In humans, SHIP mRNA expression has been shown to positively correlate with both FOXP3 and IL-10 mRNA expression, suggesting that, SHIP may be an important regulator of Treg function.349  1.7 Thesis hypothesis and objectives 1.7.1 Summary of rationale SHIP negatively regulates NFκB transcription and pro-inflammatory cytokine production. As such, SHIP-/- macrophages are hyper-responsive to immune stimuli including those found on commensal microorganisms in the gastrointestinal tract, such as LPS, and secrete high levels of IL-6 and TNFα.314,350 Moreover, SHIP-/- mice develop spontaneous CD-like intestinal inflammation that is restricted to the distal ileum.323,337 Il-1β is a critical mediator of pro-inflammatory cytokine and chemokine production since it acts on cells in an autocrine fashion to amplify its own production351,352 and drives auto-inflammation.170 The goal of Chapter 2 was to investigate the role of SHIP in macrophage IL-1β production. I also determined whether depleting intestinal macrophages was sufficient to prevent intestinal inflammation in the SHIP-/- mouse. Furthermore, I used an IL-1 receptor antagonist, Anakinra, to block IL-1 signalling to determine its role in intestinal pathology in SHIP deficient mice.  44  In humans SHIP mRNA expression is increased in inflamed colonic biopsies from people with ulcerative colitis and colonic CD, but strangely, not in ileal biopsies from subjects with ileal CD.349 The human gene encoding SHIP protein (INPP5D) is located at chromosome 2q37.1 and is upstream of the gene encoding ATG16L1.338 The goal of Chapter 3 was to determine whether SHIP mRNA expression and activity were altered in CD subjects compared to control subjects, who did not have IBD. I also determined whether SHIP mRNA expression and activity were regulated by ATG16L1, and if SHIP may contribute to increased IL-1 production in subjects with the ATG16L1 risk variant.    Humans carrying the ATG16L1, NOD2, IRGM, or XBP1 CD gene variant have increased susceptibility to CD, and macrophages from subjects homozygous for the ATG16L1 T300A-encoding gene variant have increased ability to produce IL-1.261  To clearly understand the role of autophagy in regulating SHIP expression and IL-1 production, I genotyped healthy control subjects for five autophagy and CD-related genetic variants, and determined their effects on IL-1 and other pro-inflammatory cytokine production in PBMCs in response to different PRR ligands that are present on commensal bacteria in the gut.    1.7.2 Hypothesis and objectives I hypothesize that SHIP deficiency leads to increased macrophage-derived IL-1 production that may cause, or contribute to, intestinal inflammation in people with CD. Aim 1 (Chapter 2): To determine the cause of intestinal inflammation in SHIP-/- mice and to examine the role of macrophage-derived IL-1β in intestinal pathology. 45  Aim 2 (Chapter 3): To determine whether SHIP mRNA expression and activity are regulated by ATG16L1 and autophagy, and how this affects IL-1 production in human cells. Aim 3 (Chapter 4): To determine the effects of autophagy related CD-associated gene variants on IL-1 and other pro-inflammatory cytokine production in human cells.  1.7.3 Significance These studies contribute to our understanding of the mechanisms by which ATG16L1/autophagy regulate IL-1β production and intestinal auto-inflammation, by suggesting that this may occur via SHIP up-regulation. This work also helps to identify a sub-group of CD patients with a particular genotype (homozygosity for ATG16L1 T300A-encoding gene variant, or the presence of the XBP-1 gene variant), who may be amenable to treatment with Anakinra (IL-1Ra). Moreover, these studies suggest that Anakinra could be used as a prophylactic treatment to prevent relapse in CD subjects, who are homozygous for the T300A-encoding gene variant.          46  Chapter 2: SHIP deficiency leads to increased IL-1β transcription in macrophages and intestinal autoinflammation in mice  2.1 Introduction and rationale Crohn’s disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by chronic, relapsing and remitting, or progressive inflammation along the gastrointestinal tract.353  IBD affects up to 1 in 150 people in Canada and incidence of disease is increasing in developed countries.10,353  Although the etiology of disease remains unknown, current thinking is that CD and IBD occur in genetically susceptible individuals due to an inappropriate initiation or perpetuation of an immune response to intestinal flora.106,354  Biological therapy, monoclonal antibodies directed against the pro-inflammatory cytokine TNF, is effective at inducing and maintaining remission in patients and has revolutionized the treatment for CD.355  Despite that, some patients are refractory to biological therapy and for others; biological therapy becomes ineffective because they develop antibodies to the drug.138,356    The pro-inflammatory cytokine, IL-1, acts as an alarm cytokine, initiating the inflammatory response, thus its production is tightly regulated by a two-step process.  In the first step, toll-like receptor (TLR) or endogenous ligands induce transcription of IL1B, which is translated to pro-IL-1, an inactive precursor that resides in the cell cytosol.  In the second step, numerous stimuli, typically danger associated molecular patterns, lead to assembly of the inflammasome, a hetero-oligomeric protein complex.  This complex includes one of several NOD-like receptors (NLRs), the apoptosis-associated speck-like protein (ASC) adaptor, and the zymogen, pro-caspase-1, which is cleaved, activated, and catalyzes the 47  processing of pro-IL-1, leading to its activation and secretion.190-192  Monogenic gain of function mutations leading to increased IL-1 production cause a group of autoinflammatory diseases known as periodic fever syndromes, which can be treated with anakinra, an IL-1 receptor antagonist.190,353  Intestinal inflammation is a common complication of canonical monogenic autoinflammatory diseases as well as primary immune deficiencies characterized by increased IL-1 production.353,357-359  IL-1 antagonism has been used effectively to treat some genetically defined forms of very early onset IBD360 and may be more broadly applicable for the treatment of sub-groups of IBD.    Based on previous findings from our group and others showing that SHIP-/- mice developed spontaneous CD-like intestinal inflammation, I investigated the cause of intestinal inflammation in SHIP-/- mice and determined the role and contribution of SHIP deficient macrophages to pathology.    2.2 Materials and methods Mice. Mice heterozygous for SHIP expression (Inpp5d+/-) and derived from an F2 generation of C57BL/6 X 129Sv mice were used to generate SHIP+/+ and SHIP-/- littermates for experiments. The mice were maintained in sterilized filter-top cages and were housed in the Animal Research Centre at the Child & Family Research Institute, which is specific pathogen- and Helicobacter-free.  Mice used for experiments were between 4 and 8 weeks of age and were fed with autoclaved food and water under specific pathogen-free conditions.  All experiments were performed in accordance with institutional and Canadian Council on Animal Care guidelines.  48  Subjects with Crohn’s disease and control subjects. Experiments were performed in accordance with ethical guidelines and with approval by the University of British Columbia Research Ethics Boards (protocol number H09-01826).  Subjects seen in the Division of Gastroenterology at BC Children’s Hospital were recruited into the study.  No subjects had been previously diagnosed with, or treated for, IBD or other inflammatory pathology.  Four ileal and four colonic biopsies were taken from sites of inflammation that were adjacent to tissues harvested for pathological assessment in subjects with CD.  Biopsies were taken from comparable, uninflamed sites in subjects, who were not subsequently diagnosed with IBD.  Peripheral blood was taken from the site of intravenous insertion during colonoscopy.  Diagnosis of CD with ileal inflammation or no disease was based on pathological assessment and colonoscopy.  Eight subjects diagnosed with ileal CD (no colonic involvement) and 14 subjects, who did not have IBD, were included in analyses. Biopsies were fixed for H&E staining or used immediately for analyses.  PBMCs were isolated by density gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare, Piscataway, NJ).  PBMCs were washed and resuspended at 0.5×106 cells/mL in IMDM/10% FBS for assays.    Macrophage derivation and isolation. Bone marrow macrophages (BMMфs) were generated by flushing out bone marrow aspirates from femura and tibiae of SHIP+/+ and SHIP-/- mice. Aspirates were resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum (FBS) in 75 cm2 Falcon flasks (BD Biosciences) for 1 hr. Following adherence depletion, bone marrow aspirates were then resuspended in IMDM, 10% FBS, and penicillin/streptomycin at a concentration of 0.5×106 cells/mL for 10 days in the presence of 10 ng/mL monocyte colony-stimulating factor (MCSF), granulocyte-49  monocyte colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) (StemCell Technologies, Vancouver, BC), with complete media changes at day 4 and 7. BMMф cultures were >95% Mac-1+ and F4/80+ after 10 days in culture. Peritoneal macrophages were isolated by lavaging the peritoneal cavity with 3×5 mL of complete medium containing 10 ng/mL MCSF.  To isolate ileal macrophages, lamina propria cells were prepared from whole mouse ilea, as follows: longitudinal sections of SHIP+/+ and SHIP-/- ilea were cut and rinsed with a solution of phosphate-buffered saline (PBS) containing 5% fetal calf serum (FCS), to wash off luminal contents. Ileal sections were further cut into smaller sections and washed for 3 x 15 min each, after which, tissue were shaken at 37oC for 1.5 h in a solution of PBS containing 5% FCS and 2mM ethylenediaminetetraacetic acid (EDTA). The solution was replaced every 20 min. To remove EDTA, tissue sections were washed 2 x 5 min with wash solution. Next, tissue was incubated in collagenase digestion media comprised of 20 ml RPMI (Roswell Park Memorial Institute medium), 5% FCS and 1 mg/ml collagenase (Worthington, New Jersey) at 250 rpm for 40 min at 37oC. Digested fractions were pooled and cells were selected using the mouse monocyte enrichment kit (StemCell Technologies).  Macrophage populations were ≥ 95% F4/80+ and Mac-1+.   Cell stimulations. Cells were plated at a density of 0.5×106 cells/mL and stimulated with 10 ng/mL of LPS (E. coli serotype 127:B8, Sigma Aldrich, St. Louis, MO) for 5 h, 5 mM ATP for 1 h, or LPS for 5 h with addition of ATP for the final 1 h.  After incubation, cell supernatants were harvested and clarified by centrifugation for analysis.  Inhibitors were added to cultures 30 min prior to addition of LPS or 30 min prior to addition of ATP, where indicated.  Commercially available inhibitors, controls, and final concentrations of each 50  were: glybenclamide (100 M, Sigma Aldrich), Z-YVAD-fmk (40 M, Sigma Aldrich), LY303511 (14 M, Calbiochem, San Diego CA), LY294002 (14 M, Calbiochem), DMSO (dimethyl sulphoxide) (0.1%), and wortmannin (50 nM, Calbiochem).  Isoform-specific class I PI3K inhibitors were synthesized by our collaborator as described previously and used at a concentration of 10 M.361,362   Cytokine measurements. Cytokine measurements were performed on clarified full thickness tissue homogenates from mice or cell-free tissue culture supernatants using the Mouse Cytokine Array Panel A kit (R&D Systems; Minneapolis, MN) or by enzyme-linked immunosorbent assay (ELISA). During an ELISA, cell-free supernatants are incubated with a primary antibody (capture antibody) in an ELISA plate to immobilize the antigen (Ag). Next, the Ag-Ab complex is incubated with a secondary Ab (detection Ab) that links to an enzyme. The conjugated enzyme activity is detected by incubation with a substrate solution that produces a signal, which is read as absorbance in an ELISA plate reader. The concentration of the targeted Ag is then determined from the absorbance. For Mouse Cytokine Array, ImageJ version 1.43 and GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA) were used to quantify spot densities, which were further corrected for individual background to reduce variations. ELISA kits for mouse IL-1, IL-6, and human IL- were from BD Biosciences (Mississauga, ON). The mouse IL-18 ELISA kit was from MBL International (Woburn, MA).  Gene Expression Analysis. RNA was prepared from mouse tissue or cells using the NucleoSpin RNA II Total RNA Isolation Kit (Macherey-Nagel, Bethlehem, PA) and reverse 51  transcribed using Superscript II (Invitrogen, Burlington, ON).  Gene expression was measured by quantitative polymerase chain reaction (qPCR) using the AB Applied Biosystems Taqman Universal Master Mix II (Invitrogen).  IL-1 (IL1B) gene expression was normalized to gene expression for ribosomal protein RPLP0 (Rplp0).  Primer and probe sequences were: IL1B forward 5'-ACGGACCCCAAAAGATGAAG-3' IL1B reverse 5'-TTCTCCACAGCCACAATGAG-3' IL1B probe 5'-/56-FAM/AGAGCATCC/ZEN/AGCTTCAAATCTCGCA/3IABkFQ/-3' Rplp0 forward 5'-TGACATCGTCTTTAAACCCCG-3' Rplp0 reverse 5'-TGTCTGCTCCCACAATGAAG-3' Rplp0 probe 5'-/56-FAM/TGTCTTCCC/ZEN/TGGGCATCACGTC/3IABkFQ/-3'  Macrophage Depletion. Macrophages were depleted from mouse ilea using clodronate-containing liposomes (clod-lip). 200 l of Clod-lip or PBS injection controls were administered to SHIP-/- mice intraperitoneally every two days between 4 and 6 weeks of age.  Mice were euthanized at 6 weeks of age, ilea were photographed, sections were fixed for histological analyses, and full thickness tissue homogenates were prepared for cytokine analyses.  Anakinra treatment. Anakinra was injected intraperitoneally into mice daily at a dose of 150 mg/kg between 4 and 6 weeks of age.  An equal volume of PBS was injected into mice as an injection control.  Mice were euthanized at 6 weeks of age, ilea were photographed, sections were fixed for histological analyses, and full thickness tissue homogenates were 52  prepared for cytokine analyses.  Histological analyses. Ileal and colonic biopsies from human subjects and ileal sections from SHIP+/+ and SHIP-/- mice were fixed in PBS-buffered 10% formalin at 4°C for 24 h.  Tissue sections were embedded in paraffin, and 5 m cross-sections were cut and stained with hematoxylin and eosin (H&E).  Images were acquired using a Zeiss Axiovert 200 microscope, a Zeiss AxiocamHR camera, and the Zeiss Axiovision 4.0 software imaging system.  Immune cell infiltrates were counted at 20× magnification in six H&E stained sections separated by ≥50 m, by two individuals blinded to experimental condition.  Crypt/villus length (mouse) was determined by counting epithelial cell nuclei from the base of the crypt to the villus tip on uniform horizontal ileal cross-sections.  Representative crypt/villi (10 per section) were counted in six H&E stained ileal sections for each mouse. For macrophage staining, slides were mounted and stained with F4/80. Samples were incubated with 20 μg/mL proteinase K in PBS for 15 min at room temperature and rinsed in Tris-buffered saline with 0.1% Tween 20.  Endogenous peroxidase activity was blocked with 1.5% hydrogen peroxide (H2O2) in PBS for 10 min and endogenous avidin and biotin were blocked with an avidin-biotin blocking kit, according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA).  Rat anti-F4/80 IgG (Immunoglobulin G) (AbD serotec, Oxford, UK) was the primary antibody used.  Blocking buffers, biotinylated anti-rat IgG secondary and avidin-biotin-horseradish peroxidase detection complex were prepared and used from immunohistochemistry detection kits according to the manufacturer’s instructions (Vector laboratories).  Signal was detected with a diaminobenzidine chromogen system (Dako, Carpinteria, CA) and developed sections were counter stained with Harris’ 53  hematoxylin (Sigma, St. Louis, MO).  Macrophages were counted at 20× magnification at six points in six H&E stained sections separated by ≥50 m by two individuals blinded to experimental condition. For detection of active caspase-1, F4/80 stained slides were co-stained with YVAD-FLICA (a fluorescent labeled irreversible inhibitor of caspase-1) immediately prior to counterstaining with Harris’ hematoxylin (ImmunoChemistry Technologies, Bloominton, MN).  YVAD-FLICA was resuspended in blocking buffer and tissue sections were stained for 1 h at room temperature in the dark.  Tissue sections were thoroughly rinsed and then stained with DAPI (4΄ 6-diamino-2-phenylindole) (Invitrogen).  Statistical Analyses. Unpaired two-tailed Student’s t tests were performed when indicated using GraphPad Prism version 5 software.  For multiple comparisons, the Bonferroni correction was applied.  Differences were considered significant at P < 0.05.  2.3 Results 2.3.1 Ileal macrophages from SHIP-/- mice produce high levels of IL-1 and IL-18  Our laboratory recently reported that SHIP-/- mice develop spontaneous CD-like intestinal inflammation.337 We found that full thickness tissue homogenates from SHIP-/- mice did not have elevated levels of pro-inflammatory cytokines expected, including IFN, IL-12p70, IL-23, and TNF337  However, we noted that protein levels of IL-1 family cytokines, IL-1, IL-1, and IL-1ra, were higher in SHIP-/- mice relative to their wild type littermates (Figure 2.1A).  Based on this, we measured absolute values of IL-1 and IL-18 in 54  full thickness ileal tissue homogenates from 8-week-old SHIP+/+ and SHIP-/- by ELISA and found that they were significantly higher in SHIP-/- mice (Figure 2.1B).  To investigate the cellular source of IL-1 and IL-18 in ileal tissues, fixed tissue cross sections were co-stained with YVAD-FLICA, which stains active caspase-1, and F4/80, which is a macrophage marker.  YVAD-FLICA+ cells were found in the sub-epithelial region of the distal tips of villi in SHIP-/- mice and co-stained with F4/80 (Figure 2.1C). To determine the concentration of LPS and the time point for ATP stimulation, GM-CSF-derived bone marrow macrophages were either stimulated with different concentrations of LPS for 4 hours followed by addition of ATP for 1 hour (left) or were stimulated with LPS for 4 hours followed by addition of ATP at different time points. We found that SHIP-/- macrophages produced significantly more IL-1β compared to wild type macrophages at a high concentration of LPS (10 ng/ml) and when ATP was added after 4 hours (Figure 2.2A).   To determine whether this was a cell-intrinsic effect of SHIP deficiency in macrophages, we isolated ileal macrophages from SHIP+/+ and SHIP-/- mice and stimulated them with LPS (5 h), to induce IL-1 and IL-18 transcription, ATP (1 h), to induced inflammasome activation, or LPS (5h) + ATP (for the last hour), to induce secretion of mature IL-1 and IL-18, and we measured IL-1 and IL-18 in cell culture supernatants.  Unlike their wild type counterparts, SHIP-/- ileal macrophages secreted IL-1 and IL-18 in response to LPS alone, consistent with staining for active caspase-1 observed, and SHIP-/- ileal macrophages secreted significantly more IL-1 and IL-18 in response to stimulation with LPS+ATP (Figure 2.2B).         55   Figure 2.1   IL-1β production is increased in ileal sections from SHIP-/- mice compared to SHIP+/+ mice. Full thickness ileal tissue homogenates from 8-week-old SHIP+/+ and SHIP-/- mice were assayed for IL-1 family members by cytokine array (A) and by ELISA (B). (C) Fixed ileal sections were co-stained with anti-F4/80 for macrophages and with YVAD-FLICA for active caspase-1. Data are representative of sections from 6 mice/group. Data are means SD for n=4 mice/group in (A) and n=6 mice/group in (B).  *P<0.05, **P<0.01 (unpaired Student’s t-test). 56    Figure 2.2   SHIP-/- ileal macrophages produce more IL-1β and IL-18 than wild type ileal macrophages. (A) GM-CSF-differentiated bone marrow macrophages were stimulated with different concentrations of LPS for 4 hours followed by addition of ATP (5mM) for 1 hour (left) or stimulated with 10ng/ml LPS for 4 hours followed by addition of ATP (5mM) at different time points (right). (B) Ileal mononuclear phagocytes were isolated and stimulated with LPS, ATP, or LPS+ATP.  Cell supernatants were assayed for IL-1 or IL-18 by ELISA.  Data are means SD for n=6 mice/group in (A) and (B).  **P<0.01 (unpaired Student’s t-test).  2.3.2 In vivo differentiated SHIP-/- macrophages produce high levels of IL-1 and IL-18 We next sought to establish an in vitro culture model to investigate the mechanism(s) for increased IL-1 and IL-18 secretion by SHIP-/- macrophages.  SHIP+/+ and SHIP-/- bone marrow progenitors were differentiated into macrophages in the presence of macrophage 57  growth factors, MCSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), or IL-3.  Macrophages were stimulated with LPS, ATP, or LPS+ATP and IL-1 was measured in culture supernatants.  Surprisingly, SHIP-/- bone marrow macrophages differentiated in the presence of MCSF or IL-3 did not produce more IL-1 and IL-18 than SHIP+/+ macrophages in response to stimulation.  Moreover, although GM-CSF-derived SHIP-/- macrophages produced significantly more IL-1 and IL-18 than those from SHIP+/+ mice, the effect on IL-1β production was modest compared to the differences observed for ileal macrophages (Figure 2.3A).  Given that SHIP-/- macrophages may be influenced by the complex inflammatory environment in the SHIP-/- ileum, we treated GM-CSF-derived macrophages from SHIP+/+ and SHIP-/- mice for 3 days with IFN, to induce a classically activated phenotype, IL-4, (elevated in SHIP-/- ilea)337 to induce an alternatively activated phenotype, or IFN+IL-4.  Resulting macrophages were then stimulated with LPS, ATP, or LPS+ATP.  IFN and (IFN+IL-4)-treated, GM-CSF-derived SHIP-/- macrophages produced significantly more IL-1 than SHIP+/+ macrophages in response to LPS+ATP (Figure 2.3B).  However, these in vitro culture systems did not replicate the dramatic differences in IL-1 secretion observed comparing SHIP+/+ and SHIP-/- ileal macrophages.  Reasoning that differences observed may be due to in vivo differentiation in the complex inflammatory environment in the SHIP-/- mouse, peritoneal macrophages were isolated from SHIP+/+ and SHIP-/- mice and IL-1 and IL-18 secretion were assayed in response to stimulation.  IL-1 and IL-18 secretion were significantly higher in SHIP-/- peritoneal macrophages compared to SHIP+/+ peritoneal macrophages (Figure 2.4A and B, left).  Both peritoneal and bone marrow-derived macrophages from SHIP+/+ 58  and SHIP-/- mice were also stimulated with LPS+ATP in the presence of glybenclamide, to inhibit ATP-induced potassium efflux required for inflammasome activation, or YVAD, to inhibit caspase-1.  Glybenclamide and YVAD blocked (LPS+ATP)-induced IL-1 and IL-18 secretion by SHIP-/- macrophages as effectively as in wild type macrophages (Figure 2.4A and B, middle and right panels).            59   Figure 2.3   In vivo differentiated SHIP-/- macrophages produce high levels of IL-1β and IL-18.   (A) Macrophages were differentiated from SHIP+/+ and SHIP-/- bone marrow for 10 days in the presence of 10 ng/mL of macrophage growth factors, MCSF, GM-CSF, or IL-3.  Macrophages were unstimulated or stimulated with LPS, ATP, or LPS+ATP and supernatants were assayed for IL-1β and IL-18 by ELISA.  (B) 10-day-old GM-CSF-derived bone marrow macrophages were treated for 3 days with 10 ng/mL of IFN, IL-4, or IFN+IL-4, washed, and stimulated with LPS, ATP, or LPS+ATP.  Supernatants were assayed for IL-1 by ELISA. Data are means SD for n=4 in (A) and (B). *P<0.05 (unpaired Student’s t-test). 60   Figure 2.4   IL-1β and IL-18 production are dependent on the NLRP3 inflammasome and caspase-1 activity.   Peritoneal macrophages were harvested from 8-week-old SHIP+/+ and SHIP-/- mice.  Macrophages were differentiated from SHIP+/+ and SHIP-/- bone marrow for 10 days in the presence of 10 ng/mL of GM-CSF. Cells were stimulated with LPS, ATP, or LPS+ATP (left panels).  Cells were also stimulated with LPS+ATP ± glybenclamide (NALP3 inhibitor; middle panels) or YVAD (caspase-1 inhibitor; right panels).  Cell supernatants were assayed for IL-1 (A) or IL-18 (B).  Data are means SD for n=6.  *P<0.05, **P<0.01 (unpaired Student’s t-test). 61  2.3.3 Class I PI3Kp110drives transcription of IL1B SHIP is a critical negative regulator of class I PI3Ks.  Thus, to investigate the mechanism by which SHIP deficiency increases (LPS+ATP)-induced IL-1 and IL-18 secretion, SHIP-/- peritoneal macrophages were stimulated with LPS+ATP in the presence of pan PI3K inhibitors, LY294002 (LY29) or wortmannin (Wm), or their respective controls, LY303511 (an inactive structural analogue) or DMSO (vehicle control).  Inhibitors were added to cultures 30 min prior to stimulation with LPS or 30 min prior to addition of ATP, which is added for the last hour of stimulation to activate the inflammasome.  LY29 and Wm blocked production of IL-1 when added to cultures prior to stimulation with LPS but did not block IL-1 production when added to culture prior to addition of ATP suggesting that class I PI3K is required for LPS-induced transcription of IL1B, but not for inflammasome activation by ATP (Figure 2.5A).  To determine which class I PI3K(s) was required for (LPS+ATP)-induced IL-1 secretion, inhibitors that target specific catalytic isoforms of PI3K, p110, , , and , were added to peritoneal macrophages prior to stimulation with LPS.  Class I PI3K p110 inhibitors, PIK-90 and PI-103, reduced (LPS+ATP)-induced IL-1 secretion, whereas inhibitors for other isoforms had no effect (Figure 2.5B).  To determine whether SHIP-/- peritoneal macrophages have increased LPS-induced transcription of IL1B, SHIP+/+ and SHIP-/- peritoneal macrophages were stimulated with LPS, ATP, or LPS+ATP, and total RNA was harvested for analysis of IL1B gene expression.  Peritoneal macrophages from SHIP-/- mice had higher gene expression for IL1B relative to SHIP+/+ in response to LPS or LPS+ATP (Figure 2.6A, left).  In addition, IL1B gene expression was elevated in ileal tissues from SHIP-/- mice compared to wild type littermates, where it was not detected (Figure 2.6A, 62  right).  Finally, to determine whether PI3K and specifically PI3Kp110 is required for LPS-induced IL1B gene expression, IL1Bgene expression was measured in SHIP+/+ and SHIP-/- peritoneal macrophages stimulated with LPS in the presence of pan-PI3K inhibitors, LY29 and Wm, PI3Kp110 inhibitors, PIK-90 and PI-103, or controls.  As expected, pan- and p110-specific PI3K inhibitors reduced IL1B transcription in peritoneal macrophages from both SHIP+/+ and SHIP-/- mice (Figure 2.6B).              63   Figure 2.5  IL-1β production is dependent on class I PI3K p110α activity  SHIP-/- peritoneal macrophages were stimulated with LPS for 4 hours followed by addition of ATP for 1 h ± pan-PI3K inhibitor, LY294002 (LY29), its inactive analogue LY305311 (LY30), pan-PI3K inhibitor, wortmannin (Wm), or DMSO, as a vehicle control.  PI3K inhibitors or controls were added to cultures prior to LPS (left) or 30 min prior to ATP (right) and cell supernatants were assayed for IL-1 by ELISA. (B)SHIP-/- peritoneal macrophages were stimulated with LPS+ATP ± isoform-specific inhibitors for class I PI3Ks or DMSO (vehicle).  IL-1 was assayed in cell supernatants by ELISA. Data are means SD for n=6 in (A) and n=4 in (B)-(D).  NS=not significantly different, *P<0.01, **P<0.0001 (unpaired Student’s t-test).    64  Figure 2.6   SHIP deficiency increases transcription of IL-1β (A) SHIP+/+ and SHIP-/- peritoneal macrophages were stimulated with LPS, ATP, or LPS+ATP.  RNA was isolated from cells and IL-1 transcription was assayed by Q-PCR (left).  RNA was isolated from ileal tissues of 6-week-old SHIP+/+ and SHIP-/- mice and IL-1 transcription was assayed by Q-PCR (right).  (B) SHIP+/+ (left) and SHIP-/- (right) peritoneal macrophages were stimulated with LPS+ATP ± pan-PI3K inhibitors, LY29 or Wm, or PI3Kp110 isoform-specific inhibitors, PIK-90 or PI-103, or controls, LY30 or DMSO. RNA was isolated from cells and IL-1 transcription was assayed by Q-PCR. Data are means SD for n=4. *P<0.01 (unpaired Student’s t-test).  2.3.4 Macrophage depletion reduces intestinal inflammation in SHIP-/- mice  To understand the contribution of macrophages to intestinal inflammation in SHIP-/- mice, we treated SHIP-/- mice with clodronate-containing liposomes (Clod-lip) to deplete macrophages, or injection controls, for two weeks during the development of disease and assessed pathology in mice after treatment.  SHIP-/- mice treated with Clod-lip had reduced 65  gross and histological pathology (Figure 2.7A).  Ileal tissue sections were co-stained with F4/80, for macrophages, and YVAD-FLICA, for active caspase-1, and macrophages were counted in ileal tissue sections.  Clod-lip depleted 55±5% of macrophages in the ilea of SHIP-/- mice and eliminated staining for active capase-1 (Figure 2.7B).  Macrophage depletion also reduced histological evidence of inflammation including crypt-villus hyperplasia (Figure 2.7C, left) and the number of immune cells in ileal sections (Figure 2.7C, right).  Furthermore, macrophage depletion effectively reduced the levels of IL-1 in ileal tissue homogenates (Figure 2.8, left).  The level of IL-6, an IL-1-induced pro-inflammatory cytokine that is up-regulated during intestinal inflammation, was also reduced (Figure 2.8, right).    66    Figure 2.7   Macrophage depletion reduces development of intestinal inflammation in SHIP-/- mice.  SHIP-/- mice were injected intravenously with clodronate-containing liposomes (clod-lip) or PBS, as an injection control, from 4-6 weeks of age.  (A) Gross pathology of distal ilea (left) and H&E stained ileal cross-sections (right) of 6-week-old SHIP-/- mice.  (B) Ileal cross-sections co-stained with anti-F4/80 (macrophages) and YVAD-FLICA (active caspase-1) (left) and F4/80+ macrophages in ileal cross-sections quantitated by microscopy.  (C) Crypt-villus length (left) and immune cell infiltration (right) quantitated by microscopy. Photographs in (A) and (B) are representative images from 6 mice/group.  In (B)-(C), counting was performed on 6 mice/group counting 6 fields in 6 sections separated by ≥50 μm and by two individuals blinded to experimental condition.  Data are means SD for n=6.  *P<0.01, **P<0.001(unpaired Student’s t-test). 67   Figure 2.8   Macrophage depletion reduces IL-1β and IL-6 levels in the ileum of SHIP-/- mice.  SHIP-/- mice were injected intravenously with clodronate-containing liposomes (clod-lip) or PBS, as an injection control, from 4-6 weeks of age. IL-1β (left) and IL-6 (right) were assayed in full thickness ileal tissue homogenates by ELISA. Data are means SD for n=6.  *P<0.01, (unpaired Student’s t-test).  2.3.5 Anakinra treatment reduces intestinal inflammation in SHIP-/- mice Anakinra is an IL-1 receptor antagonist that is used to treat autoinflammatory diseases.  To determine whether blocking IL-1 signalling could block intestinal inflammation in SHIP deficient mice, we treated SHIP-/- mice with anakinra or PBS, as an injection control.  SHIP-/- mice treated with anakinra had reduced gross and histological pathology (Figure 2.9A).  Anakinra did not reduce the number of macrophages in the ilea of SHIP-/- mice; however, it eliminated staining for active caspase-1 (Figure 2.9B) and dramatically reduced IL1B gene expression in ileal tissues (Figure 2.10A).  Anakinra reduced crypt-villus hyperplasia and the number of immune cells in ileal sections (Figure 2.9C).  Anakinra also reduced the levels of IL-1 and IL-6 in ileal tissue homogenates (Figure 2.10B). 68   Figure 2.9   Anakinra reduces development of intestinal inflammation in SHIP-/- mice.     SHIP-/- mice were injected intravenously with the IL-1 receptor antagonist, anakinra, or PBS, as an injection control, from 4-6 weeks of age.  (A) Gross pathology of distal ilea (left) and H&E stained ileal cross-sections (right) of 6-week-old SHIP-/- mice.  (B) Staining for macrophages (F4/80) and active caspase-1 (FLICA) in fixed ileal tissue sections (left). Quantitation of F4/80+ macrophages in ileal cross-sections by microscopy (right).  (C) Crypt-villus length (left) and immune cell infiltration (right) quantitated by microscopy. Photographs in (A) and (B) are representative images from 6 mice/group.  In (B)-(C), counting was performed on 6 mice/group counting 6 fields in 6 sections separated by ≥50 μm and by two individuals blinded to experimental condition.  Data are means SD for n=6.  *P<0.05, **P<0.0001(unpaired Student’s t-test). 69   Figure 2.10   Anakinra reduces IL-1β levels in the ileum of SHIP-/- mice. SHIP-/- mice were injected intraperitoneally with the IL-1 receptor antagonist, anakinra, or PBS, as an injection control, from 4-6 weeks of age. (A) RNA was isolated from ileal tissues and IL-1 β transcription was assayed by Q-PCR. (B) IL-1β (left) and IL-6 (right) assayed in full thickness ileal tissue homogenates by ELISA. Data are means SD for n=6.  *P<0.05,**P<0.001 (unpaired Student’s t-test).  2.3.6 SHIP activity is lower in subjects with Crohn’s disease. To determine whether SHIP may play a role in intestinal inflammation in people with CD, we compared SHIP activity in subjects with CD (no colonic involvement) to that in control subjects, who did not have IBD.  Ileal and colonic biopsies and peripheral blood were collected from treatment-naïve subjects, who were undergoing colonoscopy as part of their diagnosis.  SHIP activity was assayed in fresh ileal and colonic biopsies and tissues were fixed for H&E staining.  SHIP activity was significantly lower in ileal biopsies from subjects, 70  who were subsequently diagnosed with CD compared to control subjects (Figure 2.11A).  SHIP is hematopoietic-specific so it is interesting to note that SHIP activity is lower in ileal tissues from subjects with CD despite dramatic immune cell infiltration visible in biopsies (Figure 2.11B).  Immune cells were quantitated in biopsy sections and confirmed a 2.2-fold increase in immune cells in CD subjects (Figure 2.11C). PBMCs were prepared from subjects with CD and control subjects to measure SHIP activity and (LPS+ATP)-induced IL-1 production.  As in ileal biopsies, SHIP activity was lower in PBMCs from subjects with CD compared to control subjects (Figure 2.12A) and SHIP activity in PBMCs inversely correlated with (LPS+ATP)-induced IL-1 production by these cells (Figure 2.12B).    71   Figure 2.11   SHIP activity is lower in ileal biopsies from subjects with CD compared to control subjects. Ileal and colonic biopsies and PBMCs were collected from treatment-naïve subjects undergoing colonoscopy as part of diagnosis of IBD.  N=8 subjects were subsequently diagnosed with CD (no colonic involvement) and n=11 subjects were not diagnosed with IBD.  (A) SHIP activity in ileal and colonic biopsies from control subjects (C) and subjects with CD (CD).  (B) H&E stained ileal and colonic biopsy sections.  (C) Quantitation of immune cells in tissue sections by microscopy.  Horizontal lines indicate means ± SEM.  *P ≤ 0.05, **P ≤ 0.01, NS = not significantly different. 72    Figure 2.12   SHIP activity is lower in peripheral blood mononuclear cells (PBMCs) from subjects with CD compared to control subjects and is inversely proportional to IL-1β production.   PBMCs were collected from treatment-naïve subjects undergoing colonoscopy as part of diagnosis of IBD.  N=8 subjects were subsequently diagnosed with CD (no colonic involvement) and n=11 subjects were not diagnosed with IBD.  (D) SHIP activity in peripheral blood mononuclear cells (PBMCs) from control subjects (C) and subjects with CD (CD; left).  (B) (LPS+ATP)-induced IL-1β production by PBMCs assayed by ELISA versus SHIP activity in control subjects (black circles) and subjects with CD (open squares; right). Horizontal lines indicate means ± SEM.  *P ≤ 0.05.  2.4 Discussion We have demonstrated that chronic ileitis in SHIP-/- mice are associated with elevated levels of macrophage-derived IL-1.  We also found that increased IL-1 production occurs in in vivo differentiated SHIP-/- macrophages, which have increased Class IA PI3Kp110-driven transcription of ILIB and that SHIP deficiency does not affect inflammasome activation directly.  Development of ileitis was ameliorated by macrophage depletion or treatment with the IL-1 receptor antagonist, anakinra.  Finally, we demonstrated that SHIP activity is lower in the inflamed ileum and PBMCs from treatment-naïve subjects with ileal CD compared to control subjects and SHIP activity inversely correlated with the ability of PBMCs to produce IL-1. 73  SHIP plays pleotropic roles in macrophage activation by limiting PI3K activity downstream of receptor stimulation. Thus, SHIP deficient macrophages are hyper-responsive to immune stimuli.  Herein, we demonstrate a critical role for SHIP in transcription of ILIB in macrophages where SHIP limits PI3Kp110 activity downstream of LPS/TLR4 signalling and contributes to intestinal inflammation.  PI3K and its downstream targets, Akt and mTOR (mammalian target of rapamycin), are activated by TLR4 signalling and both contribute to increased ILIB transcription in SHIP-/- macrophages (data not shown).  Additional evidence in the literature implicates the PI3K/SHIP axis in IL-1 production and intestinal inflammation.  The micro RNA, miR-155, targets SHIP protein.363  Triptolide (a potent anti-inflammatory/ immunosuppressive plant component) ameliorates inflammation post ileocolonic anastomosis in IL-10-/- mice by decreasing miR-155 levels.364 miR-155 levels are also increased during DSS-induced colitis365 and spontaneous colitis in IL-10-/- mice366, both of which have been associated with increased IL-1 production, though a direct correlation with SHIP activity has not been investigated.  Finally, targeting mTOR, downstream of PI3K, effectively reduces inflammation during DSS-induced colitis in mice and has been used effectively to treat refractory CD.367   Our data demonstrate that SHIP-/- bone marrow macrophages derived in MCSF or IL-3 did not produce more IL-1 in response to stimulation than those derived from their wild type littermates.  GM-CSF-derived SHIP-/- bone marrow macrophages did produce significantly more IL-1 than their wild type counterparts, but the effect of SHIP deficiency on IL-1 production was modest, particularly compared with differences observed for in vivo differentiated SHIP-/- ileal and peritoneal macrophages.  Furthermore, the dramatic 74  differences in IL-1 production for in vivo differentiated SHIP-/- macrophages could not be recapitulated in vitro by addition of pro-inflammatory IFN or IL-4, which we have reported is increased in SHIP-/- ileum337, or both IFN+IL-4.  Though unexpected, this data is consistent with a recent report, which showed that LysM-cre×SHIPfl/fl mice, that is, mice deficient in SHIP in myeloid cells including macrophages, did not develop the ileal inflammation characteristic of the germ line SHIP-/- mouse.348   Taken together, these data suggest that high IL-1 production by in vivo differentiated SHIP-/- macrophages requires cell-extrinsic factors produced by other SHIP-/- cells that contribute to the complex inflammatory environment in the germ line SHIP-/- mouse.  Though the cell type that initiates the inflammatory response has not been determined, data from Kerr et al. (2011)338 suggests that bone marrow-derived hematopoietic cells drive ileitis in germ line SHIP-/- mice because disease can be transferred to wild type mice by bone marrow transplantation, but not by adoptive transfer of T cells or NK cells, and because disease can be cured in SHIP-/- mice by transplantation of wild type bone marrow.338 In our study, SHIP activity inversely correlated with IL-1 production in three model systems: in ex vivo isolated macrophages from mice, in ileal tissues from mice, and in PBMCs from human subjects.  Intriguingly, increased IL-1 production by SHIP-/- macrophages is consistent with some previously described, but unexplained features of the SHIP-/- mouse.  IL-1 production is associated with neutrophil influx368 and our laboratory previously reported that ileal inflammation in the SHIP-/- mice is associated with dramatic neutrophilia.337  IL-1 is also a potent inducer of fever and, while monitoring temperature in SHIP-/- mice, it was previously reported that SHIP-/- mice have a chronic, low-grade fever.369  75  These unexplained phenotypes may be accounted for by hyper-responsiveness to IL1B transcription in SHIP-/- mouse macrophages.  The role of IL-1 production in intestinal inflammation is considered controversial.  In a model of acute intestinal inflammation in mice, DSS-induced colitis, concurrent studies reported that loss of the NLRP3 inflammasome led to reduced370 or exacerbated236 intestinal inflammation and disease severity.  Consistent with the former report, caspase-1 inhibition370 or caspase-1 deficiency,371 which would lead to an inability to process and secrete mature IL-1, protected mice from DSS-induced colitis.  Consistent with the latter report, NLRP6239 or IL-1240 deficiency exacerbated DSS-induced colitis in mice.  These last two studies may provide insight into the discrepancies reported in the literature highlighting key roles for dysbiosis in development of intestinal inflammation239 and the central role of macrophage-derived IL-1 in colon repair post DSS-induced epithelial cell injury.240  In contrast, the role of IL-1 during chronic intestinal inflammation is not controversial and IL-1 has consistently been reported to contribute to disease.  Caspase-1 deficiency protected mice during chronic inflammation induced by DSS371; TLR5-/- mice treated with a neutralizing antibody to the IL-10 receptor developed spontaneous colitis, which was driven by IL-1 and dependent on the IL-1 receptor244; and inflammasome activation and IL-1 production contributed to the development of spontaneous intestinal inflammation in IL-10-/- mice, which was inhibited by antagonizing the IL-1 receptor or caspase-1 activity.242  Our data provide additional evidence supporting the thesis that IL-1 contributes to chronic intestinal inflammation in mice because reducing IL-1 levels by either macrophage depletion or anakinra treatment ameliorated spontaneous intestinal inflammation in SHIP-/- mice.  76  Moreover, the efficacy of anakinra treatment suggests that SHIP-/- mice suffer from autoinflammation.  Anakinra is an IL-1 receptor antagonist.  As such, it does not interfere with IL-1 production directly, but rather blocks IL-1 signalling through the IL-1 receptor, which can drive transcription of more IL1B.  Anakinra treatment in SHIP-/- mice led to a dramatic decrease in IL-1 mRNA, which is consistent with it blocking the auto amplification of IL-1.  Finally, we report that SHIP activity was reduced in ileal biopsies and PBMCs from treatment-naïve subjects with ileal CD compared to control subjects, who did not have IBD.  This is particularly compelling given that SHIP expression is restricted to hematopoietic cells and there is a dramatic influx of hematopoietic cells into intestinal tissue in patients with IBD.  Arijs et al. (2011)349 previously reported that SHIP mRNA levels are increased in colonic biopsies from IBD subjects with colonic inflammation, both ulcerative colitis and Crohn’s colitis, but remain unaffected in ileal biopsies from subjects with ileal CD.349  Our data extend these observations suggesting that SHIP protein levels are regulated post-transcriptionally thus reducing net SHIP activity in subjects with ileal inflammation.  It has previously been reported that SHIP is up-regulated in response to LPS and MyD88-dependent TLR signalling,327,369 which provides a mechanism for maintenance of SHIP mRNA levels and activity in the colon where commensal microorganisms are abundant.  Our laboratory has also reported that SHIP protein expression and activity are reduced post-transcriptionally after activation and phosphorylation by Src family tyrosine kinases, which trigger SHIP’s polyubiquitination and degradation by the proteasome.330,361  This suggests a model in which SHIP is activated to dampen down PI3K signalling during intestinal inflammation, but is ultimately degraded in the ileum leading to increased inflammation.   77  Chapter 3: The Crohn`s disease-associated polymorphism in ATG16L1 reduces SHIP gene expression and activity in human subjects  3.1 Introduction and rationale Crohn’s disease (CD) is a chronic inflammatory disease characterized by intestinal inflammation that can occur anywhere along the gastrointestinal tract.4  The most common site for inflammation is the distal part of the ileum.4  The interactions between genetic and environmental factors,16 intestinal microbes and the epithelial lining, as well as the immune system are believed to be crucial for the development of chronic inflammation during CD.17   Genome-wide association studies have identified 163 susceptibility loci associated with inflammatory bowel disease (IBD), 140 of which are associated with CD.16  A non-synonymous single nucleotide polymorphism (SNP) in the gene encoding the autophagy-related 16-like protein (ATG16L1) has been associated with increased risk for developing CD in multiple populations35,372-374 and specifically with the development of ileal CD.36 The ATG16L1 CD-associated gene variant is common in the population with 33.2% of people from Western European descent being homozygous for the risk variant.258 People, who have one copy of the ATG16L1 CD-associated gene variant, have a 1.86-fold increased risk of developing CD (95% confidence interval 1.09-3.24) and people, who have 2 copies of the ATG16L1 risk allele, have a 2.38-fold increased risk of developing CD (95% confidence interval 1.40-4.04) thus the effect of this risk allele is gene dose dependent.259 The ATG16L1 risk allele codes for a threonine to alanine amino acid substitution at position 300 in the ATG16L1 protein (T300A).36 ATG16L1 is crucial for autophagy, a multi-step, catabolic self-78  preservation process that controls clearance and re-use of intracellular components during homeostasis and provides amino acids required for cell survival under stress conditions including starvation, accumulation of unfolded proteins, or intracellular infection.245 ATG16L1 sets the platform for recruitment of the ATG12-ATG5 complex to the immature phagophore membrane, which in turn permits progression and closure of the membrane to form the autophagosome.253 ATG16L1 deficiency in mice or homozygosity for the ATG16L1 CD-associated gene variant in humans causes defects in Paneth cell granule exocytosis,80 which may be important in susceptibility to disease in response to specific infections and/or environmental influences.73 Moreover, CD patients, who are homozygous for the ATG16L1 CD risk allele, are unable to adequately clear pathosymbionts and monocytes from subjects with the ATG16L1 T300A risk protein have impaired ability to clear invading pathogens.260 In humans, defects in autophagy and the ATG16L1 CD risk variant have been associated with increased IL-1β production.254,255,261  IL-1β is a critical cytokine, which can be produced by monocytes and macrophages to initiate and amplify the innate immune responses.170  Thus, its production is tightly regulated by a two-step process.  First, innate immune stimuli induce transcription of IL1B, which is translated to pro-IL-1, an inactive precursor.  Second, danger associated molecular patterns cause assembly of the inflammasome activating caspase-1, which cleaves pro-IL-1, leading to activation and secretion of mature IL-1.190-192  The ATG16L1 T300A protein has recently been shown to be cleaved by caspase 3/7, resulting in defective autophagic control of intracellular bacteria and increased IL-1β secretion.254,255  Consistent with these results, PBMCs from patients, who are homozygous for ATG16L1 CD risk variant, produce more IL-1β in response to muramyl dipeptide (MDP) than those from subjects, who are homozygous for the variant that 79  is not associated with CD.261  Moreover, differences observed were attributed to increased ILIB transcription.261  In mice, increased IL-1 production associated with autophagy defects has been attributed to increased inflammasome activity but the molecular mechanism(s) by which autophagy and/or ATG16L1 regulate ILIB transcription in humans remains unknown.  The SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP) is a hematopoietic-specific lipid phosphatase that negatively regulates class I phosphatidylinositol 3-kinase (PI3K) activity.  SHIP antagonizes class I PI3K signalling by dephosphorylating the 5' position of class I PI3K-generated phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3).  Class I PI3K is critical in many cellular processes including immune activation, thus, SHIP deficient mice are hyper-responsive to immune stimuli, 327,337,369,375 including increased IL-1 production in response to innate immune activation.327  We, and others, have reported that SHIP deficient mice develop spontaneous CD-like intestinal inflammation that is primarily restricted to the distal ileum.337,338  In humans, SHIP mRNA expression is increased in inflamed colonic biopsies from people with ulcerative colitis and colonic CD, which is consistent with dramatic immune cell infiltration into tissues during inflammation and SHIP expression being restricted to hematopoietic cells.349   More surprisingly, SHIP mRNA expression was not elevated in inflamed biopsies from subjects with ileal CD. 349  The human gene encoding SHIP protein (INPP5D) is located at chromosome 2q37.1 and is 40 kilobases upstream of the gene encoding ATG16L1.305   INPP5D and ATG16L1 are located on the same DNA strand and are transcribed in the same direction.  Though there are SNPs in ATG16L1, which are in linkage disequilibrium with rs2241880,376 there are no SNPs in INPP5D, which co-segregate with rs2241880.    80  Humans carrying the ATG16L1 risk variant and mice deficient in SHIP have increased susceptibility to CD or ileal inflammation, respectively, and their macrophages have increased ability to produce IL-1as shown in chapter 2. Thus, we asked whether SHIP mRNA expression and activity are regulated by ATG16L1 and whether SHIP may contribute to increased IL-1 production in subjects with the ATG16L1 risk variant.  Herein, we report that SHIP mRNA expression was reduced in subjects with ileal CD.  In our patient cohort and a second cohort of healthy control subjects, both SHIP mRNA expression and activity levels were extremely low in subjects, who were homozygous for the ATG16L1 T300A-encoding risk allele.  Furthermore, we show that autophagy up-regulated SHIP protein and that SHIP up-regulation was dependent on ATG16L1 protein levels and/or autophagy, and the ATG16L1 gene variant.  Finally, we demonstrate that the ATG16L1 genotype and low SHIP mRNA expression correlated with increased IL-1β production by (LPS+ATP)-stimulated PBMCs, and was dependent, at least in part, on IL1B transcription.  Taken together, these results identify SHIP as a critical link between the ATG16L1 CD-associated gene variant and increased IL1B transcription, which may contribute to increased risk of developing intestinal inflammation.  3.2 Materials and methods Subjects with ileal Crohn’s disease and control subjects. This study was performed in accordance with ethical guidelines approved by the University of British Columbia Research Ethics Boards (H09-01826).  Written informed consent was obtained from all subjects.  Patients seen in the Division of Gastroenterology at BC Children’s Hospital and scheduled 81  for colonoscopy were recruited into the study.  None of the patients had been previously diagnosed or treated for IBD or other inflammatory disease.  Four biopsies were taken from the ileum and colon at sites of inflammation, which were adjacent to tissues harvested for pathological assessment in patients diagnosed with CD.  Similar sites were biopsied in control subjects.  During the procedure, peripheral blood was drawn from the site of intravenous insertion.  The diagnosis of CD with inflammation in the ileum, or absence of IBD, was based on colonoscopy and pathological assessment.  Twenty-one subjects were included in the analyses, 8 patients diagnosed with CD with ileal inflammation and 13 subjects with no IBD.  Biopsy samples and PBMCs isolated from peripheral blood were either stored in RNAlater (Invitrogen; Burlington, ON) for gene expression analyses, or used immediately for biochemical and immunological assays. All analyses were performed on all participants prior to diagnosis.  Healthy control subjects. Experiments were performed in accordance with ethical guidelines and with approval by the University of British Columbia Research Ethics Boards (H04-0534).  Written informed consent was obtained from all subjects.  Isolated PBMCs were used for analyses.  Eighteen subjects, 6 from each ATG16L1 genotype, were used for analyses.  PBMCs were isolated from peripheral blood and DNA was stored for genotyping, or fresh PBMCs were used for biochemical and immunological assays. All analyses were performed on all participants prior to stratification by genotype.   Cell isolation and derivation conditions. PBMCs were isolated from whole blood by density gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare, Piscataway, NJ).  Cells were washed and resuspended at a density of 0.5×106 cells/mL in Iscove’s Modified 82  Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) for assays.  DNA was prepared for genotyping using the QiaAMP DNA blood mini kit, as per the manufacturer’s instructions (Qiagen, Toronto, Ontario, Canada), and fresh PBMCs were used for analyses of gene expression, SHIP activity, autophagy, and cytokine production. THP-1 cells from the ATCC and free of mycoplasma contamination, were maintained in RPMI 1640, 10% FCS, and penicillin/streptomycin.  Cells (0.5 × 106 cells/mL) were differentiated for 16 h in 20 ng/mL phorbol 12-myristate 13-acetate (PMA) for use in autophagy assays. Bone marrow macrophages (BMMфs) were generated from bone marrow aspirates from femura and tibiae of SHIP+/+ and SHIP-/- mice. Aspirates were resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum (FBS) in 75 cm2 Falcon flasks (BD Biosciences) for 1 hr. Following adherence depletion, bone marrow aspirates were then resuspended in IMDM, 10% FBS, and penicillin/streptomycin at a concentration of 0.5×106 cells/mL for 10 days in the presence of 5 ng/mL MCSF (StemCell Technologies, Vancouver, BC), with complete media changes at day 4 and 7. At day 10, macrophages were ≥ 95% F4/80+ and Mac-1+. In THP-1 cells, ATG16L1 was knocked down using two Stealth RNAi designed against non-overlapping sequences (HSS147873 and HSS182825) or a non-silencing control (Life Technologies).  Stealth RNAi or control RNAi was transfected into THP-1 cells using Lipofectamine 2000 according to the manufacturer’s protocol (Life Technologies).  Genotyping the ATG16L1 Crohn’s disease susceptibility SNP, rs2241880. Stored DNA samples were used to genotype the ATG16L1 SNP, rs2241880, using a commercially available Taqman assay (C_9095577_20; Applied Biosystems, Burlington, ON, Canada).  83  SNPs were deemed acceptable for analysis if they had call rates > 95% and frequencies did not deviate from Hardy-Weinberg equilibrium (P > 0.05).   Gene Expression Analyses. Human tissue sections or PBMCs were stored in RNAlater at -20°C.  RNA was prepared using the either Qiagen RNeasy Mini Kit (Qiagen, Toronto, ON) or NucleoSpin RNA II Total RNA Isolation Kit according the manufacturer’s protocol (Macherey-Nagel, Bethlehem, PA).  RNA concentration was measured using the NanoDrop 1000 Spectrophotometer. RNA was reverse transcribed using Superscript II (Invitrogen, Burlington, ON). For analysis of murine SHIP (Inpp5d) and -actin (ActB) gene expression, qPCR was performed using 2× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on the CFX96 C1000 Thermal Cycler (BioRad Laboratories, Mississauga, ON).  Primers were from BioRad Laboratories, catalogue number 100-25636, with unique identifiers qMmuCED0045767 (Inpp5D), and qMmuCED0027505 (ActB). For analysis of human SHIP (INPP5D), ATG16L1 (ATG16L1), CD45 (PTPRC), and -actin (ACTB) gene expression, qPCR was performed using 2× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on the CFX96 C1000 Thermal Cycler (BioRad Laboratories, Mississauga, ON).  Primers sequences used were: INPP5D (SHIP) forward  5'-GGGAGATGTAGTGTCACTGG-3' INPP5D (SHIP) reverse  5'-TACAGGAATGAGCCCCAAAG-3' PTPRC (CD45) forward  5'-CTGACATCATCACCTAGCAG-3' PTPRC (CD45) reverse  5'-TGCTGTAGTCAATCCAGTGG-3' ACTB (-actin) forward  5'-AAATCTAGCTGCCCGTCATT-3' 84  ACTB (-actin) reverse  5'-GCATCGAGTCCCTGATTTCT-3' Primers for ATG16L1 were from BioRad Laboratories, catalogue number 100-25636, and sequences are proprietary. For analysis of IL-1β gene expression (IL1B), qPCR was performed using the AB Applied Biosystems Taqman Universal Master Mix II (Invitrogen).  Primers sequences used were: ILIB (IL-1β) forward       5'-ATGCACCTGTACGATCACTG-3' ILIB (IL-1β) reverse        5'-ACAAAGGACATGGAGAACACC-3' ILIB (IL-1β) probe          5'-/56 FAM/CCAGGGACA/ZENGGATATGGAGCAACA/3IABkFQ/-3'   ACTB forward                 5'-ACCTTCTACAATGAGCTGCG-3' ACTB reverse                5'-CCTGGATAGCAACGTACATGG-3' ACTB probe                5'-/56-FAM/ATCTGGGTC/ZEN/ATCTTCTCGCGGTTG/3IABkFQ/-3'  SHIP activity assays. To quantify SHIP activity, SHIP was immunoprecipitated with anti-human SHIP1 antibody (N-1, sc6244; Santa Cruz Biotechnology, CA).  Intestinal biopsy sections were weighed or PBMCs were counted and immunoprecipitates prepared.  Substrate, 100 μM IP4 (Echelon Biosciences, Salt Lake City, UT, USA) was incubated with immunoprecipitates for 20 min and the reaction was stopped by heating samples to 80°C for 5 min.  Inorganic phosphate released was detected using Malachite Green (Echelon Biosciences) and absorbance was read at 650 nm and compared to a standard curve.  85  Autophagy induction and Western blot analyses. Autophagy was induced in murine macrophages, THP-1 cells, or PBMCs by starvation.  Media was removed and cells were washed twice and resuspended in Hanks Balanced Salt Solution (HBSS) for 4 h.  Cells, 2.5 × 106 murine bone marrow derived macrophages or 0.5 × 106 THP-1 cells or PBMCs, were harvested for Western immunoblot analysis.  In THP-1 cells, ATG16L1 was knocked down using two Stealth RNA interference (RNAi) designed against non-overlapping sequences (HSS147873 and HSS182825) or a non-silencing control RNAi (Life Technologies).  Stealth RNAi or non-silencing control RNAi was transfected into THP-1 cells using Lipofectamine 2000, according to the manufacturer’s protocol (Life Technologies). Whole cell lysates were subjected to SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and transferred onto polyvinylidene fluoride (PVDF) for immunodetection, as described previously.377   The following antibodies were used for analyses:  anti-mouse SHIP (P1C1, Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse and -human LC3 (NB100-2220, Novus Biologicals, Oakville, ON), anti-mouse GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Fitzgerald Industries International, Acton, MA), anti-human ATG16L1 (Abcam, Toronto, ON), anti-human SHIP (N1, Santa Cruz), and anti-human GAPDH (eBiosciences, San Diego, CA).  Densitometry was performed using ImageJ software (National Institute of Health, USA).  Cell stimulations for cytokine production and analyses. Murine bone marrow macrophages or PBMCs were plated at a density of 0.5 × 106 cells/mL of complete medium.  For ILIB gene expression analysis, PBMCs were unstimulated or stimulated with 10 ng/mL of LPS (E. coli serotype 127:B8, Sigma Aldrich, St. Louis, MO) for 3 h, and RNA was 86  isolated using the Qiagen RNeasy Mini Kit (Qiagen, Toronto, ON) and gene expression measured. For IL-1 cytokine production, cells were unstimulated or stimulated with 10 ng/mL LPS for 5 h, 5 mM ATP for 1 h, or LPS for 4 h with the addition of ATP for a final 1 h.  After incubation (5 h in total), clarified cell supernatants were analyzed by ELISA.  For TNF and IL-6 production, cells were unstimulated or stimulated with 10 ng/mL LPS for 24 h.  Cytokine measurements were performed on clarified, cell-free tissue culture supernatants by ELISA, according to the manufacturers’ instructions.  ELISA kits for human IL-β, TNFα, and IL-6 were from BD Biosciences (Mississauga, ON).   Statistical Analyses. Unpaired two-tailed Student’s t tests, one-way analysis of variance (ANOVA), and linear regression analyses were performed using GraphPad Prism version 5 software.  For multiple comparisons, the Bonferroni correction was applied.  Differences were considered significant at P < 0.05.   3.3 Results 3.3.1 SHIP mRNA expression is reduced in subjects with ileal Crohn’s disease  Previous studies from our laboratory, and others, have shown that SHIP deficient mice develop spontaneous intestinal inflammation with similar features to human CD.337,338  Subsequent studies demonstrated that SHIP mRNA is not up-regulated in ileal biopsies from people with ileal CD,349 despite the fact that SHIP is hematopoietic-restricted and there is dramatic immune cell infiltration into the ileum during ileal CD.  Based on that, we asked whether SHIP mRNA expression was reduced in immune cells from subjects with ileal CD 87  compared to control subjects (C), who did not have IBD.  Ileal and colonic biopsies and peripheral blood were collected from 21 treatment-naïve subjects, who were undergoing colonoscopy as part of their diagnosis.  Subjects were subsequently diagnosed with ileal CD (CD = 8) or were control subjects, who were not diagnosed with IBD (C = 13).  Relative gene expression for genes encoding SHIP (INPP5D), CD45 (PTPRC, a hematopoietic cell marker), and β-actin (ACTB) were measured.  SHIP mRNA expression was reduced in the ileum and colon, relative to PTPRC expression, and in PBMCs, relative to ACTB, from subjects diagnosed with ileal CD compared to control subjects (Figure 3.1A).  SHIP mRNA expression in the ileum, colon, and PBMCs was associated with SHIP activity in both control and CD groups with R2 = 0.85, 0.83, and 0.92 (P < 0.0001), respectively (Figure 3.1B).   88    Figure 3.1   Subjects with ileal CD have lower SHIP mRNA expression compared to control subjects.   (A) SHIP mRNA expression in biopsies from the ileum and colon, relative to CD45 gene expression (a hematopoietic cell marker), and in PBMCs, relative to β-actin gene expression, from control subjects (C) and subjects with ileal CD (CD) were determined by qRT-PCR.  (B) Linear regression analyses were used to compare SHIP mRNA expression levels with SHIP activity, from control subjects (black circles) and subjects with ileal CD (open squares).  For (A) and (B), each symbol represents an individual subject (n = 13 control subjects, n = 8 subjects with ileal CD).  In (A), horizontal lines indicate means ± SEM, *P ≤ 0.05 (unpaired Student’s t-test); in (B), R2 = 0.85, 0.91, and 0.92 with P < 0.0001.  3.3.2 Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with low SHIP mRNA expression and activity SHIP mRNA expression varied more than 10-fold in PBMCs from different subjects.  Intriguingly, some subjects in both control and CD groups had very low SHIP mRNA 89  expression and activity in all tissues samples, suggesting that there may be a genetic cause, which predisposes individuals to express low levels of SHIP mRNA.  We next asked whether low SHIP mRNA and activity were associated with the ATG16L1 genotype.  As shown in Figure 3.2, both SHIP mRNA expression (Figure 3.2A) and SHIP activity (Figure 3.2B) were significantly lower in biopsies from the ileum and colon and in PBMCs from all subjects (controls and CD), who were homozygous (GG, n=5) for the ATG16L1 CD susceptibility allele, compared to subjects, who were heterozygous (AG, n=10) or did not have the susceptibility allele (AA, n=6).  There was no difference in SHIP mRNA expression or activity between subjects with the AA or AG genotypes.    90   Figure 3.2   Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with low SHIP mRNA expression and activity.    (A) SHIP mRNA expression and (B) SHIP activity in biopsies from the ileum and colon and in PBMCs from control subjects (black circles) and subjects with ileal CD (open squares), who did not have (AA), were heterozygous (AG), or were homozygous (GG) for the ATG16L1 CD susceptibility SNP (rs2241880).  For (A) and (B), each symbol represents an individual subject (n = 13 control subjects, n = 8 subjects with ileal CD).  Horizontal lines indicate means ± SEM.  *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, NS = not significantly different (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes).  3.3.3 SHIP mRNA expression and activity are lower in PBMCs from healthy control subjects, who are homozygous for the ATG16L1 T300A-encoding gene variant To determine whether low SHIP mRNA expression and activity were independent of inflammation in CD, we recruited a second cohort of 18 healthy control subjects, who were genotyped for the ATG16L1 SNP (rs2241880) (n = 6 subjects per genotype).  We measured 91  SHIP mRNA expression and activity in their PBMCs.  Healthy control subjects, who were homozygous for the ATG16L1 CD susceptibility allele, had significantly lower SHIP mRNA expression (Figure 3.3A) and lower SHIP activity (Figure 3.3B) compared to subjects, who did not have the susceptibility allele.  SHIP mRNA expression and activity in subjects, who were heterozygous for the ATG16L1 genotype (AG), were not significantly different from the AA and GG groups (Figures 3.3A and 3.3B).     Figure 3.3   SHIP mRNA expression and activity are lower in PBMCs from healthy control subjects, who are homozygous for the ATG16L1 T300A-encoding gene variant.     (A) SHIP mRNA expression and (B) SHIP activity in healthy control subjects stratified for ATG16L1 SNP (rs2241880) genotype.  For (A) and (B), horizontal lines indicate means ± SEM for n = 6 subjects per genotype.  *P ≤ 0.01, NS = not significantly different (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes).     3.3.4 SHIP and ATG16L1 are not transcriptionally co-regulated Low SHIP mRNA expression in subjects, who were homozygous for the ATG16L1 risk variant, suggested that SHIP (INPP5D) gene transcription may be co-regulated with ATG16L1 and expressed at lower levels in subjects, who were homozygous for the ATG16L1 92  risk allele.  To investigate this possibility, we measured ATG16L1 gene expression in our subject cohorts.  In our patient group, both control (C) and CD subjects had similar mRNA expression levels for ATG16L1 in intestinal tissues (Figure 3.4A, left), which did not vary linearly with SHIP mRNA expression (R2 = 0.065; P = 0.356) (Figure 3.4A, right).  Similarly, in our healthy control subject cohort, there was no difference in ATG16L1 mRNA expression in PBMCs from subjects with different ATG16L1 SNP genotypes (Figure 3.4B, left) and no relationship between ATG16L1 and SHIP mRNA expression (R2 = 0.005; P = 0.812) (Figure 3.4B, right).         93   Figure 3.4   ATG16L1 mRNA expression does not vary linearly with SHIP mRNA expression.   (A) ATG16L1 and SHIP mRNA expression in biopsies from the ileum of control subjects (C, black circles) and subjects with ileal CD (CD, open squares) were determined by qRT-PCR.  Linear regression analyses were used to compare ATG16L1 mRNA expression with SHIP mRNA expression.  Each symbol represents an individual subject (n = 8 control subjects, n = 7 subjects with ileal CD).  Horizontal lines indicate means ± SEM.  NS = not significantly different (unpaired Student’s t-test); R2 = 0.065 with P = 0.356.  (B) ATG16L1 mRNA expression in PBMCs from healthy control subjects that did not have (AA), were heterozygous (AG), or were homozygous (GG) for the ATG16L1 CD susceptibility SNP (rs2241880) were measured by qRT-PCR.  Horizontal lines indicate means ± SEM for n = 6 subjects per genotype.  NS = not significantly different (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes).  Linear regression analyses were used to compare ATG16L1 mRNA expression and SHIP mRNA expression in all genotypes.  Each symbol represents an individual subject (n = 5 subjects with each genotype; AA open circles, AG half-filled circles, GG black circles).  R2 = 0.005 with P = 0.812.    94  3.3.5 Starvation-induced autophagy up-regulates SHIP protein, which is dependent on ATG16L1 or autophagy, and the risk allele genotype  ATG16L1 is required for autophagy so we next asked whether autophagy affected SHIP protein levels.  First, we compared SHIP protein levels during starvation-induced autophagy in murine MCSF-derived bone marrow macrophages from wild type (SHIP+/+) and SHIP deficient mice (SHIP-/-).  Intriguingly, SHIP protein levels were 4.3-fold higher during starvation-induced autophagy in macrophages from wild type mice (Figure 3.5A, left, top panel).  Also, in contrast to wild type macrophages, SHIP deficient macrophages did not express LC3-II protein upon starvation, a marker of autophagosome formation (Figure 3.5A, left, middle panel, lanes 2 and 5).  In the presence of bafilomycin A1, which blocks autophagosome-lysosome fusion and flux through the autophagy pathway, LC3-II was detected in SHIP deficient macrophages, albeit at lower levels than in comparably treated wild type macrophages (Figure 3.5A, left, middle panel, lanes 3 and 6).  To determine whether SHIP was transcriptionally up-regulated during starvation-induced autophagy, SHIP mRNA expression was measured in wild type (SHIP+/+) murine bone marrow macrophages during starvation.  SHIP mRNA expression was increased after 4 h in starvation medium (Figure 3.5A, right). To determine whether SHIP was up-regulated during autophagy in human cells, we compared SHIP protein levels during autophagy in PMA-differentiated THP-1 cells, a human myelomonocytic cell line.  SHIP protein levels were induced 4.8-fold during starvation-induced autophagy in THP-1 cells (Figure 3.5B, left, 2nd panel).  To determine whether ATG16L1 was required for autophagy-induced up-regulation of SHIP protein, autophagy was induced by starvation in differentiated THP-1 cells in the presence of non-silencing 95  RNA (nsRNA) or a small interfering RNA (siRNA) construct targeting ATG16L1.  The siRNA construct reduced ATG16L1 protein levels by 83% in untreated THP-1 cells (Figure 5b, left, top panel).  Knockdown of ATG16L1 by siRNA was sufficient to block autophagy, indicated by reduced LC3-II expression, and blocked autophagy-induced up-regulation of SHIP protein, whereas the control nsRNA had no effect (Figure 3.5B, left).  As in bone marrow derived macrophages, SHIP protein induction by starvation correlated with increased SHIP mRNA expression in THP-1 cells.  Moreover, siRNA targeting ATG16L1, which blocked SHIP protein induction, prevented SHIP mRNA induction during autophagy compared to the nsRNA control (Figure 3.5B, right).  Finally, we measured SHIP induction in response to starvation in PBMCs from six healthy control subjects with each ATG16L1 SNP genotype.  Figure 3.6, left, shows a representative Western blot analysis of SHIP and GAPDH protein levels in one subject for each ATG16L1 genotype; AA, AG, and GG.  Average densitometry from six subjects in each group demonstrated that SHIP protein levels were 50% lower in subjects, who were homozygous for the ATG16L1 CD risk allele, compared to subjects, who did not have the risk allele (Figure 3.6, right). Together, these data demonstrate that SHIP mRNA and protein are up-regulated during autophagy in three different cell models; murine bone marrow macrophages, a human myelomonocytic cell line, and in primary PBMCs from human subjects.  Though SHIP induction is compromised during siRNA knockdown of ATG16L1 in THP-1 cells, it is not possible to distinguish whether this is cause by reduced ATG16L1 protein levels directly, or by diminished autophagy.  Notably, SHIP protein levels are lower in PBMCs from subjects, who are homozygous for the ATG16L1 CD susceptibility genotype at baseline and upon induction of autophagy by starvation. 96   Figure 3.5   Autophagy up-regulates SHIP protein and SHIP up-regulation is dependent on ATG16L1 expression and/or autophagy.  (A) Left, immunoblot analysis of SHIP and LC3 in wild type (SHIP+/+) and SHIP deficient (SHIP-/-) bone marrow macrophages that were untreated or incubated in starvation medium for 4 h in the absence or presence of bafilomycin A1.  Densitometry for SHIP and LC3-II, normalized to GAPDH, averaged from four independent experiments are shown below each band.  Right, relative SHIP mRNA expression in SHIP+/+ bone marrow macrophages that were untreated or incubated in starvation medium for 4 h. Data are mean + SEM for n = 9 independent experiments. (B) Left, immunoblot analysis of SHIP and LC3 in THP-1 cells that was untreated or incubated in starvation medium in the absence or presence of non-silencing control RNA (nsRNA) or siRNA to ATG16L1. Densitometry for ATG16L1, SHIP, and LC-II; normalized to GAPDH, averaged from four independent experiments are shown below each band. Right, relative SHIP mRNA expression in THP-1 cells incubated in starvation medium in the absence or presence of nsRNA or siRNA targeting ATG16L1. Data are means + SEM for n = 3 independent experiments, *P ≤ 0.05, **P < 0.01 (unpaired Student’s t-test).  97   Figure 3.6   SHIP up-regulation is dependent on the ATG16L1 genotype. Left, representative immunoblot analysis of SHIP in PBMCs from one healthy control subject with each ATG16L1 genotype (AA, AG, or GG) that were untreated or incubated in starvation medium. Densitometry for ATG16L1 and SHIP, normalized to GAPDH, averaged from six independent experiments are shown below each band. Right, densitometry for SHIP protein levels, normalized to GAPDH, in PBMCs from healthy control subjects from each ATG16L1 genotype (AA, AG, or GG) that were untreated or incubated in starvation medium. N = 6 subjects for each genotype; AA, AG, or GG; **P ≤ 0.001 (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes).     3.3.6 Homozygosity for the Crohn’s disease-associated ATG16L1 gene variant is associated with high IL-1β production and IL1B transcription The CD-associated ATG16L1 gene variant has been associated with defective autophagy49 and high IL-1β production in response to NOD2 activation.261  In a previous study, stimulation with MDP increased IL-1β and IL-6 in PBMCs from subjects, who carry the ATG16L1 risk variant.261  Along with IL-1β and IL-6, TNFα is a critical cytokine that is up-regulated and contributes to immune mediated inflammation in Crohn’s disease.378 Thus, we next measured production of these pro-inflammatory cytokines in (LPS+ATP)-stimulated 98  PBMCs from control (C) and CD subjects as well as in our cohort of healthy control subjects, who were homozygous (GG), heterozygous (AG), or did not have the ATG16L1 T300A-encoding gene variant (AA).  In our patient cohort, PBMCs from both control and CD subjects, who were homozygous for the ATG16L1 CD susceptibility gene variant, produced more IL-1β compared to subjects, who were heterozygous or did not carry the susceptibility allele (Figure 3.7A).  In contrast, there was no difference for TNFα or IL-6 production between the genotypes (Figure 3.7A).  In our second cohort of healthy control subjects, subjects, who were homozygous (GG) for the ATG16L1 T300A-encoding gene variant, produced high levels of IL-1β compared to subjects, who were heterozygous (AG) or did not carry the risk variant (AA) (Figure 3.7B). TNFα and IL-6 production were not affected by the ATG16L1 CD-associated gene variant in healthy control subjects (Figure 3.7B).  To determine whether increased transcription of ILIB contributes to increased IL-1β production by human PBMCs, IL1B transcription was measured in LPS-stimulated PBMCs from healthy control subjects with each genotype.  Healthy control subjects, who were homozygous (GG) or heterozygous (AG) for the ATG16L1 risk variant, had significantly higher IL-1β mRNA expression compared to subjects without the risk variant (AA) (Figure 3.7C).  There was no difference in IL-1β mRNA expression levels between subjects with AG and GG genotypes (Figure 3.7C).    99   Figure 3.7   Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased (LPS+ATP)-induced IL-1β production.  (A and B) IL-1β (left), IL-6 (middle), and TNFα (right) assayed by ELISA in clarified tissue culture supernatants from (LPS+ATP)-stimulated PBMCs, stratified for the ATG16L1 CD susceptibility SNP (rs2241880) genotype from (A) control subjects (black circles) and subjects with ileal CD (open squares) and (B) a second cohort of healthy control subjects.  (C) IL-1β mRNA expression, normalized to β-actin gene expression, in LPS-stimulated PBMCs from healthy control subjects stratified for the ATG16L1 CD susceptibility SNP (rs2241880) genotype was determined by qRT-PCR.  In (A), each symbol represents an individual subject (n = 13 control subjects, n = 8 subjects with ileal CD).  In (A) - (C), horizontal lines indicate means ± SEM.  In (B) and (C), n = 6 subjects per genotype.  *P ≤ 0.05, **P ≤ 0.01, NS = not significantly different (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes). 100  3.3.7 Reduced SHIP mRNA expression is associated with high IL-1β production and IL1B transcription Next, we determined whether increased IL-1 production and IL1B transcription was associated with SHIP mRNA expression.  Indeed, IL-1 production inversely related to SHIP mRNA expression in (LPS+ATP)-stimulated PBMCs from control subjects (black circles) and subjects with CD (open squares; R2 = 0.3723, P = 0.0033) (Figure 3.8A).  Similarly, in our second cohort of healthy control subjects, IL-1 production by (LPS+ATP)-stimulated PBMCs was inversely related to SHIP mRNA expression (R2 = 0.4438, p = 0.0129) (Figure 3.8B) and IL1B transcription also inversely correlated with SHIP mRNA expression (R2 = 0.4896, p = 0.0078) (Figure 3.8C).  Taken together, these data suggest that low SHIP expression contributes to increased IL1B transcription and thereby regulates the level of IL-1 secreted by human PBMCs. 101   Figure 3.8   Reduced SHIP mRNA expression is associated with increased LPS-induced IL-1β production. (A and B) IL-1β assayed by ELISA in clarified tissue culture supernatants from (LPS+ATP)-stimulated PBMCs versus SHIP mRNA expression, normalized to ACTB expression, determined by qRT-PCR in (A) control subjects (black circles) and subjects with ileal CD (open squares) and (B) a second cohort of healthy control subjects.  (C) IL-1β mRNA expression, normalized to ACTB expression, in LPS-stimulated PBMCs from healthy control subjects versus SHIP mRNA expression, both determined by qRT-PCR.  In (A) – (C), each symbol represents an individual subject.  In (A), n = 13 control subjects (black circles) and n = 8 subjects with ileal CD (open squares).  In (B) and (C), n = 5 subjects per genotype.  In (A) – (C), R2 = 0.3723, 0.4438, and 0.4896; with P = 0.0033, 0.0129, and 0.0078; respectively.  102  3.4 Discussion Herein, I demonstrate that SHIP mRNA expression and activity are reduced in subjects with ileal CD and are associated with homozygosity for the ATG16L1 CD-associated gene variant.  Furthermore, I also show that SHIP is up-regulated during autophagy and that SHIP protein levels and up-regulation are compromised in subjects, who are homozygous for the ATG16L1 CD-associated gene variant.  Finally, I report that IL-1 production and IL1B transcription are higher in PBMCs from subjects, who are homozygous for the ATG16L1 CD-associated gene variant and is associated with reduced SHIP mRNA expression in PBMCs from our subject cohorts.   The fact that we see reduced SHIP mRNA expression in subjects with ileal CD, confirms previous report by Arijs et al, 2012, in which they showed that SHIP mRNA is not up-regulated in the ileum during ileal CD.349 We extend that finding, demonstrating that SHIP mRNA expression is actually decreased during ileal CD, when normalized to infiltrating immune cells, and that reduced SHIP mRNA expression correlates with reduced SHIP activity.  SHIP mRNA expression may be up-regulated maintaining SHIP protein levels and activity in the colon, but not the ileum, due to higher abundance of commensal microorganisms in the colon379 as SHIP mRNA and protein are induced by MyD88-dependent Toll-like receptor signalling.327,369  In two independent cohorts, I demonstrate that homozygosity for the ATG16L1 CD-associated gene variant was associated with low SHIP mRNA expression and activity.  Moreover, the relationship between homozygosity for the ATG16L1 CD-associated gene variant and low SHIP mRNA and activity is independent of inflammation.  In healthy control subjects who were heterozygous for the SNP (AG genotype), SHIP mRNA expression and 103  activity varied widely, ranging from low levels, comparable to that for people with the GG genotype, to high levels, comparable to that for people with the AA genotype.  This suggests that additional environmental and/or genetic factors may contribute to low SHIP mRNA expression and activity in some individuals.  SHIP protein levels are reduced by the type II inflammatory cytokines, IL-4 and IL-13.361  In addition, 140 CD-associated risk variants have been identified,16 including some that code for non-synonymous SNPs in proteins that cause autophagy defects (NOD2)49 or that are predicted to affect autophagy (IRGM, ATG5, ORMDL3, and XBP-1).16  There was no association between SHIP mRNA expression and ATG16L1 mRNA expression in the two cohorts studied. These data are consistent with a previous report demonstrating that ATG16L1 mRNA expression is not influenced by the rs2241880 genotype.35  This does not, however, preclude the possibility that SHIP gene expression may be reduced epigenetically by the presence of the ATG16L1 CD risk allele. Intriguingly, SHIP deficient macrophages had a defect in starvation-induced autophagy. Furthermore, SHIP protein expression was up-regulated during starvation conditions in both PBMCs from our healthy cohort and THP-1 cells, but was compromised in the presence of the T300A CD risk variant and siRNA constructs targeting ATG16L1 respectively. These results suggest that SHIP may be required for starvation-induced autophagy and that fully functional ATG16L1 protein was required for autophagy-induced up-regulation of SHIP protein expression. Class I PI3K is a positive regulator of autophagy380 and rapamycin-mediated inhibition of mTOR, which is activated downstream of the PI3K/SHIP axis, is frequently used to induce autophagy.381  Thus, SHIP may regulate autophagy in hematopoietic cells and increased SHIP expression may be required for the 104  induction and/or maintenance of autophagy.  In addition, a defect in autophagy could cause and/or contribute to the intestinal inflammation observed in SHIP deficient mice. IL-1 production and IL1B transcription were higher in PBMCs from subjects, who are homozygous for the ATG16L1 CD-associated gene variant and was associated with reduced SHIP mRNA expression. In a previous study by Plantinga et al, stimulation with MDP increased IL-1 and IL-6 in PBMCs from subjects, who carry the ATG16L1 risk variant.261  I did not see any difference in IL-6 between the genotypes in our cohorts. Higher IL-6 production by PBMCs may be specific to MDP stimulation and may be induced downstream of IL-1β production.382,383  TNFα production was also not elevated in PBMCs from subjects, who carry the ATG16L1 risk variant, which may reflect differences in the signalling pathways required for TNFα versus IL-1β transcription.384,385  Production of both IL-6 and TNF may be increased by PBMCs at later time points, downstream of IL-1 production.  IL-1 is an important cytokine in IBD.  It is secreted from intestinal tissues and macrophages isolated from patients with IBD and IL-1 levels correlate with disease severity.232,386-388  IL-1 antagonism has been used effectively to treat some genetically defined forms of very early onset IBD389 and may be more broadly applicable for the treatment of sub-groups of IBD.  To our knowledge, this is the first mechanistic insight into the regulation of IL1B transcription by autophagy and ATG16L1.  Evidence suggests that autophagy modulates the inflammatory response by regulating pro-inflammatory IL-1β production in both mice and in humans.85  In mice, autophagy blocks inflammasome activation by degrading cytosolic danger associated molecular patterns (DAMPs) that activate inflammasomes, mitochondrial DNA, or reactive oxygen species,249 or by degrading 105  inflammasomes/caspase-1250 or pro-IL-1β directly.251  Thus, in mice, inhibition or defects in autophagy may lead to uncontrolled IL-1β production by increasing inflammasome activation, rather than by affecting IL1B transcription.249-251,257,390  In contrast, in humans, increased IL-1β production in people with the ATG16L1 risk variant has been attributed to increased ILIB transcription.261  Our human data are consistent with a model in which autophagy up-regulates SHIP, and SHIP limits the transcription of IL1B.   In summary, we have demonstrated that SHIP mRNA expression and activity are reduced in subjects with ileal CD and are also lower in subjects, who are homozygous for the ATG16L1 CD-associated gene variant, in two independent human cohorts.  SHIP is up-regulated during autophagy and SHIP protein levels and induction are compromised in subjects, who are homozygous for the ATG16L1 CD-associated gene variant.  IL-1 production and IL1B transcription are higher in PBMCs from subjects, who are homozygous for the ATG16L1 CD-associated gene variant.  Importantly, SHIP mRNA expression inversely correlates with IL-1 production and IL1B transcription induced in PBMCs from our subject cohorts.  These results identify SHIP as a critical link between the ATG16L1 CD-associated gene variant and increased IL1B transcription, which may contribute to the increased risk of developing intestinal inflammation in subjects, who carry the ATG16L1 CD-associated gene variant.     106  Chapter 4: Autophagy-related CD genetic variants modulate IL-1β production upon selective action of PRRs   4.1 Introduction and rationale CD is a chronic polygenic disease of the gastrointestinal tract characterized by painful intestinal inflammation. Current evidence indicates that CD arises from a disproportionate immune response to intestinal microflora in genetically susceptible individuals. GWAS have identified several genetic variants encoding proteins linked to the cell homeostatic process, autophagy, as associated with increased susceptibility to CD. Despite that, very little is known of how these genetic variants affect commensal-driven inflammation.  SNPs in genes encoding ATG16L1,35,37,391 NOD2,48 and IRGM38,392 that are associated with CD, result in defective autophagy. Defective autophagy results in enhanced bacterial persistence and intestinal inflammation, 256 and has been associated with increased IL-1β production in both mouse and human cells. In humans, homozygosity for the ATG16L1 T300A-encoding  gene variant is associated with increased IL-1β and IL-6 production in response to muramyl dipeptide.261 In mice, ATG16L1 deletion leads to increased LPS-induced IL-1β production.253 Also, the CD-associated NOD2 variants are associated with reduced NFκB activation in response to MDP.281 Furthermore, TLR2/4-induced NFκB activation is reduced in subjects homozygous for the CD-associated NOD2 variants, R702W, G908R, and L1007fsinsC.282 393 Accumulation of misfolded or unfolded proteins leads to endoplasmic reticulum (ER) stress that initiates the unfolded protein response (UPR) to restore homeostasis.50,51 UPR 107  induces autophagy genes that are important for cell survival and selectively sequester ER membranes into autophagosome-like structures.50 SNPs in XBP1 and ORMDL3, two CD-associated genes required for the UPR have also been implicated in autophagy-related defects in CD,49 but their effects on autophagy and the regulation of IL-1β production in response to PRRs are unknown.  IL-1β has been shown to mediate intestinal inflammation in many mouse models as well as in human CD. In IL-10 KO mice, IL-1β is increased in the small intestine and the colon compared to similar tissue sections from WT mice.242 IL-1β also drives intestinal pathology in both Helicobacter hepaticus-induced and the T cell transfer model of colitis.241 Moreover, colitis in TLR5 KO mice is associated with increased levels of IL-1β in the inflamed tissues.244  We have previously demonstrated that homozygosity for the ATG16L1 T300A-encoding gene variant is associated with reduced mRNA expression and activity of the lipid phosphatase, SHIP, and that, in turn, results in increased IL1B transcription. Based on these findings, we hypothesized that autophagy-related CD genetic variants may be associated with increased IL-1β production. Herein, we measured the effects of five autophagy and CD-related gene variants on cytokine production by human peripheral blood mononuclear cells (PBMCs) in response to TLR and NOD2 ligands. DNA samples were obtained from 45 healthy control subjects, which we genotyped for gene variants in five CD-related genes (ATG16L1, ATG5, NOD2, IRGM, and XBP1), encompassing six SNPs that may be associated with defects in autophagy. Since PBMCs circulate in blood and infiltrate intestinal tissues during inflammation, we measured cytokine production by PBMCs in response to eight TLR ligands and NOD2 ligand comparing responses of the different genotypes.  108  4.2 Materials and methods Subject recruitment and blood collection  The study was performed according to the ethical guidelines approved by the University of British Columbia Research Ethics Boards (H14-00622). All subjects provided written informed consent for the study. Peripheral blood was obtained from 45 healthy control subjects and DNA was prepared for genotyping using the QiaAMP DNA blood mini kit (Qiagen, Toronto, Ontario, Canada). PBMCs were isolated by density gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare, Piscataway, NJ).  Cells were washed and resuspended at a density of 0.5 × 106 cells/mL in RPMI supplemented with 10% FBS.   Genotyping Stored DNA samples were used to genotype CD-associated SNPs (Table 4.1), using commercially available Taqman assay (Applied Biosystems, Burlington, ON, Canada). Table 4.1 also shows the allelic frequency (as percentage) in subjects of European descent for each of the SNPs examined. SNPs were accepted at call rates >95% and frequencies did not deviate from Hardy-Weinberg equilibrium (P>0.05). Subject numbers (n) per genotype are shown in Table 4.2. Total number of subjects per SNP was less than 45 (total number of subjects whose DNA were collected) because the genotyping for some of the subjects for specific SNPs did not work.     109  Table 4.1   Crohn’s disease-associated single nucleotide polymorphisms       Gene  SNP identifier Frequency (%)  Applied Biosystems assay cat # Non-disease homozygous heterozygous CD-associated homozygous ATG16L1 rs2241880 21.6 (AA) 45.2 (AG) 33.2 (GG)   c_9095577_20  ATG5 rs510432 31.9 (AA) 44.2 (AG) 23.9 (GG) c_910351_10 rs12201458 81.7 (CC) 16.7 (CA) 01.7 (AA)   c_30905134_10 NOD2 rs2066844 94.3 (CC) 05.6 (CT) 00.1 (TT) c_11717468_20 IRGM rs7714584 91.2 (AA) 08.8 (AG) 00.0 (GG) c_29565819_10 XBP1 rs35873774 95.1 (TT) 04.9 (TC) 00.0 (CC) c_25625445_10  Table 4.2   Subject numbers (n) per genotype        Gene  SNP  Non-disease homozygous (n)  Heterozygous (n)  CD-associated homozygous (n) ATG16L1 rs2241880 AA (n=9) AG (n=17) GG (n=11) ATG5 rs510432 AA (n-10) AG (n=20) GG (n=8) ATG5 rs12201458 CC (n=26) CA (n=9) AA (n=0) NOD2 rs2066844 CC (n=26) CT (n=5) TT (n=0) IRGM Rs7714584 AA (n=26) AG (n=11) GG (n=4) XBP1 rs35873774 TT (n=33) TC (n=5) CC (n=0)    110  Cell stimulations  For IL-1β production, PBMCs were unstimulated or stimulated with TLR/NOD2 ligands for 5 h, 5 mM ATP for 1 h, or ligands for 4 h with the addition of ATP for the final 1 h. For IL-6, TNFα, and IL-10 production, cells were unstimulated or stimulated with TLR/NOD2 ligands for 24 h. TLRs and NOD2 ligands (InvivoGen, San Diego, CA) used and the concentrations at which they were used are shown in Table 4.3. After incubation (5 h in total for IL-1β and 24 h in total for IL-6, TNFα, and IL-10), supernatants were collected, centrifuged at 10 000 rpm for 5 min to remove floating cells, and were stored at -80°C until they were analyzed by ELISA.  Cytokine measurements Cytokine measurements were performed on clarified, cell-free tissue culture supernatants by ELISA according to the manufacturers΄ instructions. ELISA kits for human IL-β, TNFα, IL-6, and IL-10 were from BD Biosciences (Mississauga, ON).   Statistical Analyses  Unpaired two-tailed Student’s t tests and one-way ANOVA were performed using GraphPad Prism version 5 software.  For multiple comparisons, the Bonferroni correction was applied.  Differences were considered significant at P < 0.05.      111  Table 4.3   PRR ligands and concentrations used for stimulations    PRR Ligand Concentration TLR1/2 Pam3CysSerLys4 (Pam3CSK4) 50 ng/mL TLR2 Heat-killed Listeria monocytogenes (HKLM) 108 cells/mL TLR3 Poly (I:C) low molecular weight (LMW) 10 μg/mL TLR4 E. coli K12 lipopolysaccharide (LPS) 10 ng/mL TLR5 S. typhimurium flagellin 100 ng/mL TLR6 Pam2CGDPKHPKSF (FSL) 100 ng/mL TLR7 Imiquimod (R837) 5 μg/mL TLR8 Single stranded RNA40 (ssRNA40) 5 μg/mL TLR9 CpG ODN 2006 (type B)  5 μM NOD2 Muramyl dipeptide (MDP) 10 μg/mL   4.3 Results 4.3.1 Determination of PRR ligand concentrations to be used for stimulations To determine the concentrations of ligands used for PBMC stimulations, cells were stimulated with different concentrations of ligands for 4 h followed by addition of ATP (5 mM) for 1 h and IL-1β production measured. We found that, all ligands except TLR9 ligand (CpG ODN 2006 type B) activated PBMCs to produce IL-1β (Figure 4.1). TLR9 ligand did not stimulate IL-1β production at low or high doses. As such, TLR9 ligand was not used in the rest of the study. Concentrations used for the different TLR/NOD2 ligands indicated in Table 4.3 are highlighted in red (Figure 4.1). 112   Figure 4.1   TLRs/NOD2 ligand dose response assays  IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs of 4 healthy control subjects stimulated with PRR ligands for 4 h followed by addition of ATP (5 mM) for 1 h. Horizontal lines indicate means ± SEM for PBMCs from 4 subjects.   4.3.2 Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased IL-1β production in response to TLR4 and TLR5 ligands The ATG16L1 T300A-encoding gene variant is associated with increased susceptibility to CD.35-37 Subjects homozygous for the risk allele (GG) have an odds ratio (OR) of 2.38 while heterozygotes have an OR of 1.86,35,259 suggesting that subjects with the ATG16L1 CD-associated gene variant are more likely to develop CD than subjects, who do not have the gene variant. The T300A gene variant significantly increases ATG16L1 susceptible to cleavage by Caspase 3/7 released during cellular/metabolic stress, death receptor ligation, or bacterial infection, resulting in a reduction in full-length, functional ATG16L1, and defective autophagy.254,255 Autophagy acts as a self-regulatory mechanism to 113  limit secretion of inflammatory cytokines, such as IL-1β and IL-23,257 as such, the presence of ATG16L1 T300A-encoding gene variant enhances proinflammatory cytokine secretion.261 To determine the effect of the ATG16L1 rs2241880 risk allele (G) on cytokine production in response to PRR ligands, PBMCs obtained from healthy control subjects with different ATG16L1 genotypes were stimulated with the following ligands (receptors): Pam3CSK4 (TLR1/2), HKLM (TLR2), polyI:C LMW (TLR3), LPS (TLR4), flagellin (TLR5), FSL (TLR6/2), Imiqimod (TLR7), ssRNA40 (TLR8), and MDP (NOD2) in the presence or absence of ATP, and cytokine production was measured in cell-free supernatants (Figure 4.2). Data showing significant differences in cytokine production between genotypes are shown in a red box. Significantly higher IL-1β production was observed in subjects homozygous (GG) for the gene variant associated with CD in response to LPS (TLR4) and flagellin (TLR5) (Figure 4.2A). The rs2241880 genotype did not affect IL-1β production in response to TLR1/2, TLR2, TLR3, TLR6/2, TLR7, TLR8, or NOD2 ligands (Figure 4.2A).  Both IL-6 (Figure 4.2B) and TNFα (Figure 4.2C) production were not affected by the ATG16L1 rs2241880 genotype. Interestingly, homozygosity (GG) for the ATG16L1 gene variant was associated with increased IL-10 production in response to LPS (Figure 4.2D). There was no TNFα (Figure 4.2C) or IL-10 (Figure 4.2D) production in response to TLR3, TLR6/2, and NOD2 ligands, and the TLR7 ligand did not stimulate TNFα production (Figure 4.2C).   114    Figure 4.2   Homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased IL-1β production in response to TLR4/5 ligands+ATP.   (A) IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects and stimulated with TLR/NOD2 ligands+ATP, stratified for the ATG16L1 rs2241880 genotype. Each symbol represents an individual subject. (B) IL-6, (C) TNFα, and (D) IL-10 measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects stimulated with TLR/NOD2 ligands, stratified for the ATG16L1 rs2241880 genotype. Each symbol represents an individual subject. Number of subjects (n) per genotype was: AA (n=9), AG (n=17), and GG (n=11). In (A) - (D), horizontal lines indicate means ± SD. *P ≤ 0.05 (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes).115  4.3.3 ATG5 rs12201458 gene variant is associated with an anti-inflammatory response to TLR4 ligand ATG5 forms a complex with ATG12 and is important for the conjugation of LC3-I with phosphatidylethanolamine to form LC3-II, and formation of the autophagosome. ATG5 is a key component of the autophagy machinery, and enhances autophagic clearance of viruses262 and antigen presentation.263 In mice, Atg5 deletion leads to impaired autophagy and defective lysozyme secretion by Paneth cells.80 The ATG5 rs510432 gene variant is associated with increased ATG5 mRNA expression in human nasal epithelial cells from asthma patients, 267 suggesting that, the ATG5 rs510432 gene variant may increase autophagy, which in turn, may lead to reduced inflammatory cytokine production. We next determined whether the presence of the ATG5 rs510432 or rs12201458 gene variants affected cytokine production in response to PRR ligands. The rs510432 gene variant did not affect cytokine production in response to PRR ligands (Figure 4.3). Intriguingly, the ATG5 rs12201458 gene variant (CA genotype), which is associated with CD, was associated with increased IL-10 production (Figure 4.4D) in response to a TLR4 ligand. The rs12201458 gene variant did not affect IL-1β production (Figure 4.4A), IL-6 production (Figure 4.4B), or TNFα production (Figure 4.4C) in response to PRR ligands. All other ligands tested did not show any difference in IL-10 production (Figure 4.4D) between genotypes.   116   Figure 4.3   The ATG5 rs510432 gene variant does not affect IL-1β production in response to TLRs and NOD2 ligands.  (A) IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects stimulated with TLR/NOD2 ligands+ATP, stratified for the ATG5 rs510432 genotype. (B) IL-6, (C) TNFα, and (D) IL-10 measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects and stimulated with TLR/NOD2 ligands, stratified for the ATG5 rs510432 genotype. Each symbol represents an individual subject. Number of subjects (n) per genotype was: AA (n=10), AG (n=20), and GG (n=8). In (A) - (D), horizontal lines indicate means ± SD. *P ≤ 0.05 (one-way ANOVA with Bonferroni correction for multiple pairwise comparisons between genotypes). 117    Figure 4.4   The ATG5 rs12201458 gene variant is associated with an increase in IL-10 production in response to TLR4 ligand.    (A) IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects stimulated with TLR/NOD2 ligands+ATP, stratified for the ATG5 rs12201458 genotype. (B) IL-6, (C) TNFα, and (D) IL-10 measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects and stimulated with TLR/NOD2 ligands, stratified for the ATG5 rs12201458 genotype. Each symbol represents an individual subject. Number of subjects (n) per genotype was: CC (n=26), CA (n=9), and AA (n=0). In (A) - (D), horizontal lines indicate means ± SD. *P ≤ 0.05 (unpaired Student’s t-test.118  4.3.4 The NOD2 rs2066844 gene variant is associated with increased IL-1β production in response to TLR4 ligand The NOD2 rs2066844 SNP is strongly associated with increased susceptibility to CD.32,33 The OR for individuals homozygous for the SNP (TT) ranges from 20-40, while the OR for heterozygotes (CT) ranges from 2-4,32,33,279,280 suggesting that individuals with the NOD2 rs2066844 risk allele are more likely to develop CD. In the presence of the CD-associated NOD2 gene variants, NOD2 fails to recruit ATG16L1 to the cell membrane at the site of bacterial entry, resulting in defective autophagy, and clearance of intracellular bacteria.284 To determine whether the NOD2 CD-associated risk allele (T) affected cytokine production in response to PRR ligands, we measured cytokine production in PBMCs stimulated with various PAMPs. The NOD2 rs2066844 gene variant was associated with increased IL-1β production in response to LPS+ATP (Figure 4.5A). Activation of TLR1/2 and NOD2 resulted in reduced IL-1β production (Figure 4.5A). No significant differences were observed for IL-6 production (Figure 4.5B), TNFα production (Figure 4.5C), or IL-10 production (Figure 4.5D) between the genotypes.119   Figure 4.5    The NOD2 rs2066844 gene variant is associated with increased IL-1β production in response to TLR4 ligand.  (A) IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects stimulated with TLR/NOD2 ligands+ATP, stratified for the NOD2 rs2066844 genotype. (B) IL-6, (C) TNFα, and (D) IL-10 measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects and stimulated with TLR/NOD2 ligands, stratified for the NOD2 rs2066844 genotype. Each symbol represents an individual subject. Number of subjects (n) per genotype was: CC (n=30), CT (n=5), and TT (n=0). In (A) - (D), horizontal lines indicate means ± SD. *P ≤ 0.05 (unpaired Student’s t-test)120  4.3.5 The IRGM rs7714584 gene variant does not affect IL-1β production in response to TLRs and NOD2 ligands  IRGM is a mitochondrial protein that regulates the initial phase of anti-bacterial autophagy and cellular homeostasis.53,268-270 The IRGM rs7714584 SNP has been strongly associated with increased susceptibility for developing CD.38,272,273 Individuals carrying the IRGM rs7714584 CD-associated gene variant have an OR of 1.37,39 suggesting that the presence of this SNP increases the risk of developing CD. The IRGM SNP has been reported to reduce expression levels of IRGM, there by affecting conversion of LC3-I to LC3-II and results in defective autophagy.272 CD patients harboring the IRGM CD-associated gene variant have reduced autophagy in the inflamed intestinal mucosa,273 as such, the presence of the IRGM rs7714584 gene variant may be associated with increased pro-inflammatory cytokine production. We next determine whether the CD-associated IRGM rs7714584 gene variant altered cytokine production in PBMCs. IL-1β (Figure 4.6A), IL-6 (Figure 4.6B), TNFα (Figure 4.6C), and IL-10 (Figure 4.6D) production were not affected by the presence of rs7714584 gene variant.         121   Figure 4.6   The IRGM rs7714584 gene variant does not affect IL-1β production in response to TLRs and NOD2 ligands.  (A) IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects and stimulated with TLR/NOD2 ligands+ATP, stratified for the IRGM rs7714584 genotype. Each symbol represents an individual subject. (B) IL-6, (C) TNFα, and (D) IL-10 measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects stimulated with TLR/NOD2 ligands, stratified for IRGM rs7714584 genotype. Each symbol represents an individual subject. Number of subjects (n) per genotype was: AA (n=26), AG (n=11), and GG (n=4). In (A) - (D), horizontal lines indicate means ± SD.122  4.3.6 The XBP-1 rs35873774 gene variant is associated with increased IL-1β production in response to PRR ligands XBP-1 is a transcription factor that is activated downstream of the UPR during ER stress.292,293 Accumulation of misfolded proteins within the ER causes ER stress that initiates the UPR.285,286 UPR enhances production of the spliced variant of XBP1 (XBP1s), which is translocated to the nucleus, and up regulates transcription of XBP1 and other UPR target genes to restore protein folding.292-294 TLR2 or TLR4 have also been shown to activate XBP1 in macrophages.294 XBP1 in turn increases transcription of pro-inflammatory genes, such as IL6 and TNFα.294 The XBP1 rs35873774 SNP has been strongly associated with both CD and UC.295-297 The OR for individuals carrying the XBP1 rs35873774 risk variant, associated with IBD is 0.74 (95% confidence interval 0.66-0.84), suggesting that, the risk allele may confer protection from IBD.298 We next determined whether the XBP-1 rs35873774 gene variant associated with IBD affects cytokine production in PBMCs from healthy control subjects. Increased IL-1β production was associated with the XBP-1 gene variant in response to TLR1/2, TLR3, TLR4, TLR7, TLR8, and NOD2 ligands (Figure 4.7A). IL-1β production in response to TLR5 and TLR6/2 agonists were not significantly different (Figure 4.7A). The XBP1 gene variant was also associated with increased IL-6 production (Figure 4.7B) in response to TLR1/2 and TLR8 ligands, and increased TNFα production (Figure 4.7C) in response to TLR5 and TLR8 ligands. There was no effect of the XBP1 rs35873774 genotype on IL-6 production in response to TLR2, TLR3, TLR4, TLR5, TLR6/2, TLR7, and NOD2 ligands (Figure 4.7B); on TNFα production in response to TLR1/2, TLR2, and TLR4 ligands (Figure 4.7C); or on IL-10 production in response to ligands for TLR1/2, TLR2, TLR4, 123  TLR5, and TLR8. TNFα (Figure 4.7C), and IL-10 (Figure 4.7D) were not produced in response to TLR3, TLR6/2, TLR7, or NOD2 ligands. 124   Figure 4.7   The XBP1 rs35873774 gene variant is associated with increased IL-1β production in response to PRR ligands.  (A) IL-1β measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects and stimulated with TLR/NOD2 ligands+ATP, stratified for the XBP1 rs35873774 genotype. Each symbol represents an individual subject. (B) IL-6, (C) TNFα, and (D) IL-10 measured by ELISA in clarified tissue culture supernatants from PBMCs from healthy control subjects stimulated with TLR/NOD2 ligands, stratified for XBP1 rs35873774 genotype. Each symbol represents an individual subject. Number of subjects (n) per genotype was: TT (n=33), TC (n=5), and CC (n=0). In (A) - (D), horizontal lines indicate means ± SD. *P ≤ 0.05 (unpaired Student’s t-test).125  4.4 Discussion The effects of CD-associated genetic variants in ATG16L1,253,261 and NOD2394,395 on IL-1β production have been shown in mouse and human studies. These studies have assessed IL-1β production in response to LPS (TLR4), Pam3CSK4 (TLR2), or MDP (NOD2). No studies have reported the effects of these polymorphisms on IL-1β production in response to other TLR ligands, which are also represented in intestinal flora. Herein, I extend these observations by showing that increased IL-1β production is associated with the ATG16L1 T300A-encoding gene variant in response to flagellin, and the XBP1 rs35873774 CD variant in response to Pam3CSK4 (TLR1/2), HKLM (TLR2), polyI:C (LMW) (TLR3), LPS (TLR4), Imiquimod (TLR7), ssRNA (TLR8), and MDP (NOD2). Furthermore, the XBP1 rs35873774 variant was also associated with increased IL-6 and TNFα production in response to ssRNA (TLR8). Increased IL-10 production by PBMCs was associated with both the ATG16L1 rs2241880 and the ATG5 rs12201458 gene variants in response to LPS. In the intestine, autophagy is an important component of the innate immune response to invading pathogens.396 As such, defects in autophagy due to the presence of the ATG16L1 T300A-encoding gene variant, 35,37,254,255,391 have been associated with defective bacterial clearance, 80,245,260 increased IL-1β production, 253  and CD.16,42,254 We, and others,261 have demonstrated that homozygosity for the ATG16L1 rs2241880 gene variant is associated with increased IL-1β in response to LPS+ATP and MDP, respectively. The present data is consistent with our previous finding and demonstrates that IL-1β is increased in subjects homozygous for the T300A expressing gene variant in response to LPS+ATP in a larger cohort. In addition, our results indicate that homozygosity for the ATG16L1 T300A-encoding gene variant is associated with increased IL-1β production in response to 126  flagellin+ATP. These results suggest that the effect of the rs2241880 gene variant is TLR-specific and affects IL-1β production only when certain PRRs (TLR4, TLR5, and NOD2) are involved. Increased IL-10 production in subjects homozygous for the ATG16L1 T300A-encoding gene variant in response to TLR4 ligand could be associated with a compensatory response aimed at dampening down increased IL-1β and/or a robust immune response leading to the resolution of inflammation.  The ATG5 SNPs (rs510432 and rs12201458) have been associated with increased susceptibility to CD.16,52 However, the effect of these CD-associated SNPs in ATG5 on autophagy and IL-1β remains to be determined. In this study, we show an association between the ATG5 rs12201458 gene variant and increased IL-10 production. We also saw a trend towards increased IL-10 production in response to a TLR5 ligand. These results suggest that the CD-associated ATG5 rs12201458 SNP is associated with an anti-inflammatory response and hence its potential involvement in CD susceptibility needs to be clarified. Based on our observations, we hypothesize that the ATG5 rs12201458 SNP may increase autophagy. This, in turn, may up-regulate the lipid phosphatase, SHIP, and result in reduced IL-1β production in response to stimulation. The effects of the ATG5 rs12201458 SNP on increased susceptibility to CD may be independent of its role in autophagy. The fact that we did not see a difference in cytokine production in the presence of the ATG5 rs510432 SNP, suggests that the effects of this SNP may be cell specific.  The NOD2 gene variants result in defective autophagosome formation and autophagic clearance of intracellular bacteria.256,284 Our data for the NOD2 gene variant are consistent with previous findings. NOD2 variants are associated with increased pro-inflammatory cytokine production in mice due to a gain of function phenotype, which leads to higher IL-1β 127  production after MDP stimulation.397 Previous studies in mice have shown that homozygosity for the Nod22939ic mutation (the mouse homolog of the CD susceptibility allele, 3020insC) does not affect responses to TLR ligands.397 In contrast, in humans, reduced IL1β transcription has been associated with reduced LPS-induced NFκB activation resulting from a loss of function phenotype.398 Consistent with that, we show that IL-1β production is increased in subjects with the CD-associated NOD2 SNP in response to TLR4 ligand, but reduced in response to TLR1/2 and NOD2 ligands. In addition, the limited number of subjects with the NOD2 rs2066844 gene variant within our subject cohort makes it extremely difficult to draw conclusions about its effects in our study. The IRGM rs7714584 gene variant did not affect cytokine production in our cohort. The CD-associated IRGM rs7714584 gene variant has been shown to alter IRGM expression levels leading to defective autophagy.272 In mice, autophagy negatively regulates IL-1β production by degrading cytosolic DAMPs, ROS, and mitochondrial DNA that are known activators of the NLRP3 inflammasome, or by directly degrading pro-IL-1β or active caspase-1.49 Furthermore, we have previously reported that starvation-induced autophagy up regulates SHIP mRNA expression and protein levels in both mouse and human cells, leading to reduced IL-1β production (Chapter 3). Our results suggest that autophagy may not be affected in PBMCs from subjects with the IRGM gene variant, which may result in no change in SHIP mRNA expression and protein levels, and consequently no difference in IL-1β production.  The UPR is triggered upon accumulation of misfolded proteins in the ER and induces autophagy to restore homeostasis.49,50 Accumulation of misfolded proteins in the ER results in unconventional XBP1 mRNA splicing by endonuclease inositol-requiring enzyme 1 128  (IRE1) in the cytoplasm.399 The spliced form of XBP1 (XBP1s) that is produced, up regulates the transcription of proinflammatory cytokine genes, such as IL-6 and TNFα in the nucleus.294 SNPs in genes encoding XBP1 and ORMDL3, key proteins involved in the UPR, have been associated with increased susceptibility to CD,49 but their role in autophagy and IL-1β-mediated intestinal inflammation have not been assessed. Interestingly, our results show that, the XBP1 rs35873774 CD-associated gene variant is associated with increased IL-1β production in response to TLR and NOD2 ligands. Despite the small number of subjects with the XBP1 gene variant used in these experiments, our results demonstrate an important role for the XBP1 gene variant in regulating innate immune responses. In mice, XBP1 deficiency leads to impaired antimicrobial function of Paneth cells and increased Paneth cell apoptosis.298 XBP1 deficiency also induces ER stress and increases susceptibility to DSS-induced colitis.298 TNFα is elevated in colonic tissues of DSS-treated XBP1 deficient mice compared to WT.298 Furthermore, it has been demonstrated  that more than 60% of XBP1-/- mice and 30% of XBP1+/- mice develop spontaneous ileitis associated with ER stress, highlighting a critical role for mono-allelic expression of XBP1 in the onset of intestinal inflammation.298  In humans, expression of the spliced variant of XBP1 (XBP1s), which is induced during ER stress, is increased in both inflamed and non-inflamed ileal and colonic biopsies from CD and UC patients.298 The CD-associated XBP1 rs35873774 gene variant is hypomorphic, causing a partial loss of the gene function, and reduced expression of the UPR and XBP1 target genes.298 Autophagy is induced as a compensatory mechanism during ER stress to clear misfolded proteins. As such, SNPs in genes that are involved in proper functioning of the ER could result in defective autophagy. This may lead to enhanced bacterial persistence in the LP and intestinal inflammation. In addition, ER stress results in 129  the release of mitochondrial ROS and potassium (K+) efflux, which activate the NLRP3 inflammasome, and IL-1β/IL-18 production independent of the UPR.209 Thus, we speculate that the XBP1 SNP causes increased ER activity and ER stress, which leads to release of excess amounts of endogenous DAMPs that activate inflammasomes, and lead to increased IL-1β production. The protective effects of the XBP1 rs35873774 IBD-associated risk variant may be mediated by increased IL-18 production in IECs during ER stress. Previous studies have reported that acute exposure of IECs to DAMPs result in IL-18 production, which stimulates epithelial stem cell differentiation and proliferation, and promotes tissue restitution after injury.237 In addition, SHIP levels may be reduced in subjects with the XBP1 rs35873774 IBD-associated risk variant, resulting in increased IL-1β production.    In summary, our data suggest that the association of ATG16L1 gene variant with increased IL-1β production is PRR specific and that the CD-associated XBP1 gene variant enhances IL-1β production in response to PAMPs. Furthermore, CD is a polygenic disease, as such, it is most likely that genetic variants that alter autophagy and affect SHIP expression may impact the inflammatory response to commensal microbes, and hence predispose to intestinal inflammation.  130  Chapter 5: Concluding remarks and future directions  5.1 Concluding remarks CD may encompass multiple subtypes of disease.  An inappropriate or uncontrolled immune response to commensal bacteria and bacterial antigens in genetically susceptible individuals is believed to be crucial for the development of CD. Inappropriate immune responses could be due to an overreaction of the innate immune system to commensal or pathogenic bacteria, or a shift in the balance in favor of T helper cell responses over regulatory T cell responses. IL-1β is a central mediator of inflammatory diseases, but strangely, IL-1 targeted therapy has not been used in the treatment of IBD. This thesis addresses two very important questions related to CD: (1) can macrophage-derived IL-1β contribute to intestinal inflammation and CD, and (2) are there cellular processes and specific cell signalling pathways that link previously identified CD-associated genetic variants to autoinflammation in CD subjects?  In Chapter 2 we show that SHIP suppresses intestinal macrophage-derived IL-1 production and SHIP deficiency contributes to ileal inflammation. Moreover, depleting intestinal macrophages prevents development of intestinal inflammation in the SHIP-/- mice. Furthermore, we demonstrate that prophylactic treatment with anakinra reduced active caspase-1 and IL-1β production, and prevented development of intestinal pathology in SHIP-/- mice. In Chapter 3, we show that homozygosity for the CD-associated ATG16L1 T300A-encoding gene variant was associated with reduced SHIP mRNA and activity, and increased IL-1β production in healthy control subjects and CD subjects. We also show that autophagy up-regulates SHIP expression, which was compromised in the presence of the 131  T300A-encoding gene variant. SHIP is a critical negative regulator of inflammatory cytokine production including IL-1β, and SHIP deficiency results in increased pro-inflammatory cytokine production. Data from Chapter 3 suggest that, IL-1β is increased in subjects homozygous for the T300A-encoding gene variant because they fail to up-regulate the negative regulator, SHIP.  Finally, in Chapter 4, we confirm the association between homozygosity for the ATG16L1 CD-associated gene variant and increased IL-1β production, and identify a possible association between the XBP1 rs35875774 gene variant and increased IL-1β production. Indeed, the XBP1 rs35875774 gene variant was associated with increased IL-1β production in response to several intestinal microbial-associated molecular patterns (MAMPs) that activate different PRRs in the presence of ATP.  SNPs in genes encoding proteins that play key roles in the regulation of both innate and adaptive immune responses have been associated with IBD. We have shown that these SNPs could serve as important elements in the identification of different subtypes of CD. We have identified a unique disease phenotype in CD patients that is associated with homozygosity for the ATG16L1 T300A-encoding gene variant. Recent studies have shown that the presence of the CD risk variant in ATG16L1 increases its degradation by Caspase 3/7 activated during metabolic stress, death receptor ligation or bacterial infection, 254,255 and result in defective autophagy. We have shown that subjects homozygous for the CD risk variant in ATG16L1 not only have defective autophagy, but also have reduced SHIP expression and activity. The T300A-encoding gene variant and defective autophagy have been associated with increased IL-1β production in both mice and humans.48,250-252,261 Hence, subjects homozygous for the CD-associated ATG16L1 gene variant have defective 132  autophagy, which may lead to reduced SHIP expression and activity, and in turn increase IL-1β production.  CD has been included among polygenic diseases associated with autoinflammation due to overlapping features, which include its periodicity, strong association with environmental triggers, and a failure to respond to therapy directed against TNF.352  Identification of disease subtypes for personalized therapeutic intervention is the new goal of current IBD research. In the past two decades, biological therapies, such as infliximab and adalimumab, which are monoclonal antibodies against TNFα, have been used to manage IBD. Studies have shown that, treating CD patients early in the course of disease with infliximab is the most efficacious way to induce mucosal healing in patients.400 In addition, adult IBD patients, who not responsive to infliximab, have been treated with adalimumab, which can induce and maintain remission.401 However, the use of anti-TNFα drugs increases the possibility of opportunistic infections, since anti-TNFα may reduce the body’s ability to fight infectious diseases. Also, the large number (30%) of CD patients that are predicted to become refractory to current therapy and the fact that some patients develop antibodies against these biological therapies, underscores the urgent need to develop novel therapeutic strategies to target intestinal inflammation in CD. IL-1 antagonism with anakinra, the synthetic IL-1 receptor antagonist, rilonacept, the soluble IL-1 decoy receptor, or canakinumab, a human monoclonal Ab against IL-1 has been used to treat many different autoinflammatory diseases in humans, but not CD. Anakinra is currently used to treat autoinflammatory diseases that I have previously discussed in Chapter 1, but has a very short half-life and causes transient injection site reactions in most patients. It was approved by the US Food and Drug Administration (FDA) in 2001 for the treatment of 133  rheumatoid arthritis. Rilonacept, on the other hand, is a soluble decoy receptor that binds IL-1, and prevents IL-1 signalling events. The US FDA approved the use of this drug in 2008 for the treatment of CAPS in both adults and children, 12 years and older. Canakinumab is a human monoclonal antibody that specifically binds free floating human IL-1β, forming a complex that does not bind the IL-1 receptor, and hence prevents IL-1β-dependent signalling. It was approved in 2009 by the US FDA for the treatment of CAPS in both adults and children. Canakinumab has a long half-life and does not require daily or weekly injections, as is the case with the other approved anti-IL-1β therapies. IL-1-targeted therapy could serve as an important option for CD patients with the T300A gene variant. As shown in our in vivo studies in SHIP-/- mice, anakinra prevents CD-like intestinal pathologies by directly targeting IL-1 signalling, reducing up-regulation of IL-1 mRNA, IL-1β, and IL-6. In addition, evidence suggests that anti-IL-1β therapies reduce neutrophil recruitment, and accumulation of IL-17-producing cells in the inflamed mucosal tissue.241 A recent study by Antonella de Luca et al demonstrated that anakinra restores autophagy and significantly reduces IL-1β production, and inflammation in chronic granulomatous disease (CGD) patients and in mice.220 Our results indicate that active caspase 1 is reduced in ilea from SHIP-/- mice treated with anakinra, suggesting that this reduction might be associated with increased autophagy. Previous studies have shown that autophagy increases sequestration and degradation of inflammasomes and caspase-1 in autophagosomes when they fuse with lysosomes.250,251 Clinically, anakinra treatment could also be useful for CD patients, who have undergone small bowel resection for segmental disease, and, who are at high risk for recurrence. In this group of patients, anakinra could delay or prevent recurrence. Further 134  studies and clinical trials in subjects, who have failed current biological therapies, will shed more light on the future use of IL-1-targeted therapy in the treatment of CD.   Owing to the central role of inflammasomes in the pathogenesis of IBD, therapy that targets these multi-protein complexes may serve as therapeutic options for the treatment of these diseases. Our in vitro data suggest that, blocking the NLRP3 inflammasomes significantly reduces macrophage-derived IL-1β production, and could be a therapeutic target to reduce intestinal pathology. While this may be an important therapeutic target, identifying the specific inflammasome involved in IL-1β production during IBD will help increase the efficacy of a therapy of this nature. We also identify a reduction in SHIP expression and activity as an important immune-regulatory defect associated with the ATG16L1 CD SNP. Intriguingly, despite its up-regulation by autophagy, SHIP is also required for effective autophagy, suggesting that, enhancing SHIP expression, and/or activity could also be a possible therapeutic strategy to control IL-1β production and reduce intestinal inflammation in CD subjects with the T300A-encoding gene variant. SHIP agonists, such as Pelerol have been developed and enhance SHIP activity by two fold.402 Despite these findings, much still needs to be done to test the efficacy of these SHIP agonists at reducing intestinal inflammation.  We have demonstrated that depletion of phagocytic cells was sufficient to prevent development of CD-like pathology in SHIP deficient mice. Phagocytic cells are the main source of IL-1β that promotes intestinal inflammation. As such, specifically targeting these cells may serve as a therapeutic option for the treatment of IBD. Histological analysis of mucosal biopsies from IBD patients has shown that, the presence of massive granulocyte infiltrates correlates with disease severity.403 Leukocyte apheresis, which involves 135  extracorporeal purification of the blood by selectively removing undesired granulocytes and monocytes [granulocyte and monocyte adsorptive (GMA) apheresis] from the blood stream has been successfully used to treat IBD patients.404-406 Patients had relatively fewer side effects and significantly higher remission rates compared to patients on conventional IBD therapy.407 Further studies are required to fully determine if this therapeutic option could be used for patients with severe and/or refractory CD. Targeting Caspase activity, especially caspase-1, -3, or -7, could serve as a therapeutic option for treatment of IBD. Uncontrolled caspase activity is associated with development of IBD and colorectal cancer.408 We have shown that, increased caspase-1 activity in the inflamed ileum correlates with intestinal pathology in the SHIP deficient mice. Therapy that regulates caspase-1 activity could be an important therapeutic option for IBD patients. Furthermore, caspase 3/7 is required for the degradation of ATG16L1 containing the T300A-encoding gene variant, resulting in defective autophagy, pathogen expansion, and intestinal inflammation.254,255 As such, targeting caspase 3 or 7 could restore autophagy in subjects with the ATG16L1 gene variant, increase pathogen clearance, and prevent development of CD. In this regard, current biological therapy for IBD that targets TNFα, prevent caspase activation, and IEC and Paneth cell death.409 Pralnacasan, a potent non-peptide inhibitor of caspase-1 was shown to pharmacologically block caspase-1 activity during DSS-induced colitis, effectively reducing clinical disease in these mice.370,410 Nevertheless, owing to demonstrated liver abnormalities in certain animal studies, phase II clinical trials to test the efficacy of this drug was halted by the producing companies. Furthermore, current inhibitors of caspase activity are non-specific and may also inhibit the activity of caspases required for certain organ development. Future research could be aimed at developing small molecule 136  inhibitors that specifically target inflammatory caspases, such as caspase-1, -3, or -7 that have been strongly implicated in the development of CD. Finally, our data identify defective autophagy as central for the development of auto-inflammatory CD. Drugs that induce autophagy have been used to prevent or delay the onset of neurodegenerative diseases, such as Huntington’s disease.411 Treatment options aimed at inducing autophagy in cells may prove clinically useful in the treatment of CD patients with the ATG16L1 gene variant. Increasing autophagy in these patients will increase bacterial clearance, and reduce the presence of inflammasomes, caspase 1, and IL-1β production, and may reduce inflammation. Recently, sirolimus (rapamycin), which induces autophagy, has been shown to induce clinical remission and mucosal healing in children with severe IBD that were refractory to conventional anti-TNFα therapy.412  Taken together, data in this thesis suggest that, SHIP is reduced in CD subjects with the ATG16L1 gene variant because they have defective autophagy. Our data also demonstrate that autophagy up-regulates SHIP, and as such, defective autophagy compromises SHIP up-regulation in CD subjects. Furthermore, reduced SHIP expression results in increased IL-1β production, which drives intestinal auto-inflammation. Blocking IL-1 signalling with an IL-1Ra, anakinra, prevents development of intestinal pathology.  Figure 5.1 summarizes our model of how subjects with the ATG16L1 gene variant develop intestinal inflammation in CD and how anakinra can be used to reduce inflammation.    5.2 Future directions Future studies will focus on examining the impact of additional autophagy-related CD susceptibility SNPs on autophagy, SHIP mRNA expression/activity, and IL-1 production in 137  a larger cohort to confirm this CD phenotype that is associated with defective autophagy and also identify subjects that could be amenable to treatment with IL-1 targeted therapy. We will also examine the impact of the ORMDL3 rs2872507 gene variant on cytokine production in our cohort. Furthermore, it will be interesting to determine if some of the trends seen in this thesis are confirmed in a larger cohort with many subjects having the NOD2 (rs2066844), and XBP1 (rs35873774) gene variants. I demonstrated in Chapter 3 that the homozygosity for the ATG16L1 T300A-encoding gene variant is associated with reduced SHIP mRNA expression and activity, and that starvation-induced autophagy increases protein levels of SHIP. It will be important to determine if SHIP mRNA expression and protein levels, as well as SHIP activity, are affected by the presence of other CD-associated genetic variants. Autophagy will be determined by looking at LC3-II protein levels amongst the genotypes of the different SNPs to establish if defective autophagy could be associated with increased IL-1β as previously shown. The results of these studies will be a significant step forward in the identification of specific CD phenotypes necessary for development of personalized therapeutic approaches.        138  5.3 Thesis model    Figure 5.1   Autophagy regulates IL-1β production and intestinal auto-inflammation via SHIP up-regulation.  In subjects without the ATG16L1 gene variant, ATG16L1 protein is fully functional and sets the platform for autophagy. Autophagy up-regulates SHIP protein expression, which in turn blocks IL-1β production, and inflammation. In contrast, in subjects with the T300A-encoding gene variant, ATG16L1 protein is prone to caspase-3/7-mediated cleavage, resulting in defective autophagy. Defective autophagy results in reduced SHIP expression and increased IL-1β production. Increased IL-1β drives intestinal auto-inflammation and results in CD. Anakinra (IL-1Ra) restores defective autophagy, and blocks IL-1 signalling and intestinal auto-inflammation.   139  References 1. Hendrickson BA, Gokhale R, Cho JH. Clinical aspects and pathophysiology of inflammatory bowel disease. Clinical microbiology reviews 2002;15:79-94. 2. Rosenstiel P, Sina C, Franke A, Schreiber S. Towards a molecular risk map--recent advances on the etiology of inflammatory bowel disease. Seminars in immunology 2009;21:334-45. 3. Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 2007;369:1641-57. 4. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007;448:427-34. 5. Abraham C, Cho J. Interleukin-23/Th17 pathways and inflammatory bowel disease. Inflammatory bowel diseases 2009;15:1090-100. 6. Molodecky NA, Soon IS, Rabi DM, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology 2012;142:46-54 e42; quiz e30. 7. Cosnes J, Gower-Rousseau C, Seksik P, Cortot A. Epidemiology and natural history of inflammatory bowel diseases. Gastroenterology 2011;140:1785-94. 8. Bernstein CN, Wajda A, Svenson LW, et al. The epidemiology of inflammatory bowel disease in Canada: a population-based study. The American journal of gastroenterology 2006;101:1559-68. 9. Fakhoury M, Negrulj R, Mooranian A, Al-Salami H. Inflammatory bowel disease: clinical aspects and treatments. Journal of inflammation research 2014;7:113-20. 140  10. Rocchi A, Benchimol EI, Bernstein CN, et al. Inflammatory bowel disease: a Canadian burden of illness review. Canadian journal of gastroenterology = Journal canadien de gastroenterologie 2012;26:811-7. 11. Benchimol EI, Guttmann A, Griffiths AM, et al. Increasing incidence of paediatric inflammatory bowel disease in Ontario, Canada: evidence from health administrative data. Gut 2009;58:1490-7. 12. Bernklev T, Jahnsen J, Aadland E, et al. Health-related quality of life in patients with inflammatory bowel disease five years after the initial diagnosis. Scandinavian journal of gastroenterology 2004;39:365-73. 13. Graff LA, Vincent N, Walker JR, et al. A population-based study of fatigue and sleep difficulties in inflammatory bowel disease. Inflammatory bowel diseases 2011;17:1882-9. 14. Kanof ME, Lake AM, Bayless TM. Decreased height velocity in children and adolescents before the diagnosis of Crohn's disease. Gastroenterology 1988;95:1523-7. 15. Bernstein CN, Fried M, Krabshuis JH, et al. World Gastroenterology Organization Practice Guidelines for the diagnosis and management of IBD in 2010. Inflammatory bowel diseases 2010;16:112-24. 16. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491:119-24. 17. Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 2011;474:298-306. 18. Solberg IC, Vatn MH, Hoie O, et al. Clinical course in Crohn's disease: results of a Norwegian population-based ten-year follow-up study. Clinical gastroenterology and 141  hepatology : the official clinical practice journal of the American Gastroenterological Association 2007;5:1430-8. 19. Naija N, Karoui S, Serghini M, Kallel L, Boubaker J, Filali A. [Management of failure of infliximab in inflammatory bowel disease]. La Tunisie medicale 2011;89:517-21. 20. Yanai H, Hanauer SB. Assessing response and loss of response to biological therapies in IBD. The American journal of gastroenterology 2011;106:685-98. 21. Halme L, Paavola-Sakki P, Turunen U, Lappalainen M, Farkkila M, Kontula K. Family and twin studies in inflammatory bowel disease. World journal of gastroenterology : WJG 2006;12:3668-72. 22. Halfvarson J, Bodin L, Tysk C, Lindberg E, Jarnerot G. Inflammatory bowel disease in a Swedish twin cohort: a long-term follow-up of concordance and clinical characteristics. Gastroenterology 2003;124:1767-73. 23. Orholm M, Binder V, Sorensen TI, Rasmussen LP, Kyvik KO. Concordance of inflammatory bowel disease among Danish twins. Results of a nationwide study. Scandinavian journal of gastroenterology 2000;35:1075-81. 24. Thompson NP, Driscoll R, Pounder RE, Wakefield AJ. Genetics versus environment in inflammatory bowel disease: results of a British twin study. Bmj 1996;312:95-6. 25. Yang H, McElree C, Roth MP, Shanahan F, Targan SR, Rotter JI. Familial empirical risks for inflammatory bowel disease: differences between Jews and non-Jews. Gut 1993;34:517-24. 26. Bayless TM, Tokayer AZ, Polito JM, 2nd, Quaskey SA, Mellits ED, Harris ML. Crohn's disease: concordance for site and clinical type in affected family members--potential hereditary influences. Gastroenterology 1996;111:573-9. 142  27. Carbonnel F, Macaigne G, Beaugerie L, Gendre JP, Cosnes J. Crohn's disease severity in familial and sporadic cases. Gut 1999;44:91-5. 28. Orholm M, Munkholm P, Langholz E, Nielsen OH, Sorensen TI, Binder V. Familial occurrence of inflammatory bowel disease. The New England journal of medicine 1991;324:84-8. 29. Peeters M, Nevens H, Baert F, et al. Familial aggregation in Crohn's disease: increased age-adjusted risk and concordance in clinical characteristics. Gastroenterology 1996;111:597-603. 30. Ananthakrishnan AN. Epidemiology and risk factors for IBD. Nature reviews Gastroenterology & hepatology 2015. 31. Probert CS, Jayanthi V, Hughes AO, Thompson JR, Wicks AC, Mayberry JF. Prevalence and family risk of ulcerative colitis and Crohn's disease: an epidemiological study among Europeans and south Asians in Leicestershire. Gut 1993;34:1547-51. 32. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001;411:599-603. 33. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001;411:603-6. 34. Duerr RH, Taylor KD, Brant SR, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 2006;314:1461-3. 35. Hampe J, Franke A, Rosenstiel P, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nature genetics 2007;39:207-11. 143  36. Prescott NJ, Fisher SA, Franke A, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn's disease and is independent of CARD15 and IBD5. Gastroenterology 2007;132:1665-71. 37. Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nature genetics 2007;39:596-604. 38. Parkes M, Barrett JC, Prescott NJ, et al. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility. Nature genetics 2007;39:830-2. 39. Barrett JC, Hansoul S, Nicolae DL, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nature genetics 2008;40:955-62. 40. Wang K, Zhang H, Kugathasan S, et al. Diverse genome-wide association studies associate the IL12/IL23 pathway with Crohn Disease. American journal of human genetics 2009;84:399-405. 41. Imielinski M, Baldassano RN, Griffiths A, et al. Common variants at five new loci associated with early-onset inflammatory bowel disease. Nature genetics 2009;41:1335-40. 42. Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nature genetics 2010;42:1118-25. 43. International HapMap C. The International HapMap Project. Nature 2003;426:789-96. 44. Hugot JP, Laurent-Puig P, Gower-Rousseau C, et al. Mapping of a susceptibility locus for Crohn's disease on chromosome 16. Nature 1996;379:821-3. 144  45. Libioulle C, Louis E, Hansoul S, et al. Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4. PLoS genetics 2007;3:e58. 46. Franke A, Balschun T, Karlsen TH, et al. Replication of signals from recent studies of Crohn's disease identifies previously unknown disease loci for ulcerative colitis. Nature genetics 2008;40:713-5. 47. Silverberg MS, Cho JH, Rioux JD, et al. Ulcerative colitis-risk loci on chromosomes 1p36 and 12q15 found by genome-wide association study. Nature genetics 2009;41:216-20. 48. Ramjeet M, Hussey S, Philpott DJ, Travassos LH. 'Nodophagy': New crossroads in Crohn disease pathogenesis. Gut microbes 2010;1:307-15. 49. Fritz T, Niederreiter L, Adolph T, Blumberg RS, Kaser A. Crohn's disease: NOD2, autophagy and ER stress converge. Gut 2011;60:1580-8. 50. Bernales S, McDonald KL, Walter P. Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLoS biology 2006;4:e423. 51. Michallet AS, Mondiere P, Taillardet M, Leverrier Y, Genestier L, Defrance T. Compromising the unfolded protein response induces autophagy-mediated cell death in multiple myeloma cells. PloS one 2011;6:e25820. 52. Yousefi S, Perozzo R, Schmid I, et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nature cell biology 2006;8:1124-32. 53. Singh SB, Davis AS, Taylor GA, Deretic V. Human IRGM induces autophagy to eliminate intracellular mycobacteria. Science 2006;313:1438-41. 54. Loftus EV, Jr. Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences. Gastroenterology 2004;126:1504-17. 145  55. Rubin DT, Hanauer SB. Smoking and inflammatory bowel disease. European journal of gastroenterology & hepatology 2000;12:855-62. 56. Mahid SS, Minor KS, Soto RE, Hornung CA, Galandiuk S. Smoking and inflammatory bowel disease: a meta-analysis. Mayo Clinic proceedings 2006;81:1462-71. 57. Sopori M. Effects of cigarette smoke on the immune system. Nature reviews Immunology 2002;2:372-7. 58. Virta L, Auvinen A, Helenius H, Huovinen P, Kolho KL. Association of repeated exposure to antibiotics with the development of pediatric Crohn's disease--a nationwide, register-based finnish case-control study. American journal of epidemiology 2012;175:775-84. 59. Godet PG, May GR, Sutherland LR. Meta-analysis of the role of oral contraceptive agents in inflammatory bowel disease. Gut 1995;37:668-73. 60. Berg DJ, Zhang J, Weinstock JV, et al. Rapid development of colitis in NSAID-treated IL-10-deficient mice. Gastroenterology 2002;123:1527-42. 61. Mouli VP, Ananthakrishnan AN. Review article: vitamin D and inflammatory bowel diseases. Alimentary pharmacology & therapeutics 2014;39:125-36. 62. Levenstein S, Prantera C, Varvo V, et al. Stress and exacerbation in ulcerative colitis: a prospective study of patients enrolled in remission. The American journal of gastroenterology 2000;95:1213-20. 63. Collins SM. Stress and the Gastrointestinal Tract IV. Modulation of intestinal inflammation by stress: basic mechanisms and clinical relevance. American journal of physiology Gastrointestinal and liver physiology 2001;280:G315-8. 146  64. Straub RH, Dhabhar FS, Bijlsma JW, Cutolo M. How psychological stress via hormones and nerve fibers may exacerbate rheumatoid arthritis. Arthritis and rheumatism 2005;52:16-26. 65. Fortes C, Farchi S, Forastiere F, et al. Depressive symptoms lead to impaired cellular immune response. Psychother Psychosom 2003;72:253-60. 66. Danner M, Kasl SV, Abramson JL, Vaccarino V. Association between depression and elevated C-reactive protein. Psychosom Med 2003;65:347-56. 67. Goebel MU, Mills PJ, Irwin MR, Ziegler MG. Interleukin-6 and tumor necrosis factor-alpha production after acute psychological stress, exercise, and infused isoproterenol: differential effects and pathways. Psychosom Med 2000;62:591-8. 68. Bamias G, Corridoni D, Pizarro TT, Cominelli F. New insights into the dichotomous role of innate cytokines in gut homeostasis and inflammation. Cytokine 2012;59:451-9. 69. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 2014;505:559-63. 70. Gevers D, Kugathasan S, Denson LA, et al. The treatment-naive microbiome in new-onset Crohn's disease. Cell host & microbe 2014;15:382-92. 71. Kostic AD, Xavier RJ, Gevers D. The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014;146:1489-99. 72. Darfeuille-Michaud A, Boudeau J, Bulois P, et al. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease. Gastroenterology 2004;127:412-21. 73. Cadwell K, Patel KK, Maloney NS, et al. Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine. Cell 2010;141:1135-45. 147  74. Frank DN, Robertson CE, Hamm CM, et al. Disease phenotype and genotype are associated with shifts in intestinal-associated microbiota in inflammatory bowel diseases. Inflammatory bowel diseases 2011;17:179-84. 75. Podolsky DK. Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense. The American journal of physiology 1999;277:G495-9. 76. Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J. Restoration of barrier function in injured intestinal mucosa. Physiological reviews 2007;87:545-64. 77. Wallace KL, Zheng LB, Kanazawa Y, Shih DQ. Immunopathology of inflammatory bowel disease. World journal of gastroenterology : WJG 2014;20:6-21. 78. Salim SY, Soderholm JD. Importance of disrupted intestinal barrier in inflammatory bowel diseases. Inflammatory bowel diseases 2011;17:362-81. 79. Buisine MP, Desreumaux P, Debailleul V, et al. Abnormalities in mucin gene expression in Crohn's disease. Inflammatory bowel diseases 1999;5:24-32. 80. Cadwell K, Liu JY, Brown SL, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 2008;456:259-63. 81. Madsen KL, Malfair D, Gray D, Doyle JS, Jewell LD, Fedorak RN. Interleukin-10 gene-deficient mice develop a primary intestinal permeability defect in response to enteric microflora. Inflammatory bowel diseases 1999;5:262-70. 82. Zaki MH, Lamkanfi M, Kanneganti TD. The Nlrp3 inflammasome: contributions to intestinal homeostasis. Trends in immunology 2011;32:171-9. 83. Creagh EM, O'Neill LA. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends in immunology 2006;27:352-7. 148  84. Zelensky AN, Gready JE. The C-type lectin-like domain superfamily. The FEBS journal 2005;272:6179-217. 85. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nature reviews Immunology 2013;13:397-411. 86. Lavelle EC, Murphy C, O'Neill LA, Creagh EM. The role of TLRs, NLRs, and RLRs in mucosal innate immunity and homeostasis. Mucosal immunology 2010;3:17-28. 87. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004;118:229-41. 88. Elson CO, Cong Y, McCracken VJ, Dimmitt RA, Lorenz RG, Weaver CT. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunological reviews 2005;206:260-76. 89. Cario E, Podolsky DK. Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infection and immunity 2000;68:7010-7. 90. Hausmann M, Kiessling S, Mestermann S, et al. Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation. Gastroenterology 2002;122:1987-2000. 91. Buonocore S, Ahern PP, Uhlig HH, et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 2010;464:1371-5. 92. Sakuraba A, Sato T, Kamada N, Kitazume M, Sugita A, Hibi T. Th1/Th17 immune response is induced by mesenteric lymph node dendritic cells in Crohn's disease. Gastroenterology 2009;137:1736-45. 149  93. Chinen H, Matsuoka K, Sato T, et al. Lamina propria c-kit+ immune precursors reside in human adult intestine and differentiate into natural killer cells. Gastroenterology 2007;133:559-73. 94. Takayama T, Kamada N, Chinen H, et al. Imbalance of NKp44(+)NKp46(-) and NKp44(-)NKp46(+) natural killer cells in the intestinal mucosa of patients with Crohn's disease. Gastroenterology 2010;139:882-92, 92 e1-3. 95. Romagnani S. Lymphokine production by human T cells in disease states. Annual review of immunology 1994;12:227-57. 96. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235-8. 97. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nature immunology 2007;8:942-9. 98. Zhou L, Ivanov, II, Spolski R, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nature immunology 2007;8:967-74. 99. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nature immunology 2005;6:1123-32. 100. Izcue A, Coombes JL, Powrie F. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunological reviews 2006;212:256-71. 150  101. Annunziato F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. The Journal of experimental medicine 2007;204:1849-61. 102. Hue S, Ahern P, Buonocore S, et al. Interleukin-23 drives innate and T cell-mediated intestinal inflammation. The Journal of experimental medicine 2006;203:2473-83. 103. Yen D, Cheung J, Scheerens H, et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. The Journal of clinical investigation 2006;116:1310-6. 104. Noguchi M, Hiwatashi N, Liu Z, Toyota T. Enhanced interferon-gamma production and B7-2 expression in isolated intestinal mononuclear cells from patients with Crohn's disease. Journal of gastroenterology 1995;30 Suppl 8:52-5. 105. Fuss IJ, Neurath M, Boirivant M, et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. Journal of immunology 1996;157:1261-70. 106. Strober W, Fuss IJ. Proinflammatory cytokines in the pathogenesis of inflammatory bowel diseases. Gastroenterology 2011;140:1756-67. 107. Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. The Journal of clinical investigation 2007;117:514-21. 108. Monteleone G, Biancone L, Marasco R, et al. Interleukin 12 is expressed and actively released by Crohn's disease intestinal lamina propria mononuclear cells. Gastroenterology 1997;112:1169-78. 109. Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 2003;52:65-70. 151  110. McGovern D, Powrie F. The IL23 axis plays a key role in the pathogenesis of IBD. Gut 2007;56:1333-6. 111. Kamada N, Hisamatsu T, Okamoto S, et al. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis. The Journal of clinical investigation 2008;118:2269-80. 112. Wada Y, Hisamatsu T, Kamada N, Okamoto S, Hibi T. Retinoic acid contributes to the induction of IL-12-hypoproducing dendritic cells. Inflammatory bowel diseases 2009;15:1548-56. 113. Dubinsky MC, Wang D, Picornell Y, et al. IL-23 receptor (IL-23R) gene protects against pediatric Crohn's disease. Inflammatory bowel diseases 2007;13:511-5. 114. Sandborn WJ, Feagan BG, Fedorak RN, et al. A randomized trial of Ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with moderate-to-severe Crohn's disease. Gastroenterology 2008;135:1130-41. 115. Hueber W, Sands BE, Lewitzky S, et al. Secukinumab, a human anti-IL-17A monoclonal antibody, for moderate to severe Crohn's disease: unexpected results of a randomised, double-blind placebo-controlled trial. Gut 2012;61:1693-700. 116. Kamada N, Hisamatsu T, Honda H, et al. TL1A produced by lamina propria macrophages induces Th1 and Th17 immune responses in cooperation with IL-23 in patients with Crohn's disease. Inflammatory bowel diseases 2010;16:568-75. 117. Monteleone G, Monteleone I, Fina D, et al. Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease. Gastroenterology 2005;128:687-94. 152  118. Brand S, Beigel F, Olszak T, et al. IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration. American journal of physiology Gastrointestinal and liver physiology 2006;290:G827-38. 119. Yang XO, Chang SH, Park H, et al. Regulation of inflammatory responses by IL-17F. The Journal of experimental medicine 2008;205:1063-75. 120. Sugimoto K, Ogawa A, Mizoguchi E, et al. IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. The Journal of clinical investigation 2008;118:534-44. 121. Zenewicz LA, Yancopoulos GD, Valenzuela DM, Murphy AJ, Stevens S, Flavell RA. Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease. Immunity 2008;29:947-57. 122. Ye P, Rodriguez FH, Kanaly S, et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. The Journal of experimental medicine 2001;194:519-27. 123. Monteleone G, Caruso R, Fina D, et al. Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21. Gut 2006;55:1774-80. 124. Caruso R, Fina D, Peluso I, et al. A functional role for interleukin-21 in promoting the synthesis of the T-cell chemoattractant, MIP-3alpha, by gut epithelial cells. Gastroenterology 2007;132:166-75. 125. O'Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nature medicine 2004;10:801-5. 153  126. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood 2006;108:253-61. 127. Fantini MC, Becker C, Tubbe I, et al. Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis. Gut 2006;55:671-80. 128. Chamouard P, Monneaux F, Richert Z, et al. Diminution of Circulating CD4+CD25 high T cells in naive Crohn's disease. Digestive diseases and sciences 2009;54:2084-93. 129. Maul J, Loddenkemper C, Mundt P, et al. Peripheral and intestinal regulatory CD4+ CD25(high) T cells in inflammatory bowel disease. Gastroenterology 2005;128:1868-78. 130. Saruta M, Yu QT, Fleshner PR, et al. Characterization of FOXP3+CD4+ regulatory T cells in Crohn's disease. Clinical immunology 2007;125:281-90. 131. Fahlen L, Read S, Gorelik L, et al. T cells that cannot respond to TGF-beta escape control by CD4(+)CD25(+) regulatory T cells. The Journal of experimental medicine 2005;201:737-46. 132. Monteleone G, Kumberova A, Croft NM, McKenzie C, Steer HW, MacDonald TT. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease. The Journal of clinical investigation 2001;108:601-9. 133. Monteleone G, Fantini MC, Onali S, et al. Phase I clinical trial of Smad7 knockdown using antisense oligonucleotide in patients with active Crohn's disease. Molecular therapy : the journal of the American Society of Gene Therapy 2012;20:870-6. 134. Vermeire S. Oral SMAD7 antisense drug for Crohn's disease. The New England journal of medicine 2015;372:1166-7. 154  135. Sandborn WJ. How to avoid treating irritable bowel syndrome with biologic therapy for inflammatory bowel disease. Digestive diseases 2009;27 Suppl 1:80-4. 136. Day AS, Burgess L. Exclusive enteral nutrition and induction of remission of active Crohn's disease in children. Expert review of clinical immunology 2013;9:375-83; quiz 84. 137. Hazlewood GS, Rezaie A, Borman M, et al. Comparative effectiveness of immunosuppressants and biologics for inducing and maintaining remission in Crohn's disease: a network meta-analysis. Gastroenterology 2015;148:344-54 e5; quiz e14-5. 138. Lichtenstein GR. Comprehensive review: antitumor necrosis factor agents in inflammatory bowel disease and factors implicated in treatment response. Therapeutic advances in gastroenterology 2013;6:269-93. 139. Garlanda C, Dinarello CA, Mantovani A. The interleukin-1 family: back to the future. Immunity 2013;39:1003-18. 140. Mirchandani AS, Salmond RJ, Liew FY. Interleukin-33 and the function of innate lymphoid cells. Trends in immunology 2012;33:389-96. 141. Talabot-Ayer D, Lamacchia C, Gabay C, Palmer G. Interleukin-33 is biologically active independently of caspase-1 cleavage. The Journal of biological chemistry 2009;284:19420-6. 142. Prefontaine D, Nadigel J, Chouiali F, et al. Increased IL-33 expression by epithelial cells in bronchial asthma. The Journal of allergy and clinical immunology 2010;125:752-4. 143. Kondo Y, Yoshimoto T, Yasuda K, et al. Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system. International immunology 2008;20:791-800. 155  144. Kurowska-Stolarska M, Stolarski B, Kewin P, et al. IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation. Journal of immunology 2009;183:6469-77. 145. Stolarski B, Kurowska-Stolarska M, Kewin P, Xu D, Liew FY. IL-33 exacerbates eosinophil-mediated airway inflammation. Journal of immunology 2010;185:3472-80. 146. Vigne S, Palmer G, Lamacchia C, et al. IL-36R ligands are potent regulators of dendritic and T cells. Blood 2011;118:5813-23. 147. Vigne S, Palmer G, Martin P, et al. IL-36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naive CD4+ T cells. Blood 2012;120:3478-87. 148. Blumberg H, Dinh H, Trueblood ES, et al. Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation. The Journal of experimental medicine 2007;204:2603-14. 149. Towne JE, Renshaw BR, Douangpanya J, et al. Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36alpha, IL-36beta, and IL-36gamma) or antagonist (IL-36Ra) activity. The Journal of biological chemistry 2011;286:42594-602. 150. Wilson NJ, Boniface K, Chan JR, et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nature immunology 2007;8:950-7. 151. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annual review of immunology 2001;19:423-74. 152. Schmitz J, Owyang A, Oldham E, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005;23:479-90. 156  153. Rider P, Carmi Y, Guttman O, et al. IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation. Journal of immunology 2011;187:4835-43. 154. Chen CJ, Kono H, Golenbock D, Reed G, Akira S, Rock KL. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nature medicine 2007;13:851-6. 155. Nold MF, Nold-Petry CA, Zepp JA, Palmer BE, Bufler P, Dinarello CA. IL-37 is a fundamental inhibitor of innate immunity. Nature immunology 2010;11:1014-22. 156. Sharma S, Kulk N, Nold MF, et al. The IL-1 family member 7b translocates to the nucleus and down-regulates proinflammatory cytokines. Journal of immunology 2008;180:5477-82. 157. Grimsby S, Jaensson H, Dubrovska A, Lomnytska M, Hellman U, Souchelnytskyi S. Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity. FEBS letters 2004;577:93-100. 158. van de Veerdonk FL, Stoeckman AK, Wu G, et al. IL-38 binds to the IL-36 receptor and has biological effects on immune cells similar to IL-36 receptor antagonist. Proceedings of the National Academy of Sciences of the United States of America 2012;109:3001-5. 159. Maksymowych WP, Rahman P, Reeve JP, Gladman DD, Peddle L, Inman RD. Association of the IL1 gene cluster with susceptibility to ankylosing spondylitis: an analysis of three Canadian populations. Arthritis and rheumatism 2006;54:974-85. 160. Eisenberg SP, Brewer MT, Verderber E, Heimdal P, Brandhuber BJ, Thompson RC. Interleukin 1 receptor antagonist is a member of the interleukin 1 gene family: evolution of a 157  cytokine control mechanism. Proceedings of the National Academy of Sciences of the United States of America 1991;88:5232-6. 161. Dinarello CA. Anti-inflammatory Agents: Present and Future. Cell 2010;140:935-50. 162. Mertens M, Singh JA. Anakinra for rheumatoid arthritis. The Cochrane database of systematic reviews 2009:CD005121. 163. Aksentijevich I, Masters SL, Ferguson PJ, et al. An autoinflammatory disease with deficiency of the interleukin-1-receptor antagonist. The New England journal of medicine 2009;360:2426-37. 164. Horai R, Saijo S, Tanioka H, et al. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. The Journal of experimental medicine 2000;191:313-20. 165. Nicklin MJ, Hughes DE, Barton JL, Ure JM, Duff GW. Arterial inflammation in mice lacking the interleukin 1 receptor antagonist gene. The Journal of experimental medicine 2000;191:303-12. 166. Tortola L, Rosenwald E, Abel B, et al. Psoriasiform dermatitis is driven by IL-36-mediated DC-keratinocyte crosstalk. The Journal of clinical investigation 2012;122:3965-76. 167. Marrakchi S, Guigue P, Renshaw BR, et al. Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis. The New England journal of medicine 2011;365:620-8. 168. Mills KH, Dunne A. Immune modulation: IL-1, master mediator or initiator of inflammation. Nature medicine 2009;15:1363-4. 169. Joosten LA, Netea MG, Dinarello CA. Interleukin-1beta in innate inflammation, autophagy and immunity. Seminars in immunology 2013;25:416-24. 158  170. Dinarello CA. A clinical perspective of IL-1beta as the gatekeeper of inflammation. European journal of immunology 2011;41:1203-17. 171. Goto M, Katayama KI, Shirakawa F, Tanaka I. Involvement of NF-kappaB p50/p65 heterodimer in activation of the human pro-interleukin-1beta gene at two subregions of the upstream enhancer element. Cytokine 1999;11:16-28. 172. Bauernfeind FG, Horvath G, Stutz A, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. Journal of immunology 2009;183:787-91. 173. Franchi L, Eigenbrod T, Nunez G. Cutting edge: TNF-alpha mediates sensitization to ATP and silica via the NLRP3 inflammasome in the absence of microbial stimulation. Journal of immunology 2009;183:792-6. 174. Akira S, Takeda K. Toll-like receptor signalling. Nature reviews Immunology 2004;4:499-511. 175. Leipe DD, Koonin EV, Aravind L. STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer. Journal of molecular biology 2004;343:1-28. 176. Martinon F, Tschopp J. NLRs join TLRs as innate sensors of pathogens. Trends in immunology 2005;26:447-54. 177. Hornung V, Latz E. Intracellular DNA recognition. Nature reviews Immunology 2010;10:123-30. 159  178. Kanneganti TD, Lamkanfi M, Kim YG, et al. Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inflammasome independent of Toll-like receptor signaling. Immunity 2007;26:433-43. 179. Marina-Garcia N, Franchi L, Kim YG, et al. Pannexin-1-mediated intracellular delivery of muramyl dipeptide induces caspase-1 activation via cryopyrin/NLRP3 independently of Nod2. Journal of immunology 2008;180:4050-7. 180. Kahlenberg JM, Dubyak GR. Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release. American journal of physiology Cell physiology 2004;286:C1100-8. 181. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell death and differentiation 2007;14:1583-9. 182. Hornung V, Bauernfeind F, Halle A, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nature immunology 2008;9:847-56. 183. Davis MJ, Swanson JA. Technical advance: Caspase-1 activation and IL-1beta release correlate with the degree of lysosome damage, as illustrated by a novel imaging method to quantify phagolysosome damage. Journal of leukocyte biology 2010;88:813-22. 184. Halle A, Hornung V, Petzold GC, et al. The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nature immunology 2008;9:857-65. 185. Lamkanfi M, Dixit VM. Inflammasomes and their roles in health and disease. Annual review of cell and developmental biology 2012;28:137-61. 160  186. Duncan JA, Gao X, Huang MT, et al. Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome. Journal of immunology 2009;182:6460-9. 187. Tomalka J, Hise AG. Inflammasomes in aspergillosis-it takes two to tango. Cell host & microbe 2015;17:290-2. 188. Toma C, Higa N, Koizumi Y, et al. Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling. Journal of immunology 2010;184:5287-97. 189. Bulua AC, Simon A, Maddipati R, et al. Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS). The Journal of experimental medicine 2011;208:519-33. 190. Henao-Mejia J, Elinav E, Strowig T, Flavell RA. Inflammasomes: far beyond inflammation. Nature immunology 2012;13:321-4. 191. Strowig T, Henao-Mejia J, Elinav E, Flavell R. Inflammasomes in health and disease. Nature 2012;481:278-86. 192. Rathinam VA, Vanaja SK, Fitzgerald KA. Regulation of inflammasome signaling. Nature immunology 2012;13:333-42. 193. Netea MG, Nold-Petry CA, Nold MF, et al. Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages. Blood 2009;113:2324-35. 194. Gattorno M, Tassi S, Carta S, et al. Pattern of interleukin-1beta secretion in response to lipopolysaccharide and ATP before and after interleukin-1 blockade in patients with CIAS1 mutations. Arthritis and rheumatism 2007;56:3138-48. 161  195. Joosten LA, Netea MG, Fantuzzi G, et al. Inflammatory arthritis in caspase 1 gene-deficient mice: contribution of proteinase 3 to caspase 1-independent production of bioactive interleukin-1beta. Arthritis and rheumatism 2009;60:3651-62. 196. Coeshott C, Ohnemus C, Pilyavskaya A, et al. Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proceedings of the National Academy of Sciences of the United States of America 1999;96:6261-6. 197. Chernov AV, Reyes L, Xu Z, et al. Mycoplasma CG- and GATC-specific DNA methyltransferases selectively and efficiently methylate the host genome and alter the epigenetic landscape in human cells. Epigenetics : official journal of the DNA Methylation Society 2015:0. 198. Gardella S, Andrei C, Costigliolo S, Olcese L, Zocchi MR, Rubartelli A. Secretion of bioactive interleukin-1beta by dendritic cells is modulated by interaction with antigen specific T cells. Blood 2000;95:3809-15. 199. Andrei C, Dazzi C, Lotti L, Torrisi MR, Chimini G, Rubartelli A. The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Molecular biology of the cell 1999;10:1463-75. 200. Andrei C, Margiocco P, Poggi A, Lotti LV, Torrisi MR, Rubartelli A. Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes. Proceedings of the National Academy of Sciences of the United States of America 2004;101:9745-50. 201. Mills KH. Induction, function and regulation of IL-17-producing T cells. European journal of immunology 2008;38:2636-49. 162  202. Hoffman HM, Wanderer AA, Broide DH. Familial cold autoinflammatory syndrome: phenotype and genotype of an autosomal dominant periodic fever. The Journal of allergy and clinical immunology 2001;108:615-20. 203. Hoffman HM, Rosengren S, Boyle DL, et al. Prevention of cold-associated acute inflammation in familial cold autoinflammatory syndrome by interleukin-1 receptor antagonist. Lancet 2004;364:1779-85. 204. Goldbach-Mansky R, Dailey NJ, Canna SW, et al. Neonatal-onset multisystem inflammatory disease responsive to interleukin-1beta inhibition. The New England journal of medicine 2006;355:581-92. 205. Hawkins PN, Lachmann HJ, McDermott MF. Interleukin-1-receptor antagonist in the Muckle-Wells syndrome. The New England journal of medicine 2003;348:2583-4. 206. Hoffman HM, Throne ML, Amar NJ, et al. Efficacy and safety of rilonacept (interleukin-1 Trap) in patients with cryopyrin-associated periodic syndromes: results from two sequential placebo-controlled studies. Arthritis and rheumatism 2008;58:2443-52. 207. Lachmann HJ, Kone-Paut I, Kuemmerle-Deschner JB, et al. Use of canakinumab in the cryopyrin-associated periodic syndrome. The New England journal of medicine 2009;360:2416-25. 208. McDermott MF, Aksentijevich I, Galon J, et al. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 1999;97:133-44. 209. Menu P, Mayor A, Zhou R, et al. ER stress activates the NLRP3 inflammasome via an UPR-independent pathway. Cell death & disease 2012;3:e261. 163  210. Simon A, Bodar EJ, van der Hilst JC, et al. Beneficial response to interleukin 1 receptor antagonist in traps. The American journal of medicine 2004;117:208-10. 211. Gattorno M, Pelagatti MA, Meini A, et al. Persistent efficacy of anakinra in patients with tumor necrosis factor receptor-associated periodic syndrome. Arthritis and rheumatism 2008;58:1516-20. 212. Masters SL, Simon A, Aksentijevich I, Kastner DL. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease (*). Annual review of immunology 2009;27:621-68. 213. El-Shanti H, Majeed HA, El-Khateeb M. Familial mediterranean fever in Arabs. Lancet 2006;367:1016-24. 214. Telatar M, Grody WW. Molecular genetic testing for familial Mediterranean fever. Molecular genetics and metabolism 2000;71:256-60. 215. Chae JJ, Aksentijevich I, Kastner DL. Advances in the understanding of familial Mediterranean fever and possibilities for targeted therapy. British journal of haematology 2009;146:467-78. 216. Chae JJ, Wood G, Richard K, et al. The familial Mediterranean fever protein, pyrin, is cleaved by caspase-1 and activates NF-kappaB through its N-terminal fragment. Blood 2008;112:1794-803. 217. Fitzgerald AA, Leclercq SA, Yan A, Homik JE, Dinarello CA. Rapid responses to anakinra in patients with refractory adult-onset Still's disease. Arthritis and rheumatism 2005;52:1794-803. 164  218. Pascual V, Allantaz F, Arce E, Punaro M, Banchereau J. Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. The Journal of experimental medicine 2005;201:1479-86. 219. Chevalier X, Goupille P, Beaulieu AD, et al. Intraarticular injection of anakinra in osteoarthritis of the knee: a multicenter, randomized, double-blind, placebo-controlled study. Arthritis and rheumatism 2009;61:344-52. 220. de Luca A, Smeekens SP, Casagrande A, et al. IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans. Proceedings of the National Academy of Sciences of the United States of America 2014;111:3526-31. 221. Larsen CM, Faulenbach M, Vaag A, et al. Interleukin-1-receptor antagonist in type 2 diabetes mellitus. The New England journal of medicine 2007;356:1517-26. 222. Larsen CM, Faulenbach M, Vaag A, Ehses JA, Donath MY, Mandrup-Poulsen T. Sustained effects of interleukin-1 receptor antagonist treatment in type 2 diabetes. Diabetes care 2009;32:1663-8. 223. Lust JA, Donovan KA. The role of interleukin-1 beta in the pathogenesis of multiple myeloma. Hematology/oncology clinics of North America 1999;13:1117-25. 224. Lust JA, Lacy MQ, Zeldenrust SR, et al. Induction of a chronic disease state in patients with smoldering or indolent multiple myeloma by targeting interleukin 1{beta}-induced interleukin 6 production and the myeloma proliferative component. Mayo Clinic proceedings 2009;84:114-22. 165  225. Chen CJ, Shi Y, Hearn A, et al. MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals. The Journal of clinical investigation 2006;116:2262-71. 226. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006;440:237-41. 227. Singh D, Huston KK. IL-1 inhibition with anakinra in a patient with refractory gout. Journal of clinical rheumatology : practical reports on rheumatic & musculoskeletal diseases 2009;15:366. 228. So A, De Smedt T, Revaz S, Tschopp J. A pilot study of IL-1 inhibition by anakinra in acute gout. Arthritis research & therapy 2007;9:R28. 229. Terkeltaub R, Sundy JS, Schumacher HR, et al. The interleukin 1 inhibitor rilonacept in treatment of chronic gouty arthritis: results of a placebo-controlled, monosequence crossover, non-randomised, single-blind pilot study. Annals of the rheumatic diseases 2009;68:1613-7. 230. Joosten LA, Netea MG, Mylona E, et al. Engagement of fatty acids with Toll-like receptor 2 drives interleukin-1beta production via the ASC/caspase 1 pathway in monosodium urate monohydrate crystal-induced gouty arthritis. Arthritis and rheumatism 2010;62:3237-48. 231. Villani AC, Lemire M, Fortin G, et al. Common variants in the NLRP3 region contribute to Crohn's disease susceptibility. Nature genetics 2009;41:71-6. 232. Ligumsky M, Simon PL, Karmeli F, Rachmilewitz D. Role of interleukin 1 in inflammatory bowel disease--enhanced production during active disease. Gut 1990;31:686-9. 166  233. Cominelli F, Nast CC, Clark BD, et al. Interleukin 1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis. The Journal of clinical investigation 1990;86:972-80. 234. Kanai T, Watanabe M, Okazawa A, et al. Interleukin 18 is a potent proliferative factor for intestinal mucosal lymphocytes in Crohn's disease. Gastroenterology 2000;119:1514-23. 235. Al-Sadi RM, Ma TY. IL-1beta causes an increase in intestinal epithelial tight junction permeability. Journal of immunology 2007;178:4641-9. 236. Zaki MH, Boyd KL, Vogel P, Kastan MB, Lamkanfi M, Kanneganti TD. The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 2010;32:379-91. 237. Dupaul-Chicoine J, Yeretssian G, Doiron K, et al. Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases. Immunity 2010;32:367-78. 238. Allen IC, TeKippe EM, Woodford RM, et al. The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer. The Journal of experimental medicine 2010;207:1045-56. 239. Elinav E, Strowig T, Kau AL, et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 2011;145:745-57. 240. Bersudsky M, Luski L, Fishman D, et al. Non-redundant properties of IL-1alpha and IL-1beta during acute colon inflammation in mice. Gut 2014;63:598-609. 167  241. Coccia M, Harrison OJ, Schiering C, et al. IL-1beta mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4(+) Th17 cells. The Journal of experimental medicine 2012;209:1595-609. 242. Zhang J, Fu S, Sun S, Li Z, Guo B. Inflammasome activation has an important role in the development of spontaneous colitis. Mucosal immunology 2014;7:1139-50. 243. Kwon KH, Murakami A, Hayashi R, Ohigashi H. Interleukin-1beta targets interleukin-6 in progressing dextran sulfate sodium-induced experimental colitis. Biochemical and biophysical research communications 2005;337:647-54. 244. Carvalho FA, Nalbantoglu I, Ortega-Fernandez S, et al. Interleukin-1beta (IL-1beta) promotes susceptibility of Toll-like receptor 5 (TLR5) deficient mice to colitis. Gut 2012;61:373-84. 245. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008;451:1069-75. 246. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008;132:27-42. 247. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO journal 2000;19:5720-8. 248. Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. Journal of cell science 2004;117:2805-12. 168  249. Nakahira K, Haspel JA, Rathinam VA, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nature immunology 2011;12:222-30. 250. Shi CS, Shenderov K, Huang NN, et al. Activation of autophagy by inflammatory signals limits IL-1beta production by targeting ubiquitinated inflammasomes for destruction. Nature immunology 2012;13:255-63. 251. Harris J, Hartman M, Roche C, et al. Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation. The Journal of biological chemistry 2011;286:9587-97. 252. Lee J, Kim HR, Quinley C, et al. Autophagy suppresses interleukin-1beta (IL-1beta) signaling by activation of p62 degradation via lysosomal and proteasomal pathways. The Journal of biological chemistry 2012;287:4033-40. 253. Saitoh T, Fujita N, Jang MH, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 2008;456:264-8. 254. Murthy A, Li Y, Peng I, et al. A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3. Nature 2014;506:456-62. 255. Lassen KG, Kuballa P, Conway KL, et al. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proceedings of the National Academy of Sciences of the United States of America 2014;111:7741-6. 256. Cooney R, Baker J, Brain O, et al. NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nat Med 2010;16:90-7. 169  257. Jones SA, Mills KH, Harris J. Autophagy and inflammatory diseases. Immunology and cell biology 2013;91:250-8. 258. International HapMap C, Altshuler DM, Gibbs RA, et al. Integrating common and rare genetic variation in diverse human populations. Nature 2010;467:52-8. 259. Okazaki T, Wang MH, Rawsthorne P, et al. Contributions of IBD5, IL23R, ATG16L1, and NOD2 to Crohn's disease risk in a population-based case-control study: evidence of gene-gene interactions. Inflamm Bowel Dis 2008;14:1528-41. 260. Sadaghian Sadabad M, Regeling A, de Goffau MC, et al. The ATG16L1-T300A allele impairs clearance of pathosymbionts in the inflamed ileal mucosa of Crohn's disease patients. Gut 2014. 261. Plantinga TS, Crisan TO, Oosting M, et al. Crohn's disease-associated ATG16L1 polymorphism modulates pro-inflammatory cytokine responses selectively upon activation of NOD2. Gut 2011;60:1229-35. 262. Orvedahl A, MacPherson S, Sumpter R, Jr., Talloczy Z, Zou Z, Levine B. Autophagy protects against Sindbis virus infection of the central nervous system. Cell host & microbe 2010;7:115-27. 263. Lee HK, Mattei LM, Steinberg BE, et al. In vivo requirement for Atg5 in antigen presentation by dendritic cells. Immunity 2010;32:227-39. 264. Jounai N, Takeshita F, Kobiyama K, et al. The Atg5 Atg12 conjugate associates with innate antiviral immune responses. Proceedings of the National Academy of Sciences of the United States of America 2007;104:14050-5. 265. Deretic V, Levine B. Autophagy, immunity, and microbial adaptations. Cell host & microbe 2009;5:527-49. 170  266. Lopez P, Alonso-Perez E, Rodriguez-Carrio J, Suarez A. Influence of Atg5 mutation in SLE depends on functional IL-10 genotype. PloS one 2013;8:e78756. 267. Martin LJ, Gupta J, Jyothula SS, et al. Functional variant in the autophagy-related 5 gene promotor is associated with childhood asthma. PloS one 2012;7:e33454. 268. Bekpen C, Marques-Bonet T, Alkan C, et al. Death and resurrection of the human IRGM gene. PLoS genetics 2009;5:e1000403. 269. Bekpen C, Xavier RJ, Eichler EE. Human IRGM gene "to be or not to be". Seminars in immunopathology 2010;32:437-44. 270. Singh SB, Ornatowski W, Vergne I, et al. Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria. Nature cell biology 2010;12:1154-65. 271. Lapaquette P, Glasser AL, Huett A, Xavier RJ, Darfeuille-Michaud A. Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. Cellular microbiology 2010;12:99-113. 272. McCarroll SA, Huett A, Kuballa P, et al. Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease. Nature genetics 2008;40:1107-12. 273. Brest P, Lapaquette P, Souidi M, et al. A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease. Nature genetics 2011;43:242-5. 274. Gutierrez O, Pipaon C, Inohara N, et al. Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B activation. The Journal of biological chemistry 2002;277:41701-5. 171  275. Shaw MH, Reimer T, Sanchez-Valdepenas C, et al. T cell-intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii. Nature immunology 2009;10:1267-74. 276. Meylan E, Tschopp J, Karin M. Intracellular pattern recognition receptors in the host response. Nature 2006;442:39-44. 277. Kanneganti TD, Lamkanfi M, Nunez G. Intracellular NOD-like receptors in host defense and disease. Immunity 2007;27:549-59. 278. Sabbah A, Chang TH, Harnack R, et al. Activation of innate immune antiviral responses by Nod2. Nature immunology 2009;10:1073-80. 279. Mathew CG, Lewis CM. Genetics of inflammatory bowel disease: progress and prospects. Human molecular genetics 2004;13 Spec No 1:R161-8. 280. Williams CN, Kocher K, Lander ES, Daly MJ, Rioux JD. Using a genome-wide scan and meta-analysis to identify a novel IBD locus and confirm previously identified IBD loci. Inflammatory bowel diseases 2002;8:375-81. 281. Abbott DW, Wilkins A, Asara JM, Cantley LC. The Crohn's disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Current biology : CB 2004;14:2217-27. 282. Watanabe T, Kitani A, Murray PJ, Strober W. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nature immunology 2004;5:800-8. 283. Wehkamp J, Salzman NH, Porter E, et al. Reduced Paneth cell alpha-defensins in ileal Crohn's disease. Proceedings of the National Academy of Sciences of the United States of America 2005;102:18129-34. 172  284. Travassos LH, Carneiro LA, Ramjeet M, et al. Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Nature immunology 2010;11:55-62. 285. Todd DJ, Lee AH, Glimcher LH. The endoplasmic reticulum stress response in immunity and autoimmunity. Nature reviews Immunology 2008;8:663-74. 286. Schroder M, Kaufman RJ. The mammalian unfolded protein response. Annual review of biochemistry 2005;74:739-89. 287. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nature reviews Molecular cell biology 2007;8:519-29. 288. Kaser A, Blumberg RS. Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease. Seminars in immunology 2009;21:156-63. 289. Yorimitsu T, Klionsky DJ. Endoplasmic reticulum stress: a new pathway to induce autophagy. Autophagy 2007;3:160-2. 290. Yorimitsu T, Nair U, Yang Z, Klionsky DJ. Endoplasmic reticulum stress triggers autophagy. The Journal of biological chemistry 2006;281:30299-304. 291. Fujita E, Kouroku Y, Isoai A, et al. Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Human molecular genetics 2007;16:618-29. 292. Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Molecular and cellular biology 2003;23:7448-59. 173  293. Shaffer AL, Shapiro-Shelef M, Iwakoshi NN, et al. XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity 2004;21:81-93. 294. Martinon F, Chen X, Lee AH, Glimcher LH. TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages. Nature immunology 2010;11:411-8. 295. Hampe J, Shaw SH, Saiz R, et al. Linkage of inflammatory bowel disease to human chromosome 6p. American journal of human genetics 1999;65:1647-55. 296. Barmada MM, Brant SR, Nicolae DL, et al. A genome scan in 260 inflammatory bowel disease-affected relative pairs. Inflammatory bowel diseases 2004;10:513-20. 297. Vermeire S, Rutgeerts P, Van Steen K, et al. Genome wide scan in a Flemish inflammatory bowel disease population: support for the IBD4 locus, population heterogeneity, and epistasis. Gut 2004;53:980-6. 298. Kaser A, Lee AH, Franke A, et al. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 2008;134:743-56. 299. Cantero-Recasens G, Fandos C, Rubio-Moscardo F, Valverde MA, Vicente R. The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress. Human molecular genetics 2010;19:111-21. 300. Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 2010;140:900-17. 301. McGuckin MA, Eri RD, Das I, Lourie R, Florin TH. ER stress and the unfolded protein response in intestinal inflammation. American journal of physiology Gastrointestinal and liver physiology 2010;298:G820-32. 174  302. Lluis A, Schedel M, Liu J, et al. Asthma-associated polymorphisms in 17q21 influence cord blood ORMDL3 and GSDMA gene expression and IL-17 secretion. The Journal of allergy and clinical immunology 2011;127:1587-94 e6. 303. Iyer S, Margulies BS, Kerr WG. Role of SHIP1 in bone biology. Annals of the New York Academy of Sciences 2013;1280:11-4. 304. Hazen AL, Smith MJ, Desponts C, Winter O, Moser K, Kerr WG. SHIP is required for a functional hematopoietic stem cell niche. Blood 2009;113:2924-33. 305. Viernes DR, Choi LB, Kerr WG, Chisholm JD. Discovery and development of small molecule SHIP phosphatase modulators. Medicinal research reviews 2014;34:795-824. 306. Kalesnikoff J, Sly LM, Hughes MR, et al. The role of SHIP in cytokine-induced signaling. Reviews of physiology, biochemistry and pharmacology 2003;149:87-103. 307. Wisniewski D, Strife A, Swendeman S, 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 1999;93:2707-20. 308. Damen JE, Ware MD, Kalesnikoff J, Hughes MR, Krystal G. SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation. Blood 2001;97:1343-51. 309. Zhang Y, Wavreille AS, Kunys AR, Pei D. The SH2 domains of inositol polyphosphate 5-phosphatases SHIP1 and SHIP2 have similar ligand specificity but different binding kinetics. Biochemistry 2009;48:11075-83. 175  310. Tu Z, Ninos JM, Ma 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 2001;98:2028-38. 311. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655-7. 312. Hawkins PT, Stephens LR. PI3K signalling in inflammation. Biochimica et biophysica acta 2015;1851:882-97. 313. Vanhaesebroeck B, Leevers SJ, Ahmadi K, et al. Synthesis and function of 3-phosphorylated inositol lipids. Annual review of biochemistry 2001;70:535-602. 314. Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. The emerging mechanisms of isoform-specific PI3K signalling. Nature reviews Molecular cell biology 2010;11:329-41. 315. Paez J, Sellers WR. PI3K/PTEN/AKT pathway. A critical mediator of oncogenic signaling. Cancer treatment and research 2003;115:145-67. 316. Kok K, Geering B, Vanhaesebroeck B. Regulation of phosphoinositide 3-kinase expression in health and disease. Trends in biochemical sciences 2009;34:115-27. 317. Falasca M, Maffucci T. Role of class II phosphoinositide 3-kinase in cell signalling. Biochemical Society transactions 2007;35:211-4. 318. Backer JM. The regulation and function of Class III PI3Ks: novel roles for Vps34. The Biochemical journal 2008;410:1-17. 319. Catimel B, Yin MX, Schieber C, et al. PI(3,4,5)P3 Interactome. Journal of proteome research 2009;8:3712-26. 320. Kerr WG. A role for SHIP in stem cell biology and transplantation. Current stem cell research & therapy 2008;3:99-106. 176  321. Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 1997;275:665-8. 322. Ma K, Cheung SM, Marshall AJ, Duronio V. PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity. Cellular signalling 2008;20:684-94. 323. Peng Q, Malhotra S, Torchia JA, Kerr WG, Coggeshall KM, Humphrey MB. TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1. Science signaling 2010;3:ra38. 324. Kerr WG. Inhibitor and activator: dual functions for SHIP in immunity and cancer. Annals of the New York Academy of Sciences 2011;1217:1-17. 325. Valderrama-Carvajal H, Cocolakis E, Lacerte A, et al. Activin/TGF-beta induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nature cell biology 2002;4:963-9. 326. Baltimore D, Boldin MP, O'Connell RM, Rao DS, Taganov KD. MicroRNAs: new regulators of immune cell development and function. Nature immunology 2008;9:839-45. 327. Sly LM, Rauh MJ, Kalesnikoff J, Song CH, Krystal G. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity 2004;21:227-39. 328. Zhao J, Crowe DL, Castillo C, Wuenschell C, Chai Y, Warburton D. Smad7 is a TGF-beta-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis. Mech Dev 2000;93:71-81. 329. Jin HM, Kim TJ, Choi JH, et al. MicroRNA-155 as a proinflammatory regulator via SHIP-1 down-regulation in acute gouty arthritis. Arthritis research & therapy 2014;16:R88. 177  330. Ruschmann J, Ho V, Antignano F, et al. Tyrosine phosphorylation of SHIP promotes its proteasomal degradation. Experimental hematology 2010;38:392-402,  e1. 331. Helgason CD, Damen JE, Rosten P, et al. Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes & development 1998;12:1610-20. 332. Ghansah T, Paraiso KH, Highfill S, et al. Expansion of myeloid suppressor cells in SHIP-deficient mice represses allogeneic T cell responses. Journal of immunology 2004;173:7324-30. 333. Nakamura K, Kouro T, Kincade PW, Malykhin A, Maeda K, Coggeshall KM. Src homology 2-containing 5-inositol phosphatase (SHIP) suppresses an early stage of lymphoid cell development through elevated interleukin-6 production by myeloid cells in bone marrow. The Journal of experimental medicine 2004;199:243-54. 334. Rauh MJ, Ho V, Pereira C, et al. SHIP represses the generation of alternatively activated macrophages. Immunity 2005;23:361-74. 335. Kim CH, Hangoc G, Cooper S, et al. Altered responsiveness to chemokines due to targeted disruption of SHIP. The Journal of clinical investigation 1999;104:1751-9. 336. Kuroda E, Ho V, Ruschmann J, et al. SHIP represses the generation of IL-3-induced M2 macrophages by inhibiting IL-4 production from basophils. Journal of immunology 2009;183:3652-60. 337. McLarren KW, Cole AE, Weisser SB, et al. SHIP-deficient mice develop spontaneous intestinal inflammation and arginase-dependent fibrosis. The American journal of pathology 2011;179:180-8. 178  338. Kerr WG, Park MY, Maubert M, Engelman RW. SHIP deficiency causes Crohn's disease-like ileitis. Gut 2011;60:177-88. 339. Leitges M, Gimborn K, Elis W, et al. Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation. Molecular and cellular biology 2002;22:3970-80. 340. Lam V, Kalesnikoff J, Lee CW, et al. IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE + antigen but distinct from those used by Steel factor. Blood 2003;102:1405-13. 341. Gardai S, Whitlock BB, Helgason C, et al. Activation of SHIP by NADPH oxidase-stimulated Lyn leads to enhanced apoptosis in neutrophils. The Journal of biological chemistry 2002;277:5236-46. 342. Ono M, Bolland S, Tempst P, Ravetch JV. Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc(gamma)RIIB. Nature 1996;383:263-6. 343. Aman MJ, Walk SF, March ME, Su HP, Carver DJ, Ravichandran KS. Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling. Molecular and cellular biology 2000;20:3576-89. 344. Akiyama T, Maeda S, Yamane S, et al. Dependence of self-tolerance on TRAF6-directed development of thymic stroma. Science 2005;308:248-51. 345. Galandrini R, Tassi I, Mattia G, et al. SH2-containing inositol phosphatase (SHIP-1) transiently translocates to raft domains and modulates CD16-mediated cytotoxicity in human NK cells. Blood 2002;100:4581-9. 179  346. Fortenbery NR, Paraiso KH, Taniguchi M, Brooks C, Ibrahim L, Kerr WG. SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation. Journal of immunology 2010;184:5065-74. 347. Park MY, Srivastava N, Sudan R, et al. Impaired T-cell survival promotes mucosal inflammatory disease in SHIP1-deficient mice. Mucosal immunology 2014;7:1429-39. 348. Maxwell MJ, Srivastava N, Park MY, et al. SHIP-1 deficiency in the myeloid compartment is insufficient to induce myeloid expansion or chronic inflammation. Genes and immunity 2014;15:233-40. 349. Arijs I, De Hertogh G, Lemmens B, et al. Intestinal expression of SHIP in inflammatory bowel diseases. Gut 2012;61:956-7. 350. Brooks R, Fuhler GM, Iyer S, et al. SHIP1 inhibition increases immunoregulatory capacity and triggers apoptosis of hematopoietic cancer cells. Journal of immunology 2010;184:3582-9. 351. Goldbach-Mansky R. Immunology in clinic review series; focus on autoinflammatory diseases: update on monogenic autoinflammatory diseases: the role of interleukin (IL)-1 and an emerging role for cytokines beyond IL-1. Clinical and experimental immunology 2012;167:391-404. 352. Goldbach-Mansky R, Kastner DL. Autoinflammation: the prominent role of IL-1 in monogenic autoinflammatory diseases and implications for common illnesses. J Allergy Clin Immunol 2009;124:1141-9; quiz 50-1. 353. Podolsky DK. Inflammatory bowel disease. The New England journal of medicine 2002;347:417-29. 180  354. Van Limbergen J, Wilson DC, Satsangi J. The genetics of Crohn's disease. Annual review of genomics and human genetics 2009;10:89-116. 355. Hazlewood GS, Rezaie A, Borman M, et al. Comparative Effectiveness of Immunosuppressant and Biologics for Inducing and Maintaining Remission in Crohn's Disease: A Network Meta-Analysis. Gastroenterology 2014. 356. Sandborn WJ. State-of-the-art: Immunosuppression and biologic therapy. Digestive diseases 2010;28:536-42. 357. Zhou Q, Lee GS, Brady J, et al. A hypermorphic missense mutation in PLCG2, encoding phospholipase Cgamma2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. American journal of human genetics 2012;91:713-20. 358. Almeida de Jesus A, Goldbach-Mansky R. Monogenic autoinflammatory diseases: concept and clinical manifestations. Clinical immunology 2013;147:155-74. 359. Bianco AM, Girardelli M, Vozzi D, Crovella S, Kleiner G, Marcuzzi A. Mevalonate kinase deficiency and IBD: shared genetic background. Gut 2014;63:1367-8. 360. Uhlig HH, Schwerd T, Koletzko S, et al. The diagnostic approach to monogenic very early onset inflammatory bowel disease. Gastroenterology 2014;147:990-1007 e3. 361. Weisser SB, McLarren KW, Voglmaier N, et al. Alternative activation of macrophages by IL-4 requires SHIP degradation. European journal of immunology 2011;41:1742-53. 362. Williams O, Houseman BT, Kunkel EJ, et al. Discovery of dual inhibitors of the immune cell PI3Ks p110delta and p110gamma: a prototype for new anti-inflammatory drugs. Chemistry & biology 2010;17:123-34. 181  363. Costinean S, Sandhu SK, Pedersen IM, 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 2009;114:1374-82. 364. Wu R, Li Y, Guo Z, et al. Triptolide ameliorates ileocolonic anastomosis inflammation in IL-10 deficient mice by mechanism involving suppression of miR-155/SHIP-1 signaling pathway. Molecular immunology 2013;56:340-6. 365. Singh UP, Murphy AE, Enos RT, et al. miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses. Immunology 2014;143:478-89. 366. Schaefer JS, Montufar-Solis D, Vigneswaran N, Klein JR. Selective upregulation of microRNA expression in peripheral blood leukocytes in IL-10-/- mice precedes expression in the colon. Journal of immunology 2011;187:5834-41. 367. Massey DC, Bredin F, Parkes M. Use of sirolimus (rapamycin) to treat refractory Crohn's disease. Gut 2008;57:1294-6. 368. Dinarello CA. The IL-1 family and inflammatory diseases. Clinical and experimental rheumatology 2002;20:S1-13. 369. Sly LM, Hamilton MJ, Kuroda E, et al. SHIP prevents lipopolysaccharide from triggering an antiviral response in mice. Blood 2009;113:2945-54. 370. Bauer C, Duewell P, Mayer C, et al. Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome. Gut 2010;59:1192-9. 371. Siegmund B, Lehr HA, Fantuzzi G, Dinarello CA. IL-1 beta -converting enzyme (caspase-1) in intestinal inflammation. Proceedings of the National Academy of Sciences of the United States of America 2001;98:13249-54. 182  372. Yang SK, Ye BD, Song K. ATG16L1 contributes to Crohn's disease susceptibility in Koreans: overmuch concern for ethnic difference? Gut 2015;64:687-8. 373. Zhang J, Chen J, Gu J, Guo H, Chen W. Association of IL23R and ATG16L1 with susceptibility of Crohn's disease in Chinese population. Scandinavian journal of gastroenterology 2014;49:1201-6. 374. Hirano A, Yamazaki K, Umeno J, et al. Association study of 71 European Crohn's disease susceptibility loci in a Japanese population. Inflamm Bowel Dis 2013;19:526-33. 375. Weisser SB, Brugger HK, Voglmaier NS, McLarren KW, van Rooijen N, Sly LM. SHIP-deficient, alternatively activated macrophages protect mice during DSS-induced colitis. Journal of leukocyte biology 2011;90:483-92. 376. Van Limbergen J, Kabakchiev B, Stempak JM, et al. Hypothesis-free analysis of ATG16L1 demonstrates gene-wide extent of association with Crohn's disease susceptibility. Gut 2013;62:331-3. 377. Weisser SB, Brugger HK, Voglmaier NS, McLarren KW, van Rooijen N, Sly LM. SHIP-deficient, alternatively activated macrophages protect mice during DSS-induced colitis. J Leukoc Biol;90:483-92. 378. Dharmani P, Chadee K. Biologic therapies against inflammatory bowel disease: a dysregulated immune system and the cross talk with gastrointestinal mucosa hold the key. Curr Mol Pharmacol 2008;1:195-212. 379. Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nature reviews Immunology 2014;14:667-85. 380. F OF, Rusten TE, Stenmark H. Phosphoinositide 3-kinases as accelerators and brakes of autophagy. The FEBS journal 2013;280:6322-37. 183  381. Begun J, Xavier RJ. Autophagy at the crossroads of metabolism and cellular defense. Current opinion in gastroenterology 2013;29:588-96. 382. Tosato G, Jones KD. Interleukin-1 induces interleukin-6 production in peripheral blood monocytes. Blood 1990;75:1305-10. 383. Zhang YH, Lin JX, Vilcek J. Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a kappa B-like sequence. Mol Cell Biol 1990;10:3818-23. 384. Campbell J, Ciesielski CJ, Hunt AE, et al. A novel mechanism for TNF-alpha regulation by p38 MAPK: involvement of NF-kappa B with implications for therapy in rheumatoid arthritis. J Immunol 2004;173:6928-37. 385. Hsu HY, Wen MH. Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J Biol Chem 2002;277:22131-9. 386. Reinecker HC, Steffen M, Witthoeft T, et al. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease. Clinical and experimental immunology 1993;94:174-81. 387. Casini-Raggi V, Kam L, Chong YJ, Fiocchi C, Pizarro TT, Cominelli F. Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease. A novel mechanism of chronic intestinal inflammation. Journal of immunology 1995;154:2434-40. 388. McAlindon ME, Hawkey CJ, Mahida YR. Expression of interleukin 1 beta and interleukin 1 beta converting enzyme by intestinal macrophages in health and inflammatory bowel disease. Gut 1998;42:214-9. 184  389. Avitzur Y, Guo C, Mastropaolo LA, et al. Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease. Gastroenterology 2014;146:1028-39. 390. Harris J, Hartman M, Roche C, et al. Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation. J Biol Chem 2012;286:9587-97. 391. Kuballa P, Huett A, Rioux JD, Daly MJ, Xavier RJ. Impaired autophagy of an intracellular pathogen induced by a Crohn's disease associated ATG16L1 variant. PloS one 2008;3:e3391. 392. Edelblum KL, Singh G, Odenwald MA, et al. gammadelta Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice. Gastroenterology 2015. 393. Hedl M, Li J, Cho JH, Abraham C. Chronic stimulation of Nod2 mediates tolerance to bacterial products. Proc Natl Acad Sci U S A 2007;104:19440-5. 394. Eckmann L, Karin M. NOD2 and Crohn's disease: loss or gain of function? Immunity 2005;22:661-7. 395. Ferwerda G, Kramer M, de Jong D, et al. Engagement of NOD2 has a dual effect on proIL-1beta mRNA transcription and secretion of bioactive IL-1beta. European journal of immunology 2008;38:184-91. 396. Scharl M, Rogler G. Inflammatory bowel disease: dysfunction of autophagy? Digestive diseases 2012;30 Suppl 3:12-9. 397. Maeda S, Hsu LC, Liu H, et al. Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing. Science 2005;307:734-8. 185  398. Bonen DK, Ogura Y, Nicolae DL, et al. Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan. Gastroenterology 2003;124:140-6. 399. Uemura A, Oku M, Mori K, Yoshida H. Unconventional splicing of XBP1 mRNA occurs in the cytoplasm during the mammalian unfolded protein response. Journal of cell science 2009;122:2877-86. 400. Fidder HH, Hommes DW. Anti-TNF and Crohn's disease: when should we start? Curr Drug Targets 2010;11:143-7. 401. Sandborn WJ, Rutgeerts P, Enns R, et al. Adalimumab induction therapy for Crohn disease previously treated with infliximab: a randomized trial. Ann Intern Med 2007;146:829-38. 402. Yang L, Williams DE, Mui A, et al. Synthesis of pelorol and analogues: activators of the inositol 5-phosphatase SHIP. Org Lett 2005;7:1073-6. 403. Schreiber S, Nikolaus S, Hampe J, et al. Tumour necrosis factor alpha and interleukin 1beta in relapse of Crohn's disease. Lancet 1999;353:459-61. 404. Noguchi M, Hiwatashi N, Hayakawa T, Toyota T. Leukocyte removal filter-passed lymphocytes produce large amounts of interleukin-4 in immunotherapy for inflammatory bowel disease: role of bystander suppression. Ther Apher 1998;2:109-14. 405. Saniabadi AR, Hanai H, Suzuki Y, et al. Adacolumn for selective leukocytapheresis as a non-pharmacological treatment for patients with disorders of the immune system: an adjunct or an alternative to drug therapy? J Clin Apher 2005;20:171-84. 406. Saniabadi AR, Hanai H, Fukunaga K, et al. Therapeutic leukocytapheresis for inflammatory bowel disease. Transfusion and apheresis science : official journal of the 186  World Apheresis Association : official journal of the European Society for Haemapheresis 2007;37:191-200. 407. Su C, Lewis JD, Goldberg B, Brensinger C, Lichtenstein GR. A meta-analysis of the placebo rates of remission and response in clinical trials of active ulcerative colitis. Gastroenterology 2007;132:516-26. 408. Becker C, Watson AJ, Neurath MF. Complex roles of caspases in the pathogenesis of inflammatory bowel disease. Gastroenterology 2013;144:283-93. 409. Zeissig S, Bojarski C, Buergel N, et al. Downregulation of epithelial apoptosis and barrier repair in active Crohn's disease by tumour necrosis factor alpha antibody treatment. Gut 2004;53:1295-302. 410. Bauer C, Loher F, Dauer M, et al. The ICE inhibitor pralnacasan prevents DSS-induced colitis in C57BL/6 mice and suppresses IP-10 mRNA but not TNF-alpha mRNA expression. Digestive diseases and sciences 2007;52:1642-52. 411. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Molecular cancer therapeutics 2011;10:1533-41. 412. Mutalib M, Borrelli O, Blackstock S, et al. The use of sirolimus (rapamycin) in the management of refractory inflammatory bowel disease in children. J Crohns Colitis 2014;8:1730-4.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

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"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0216551/manifest

Comment

Related Items