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The role of Malt1 in macrophages and osteoclasts Monajemi, Mahdis 2019

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The role of Malt1 in macrophages and osteoclasts by  Mahdis Monajemi  M.Sc., Memorial University, 2012 B.Sc., Tehran University, 2008  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)  September 2019  © Mahdis Monajemi, 2019  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: The Role of Malt1 in Macrophages and Osteoclasts  submitted by Mahdis Monajemi in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine   Examining Committee: Dr. Laura Sly, Pediatrics Supervisor  Dr. Bruce Vallance, Pediatrics Supervisory Committee Member  Dr. Stefan Taubert, Medical Genetics University Examiner Dr. Alice Mui, Surgery University Examiner  Additional Supervisory Committee Members: Dr. Tobias Kollmann, Pediatrics Supervisory Committee Member  iii  Abstract  Combined immunodeficiency caused by a homozygous mutation in the mucosa associated lymphoid tissue 1 (MALT1) gene is associated with severe inflammation along the gastrointestinal tract and osteoporosis, which were corrected by hematopoietic stem cell transplant (HSCT). The consequences of Malt1 deficiency have largely been attributed to its role in lymphocytes, but Malt1 is also expressed in myeloid cells. The effect of Malt1 deficiency in macrophages and osteoclasts and their contribution to inflammatory bowel disease (IBD) and osteoporosis respectively have not been investigated. My objectives were to determine the contribution of Malt1-/- macrophages to dextran sodium sulfate (DSS)-induced colitis in mice and to assess the effect of innate immune stimuli on Malt1-/- macrophage inflammatory responses. I also studied the level of Malt1 expression during intestinal inflammation in humans and mice. I next asked whether Malt1-deficient mice develop an osteoporosis-like phenotype and whether it is caused by the effect of Malt1 deficiency on osteoclast differentiation and/or activity.  I found that Malt1 deficiency exacerbates DSS-induced colitis in mice, and that macrophages and IL-1 signaling contribute to pathology in Malt1-/- mice. Innate immune stimuli induced Malt1 protein levels in murine macrophages in vitro. However, intestinal inflammation did not have any effect on Malt1 expression in humans and mice in vivo. I also found that adult Malt1-deficient mice have lower bone volume, and Malt1 expression and activity is induced by receptor activator of nuclear factor κB ligand (RANKL) in preosteoclasts. Malt1 deficiency did not impact osteoclast differentiation or activity in vitro but their number was higher in Malt1-/- mice in vivo. Inhibition of Malt1 activity in macrophages after activation by inflammatory iv  stimuli induced macrophage colony-stimulating factor (MCSF) production, required for osteoclastogenesis, and decreased OPG production, an endogenous inhibitor of osteoclastogenesis, which was also lower in Malt1-/- mice serum.  Taken together, these data demonstrate that Malt1-/- mice are more susceptible to DSS-induced colitis through higher IL-1 production by Malt1-/- macrophages and these mice develop an osteoporotic phenotype with increased osteoclastogenesis in vivo, which is caused by inflammation rather than a cell-intrinsic effect of Malt1 deficiency in osteoclasts.   v  Lay Summary  A patient at BC Children’s Hospital had combined immunodeficiency (CID) caused by loss of a gene, called Malt1. This patient had extreme inflammation, a biological response when our immune system tries to protect us against microbes and injuries. Inflammation was more obvious in the patient gastrointestinal tract. In addition to that, the patient developed osteoporosis. Very little is known about how the absence of Malt1 can cause these clinical symptoms. Macrophages are a type of immune cell, important for developing gastrointestinal inflammation, and osteoclasts are bone macrophages, important for developing osteoporosis. I asked if loss of Malt in macrophages and osteoclasts cause gastrointestinal inflammation and osteoporosis. Using mice, I found that macrophages with no Malt1 produce more cytokines, which are molecules that cause inflammation by acting as signals to direct immune cells. I also found that these cytokines cause gastrointestinal inflammation and increase the number of osteoclasts leading to osteoporosis.    vi  Preface  Animal studies were conducted with approval by the University of British Columbia and according to the guidelines released by the Canadian Council on Animal Care (protocol numbers A17-0061 and A17-0071). The acquisition of human biopsies in the study was approved by Research Ethics Boards (REBs) at the University of British Columbia (protocol number H09-01826).  Chapter 1. Figure 1.1 was modified and reproduced with permission of Elsevier: Zaki, MH. et al. The Nlrp3 inflammasome: contributions to intestinal homeostasis. Trends Immunol. 2011;32(4). Figure 1.2 was modified and reproduced with permission of Springer Nature: Morrison, Sean J. et al. The bone marrow niche for haematopoietic stem cells. Nature. 2014; 505(7483). Figure 1.3 was modified and reproduced with permission of Copyright Clearance Center Ltd: Tidball, J. et al. Shared signaling systems in myeloid cell-mediated muscle regeneration. Development. 2014; 141(6). Figure 1.4 was modified and reproduced with permission of Oxford University Press: Henriksen, K. et al. Osteoclast activity and subtypes as a function of physiology and pathology--implications for future treatments of osteoporosis. Endocrine reviews. 2011;32(1). Figure 1.5 was modified and reproduced with permission of John Wiley and Sons: Ruland, J. CARD9 signaling in the innate immune response. Annals of the New York Academy of Sciences. 2008;1143. Chapter 2. An earlier version of this chapter has been published and was added to the thesis with permission of Copyright Clearance Center Ltd. Mahdis Monajemi, Yvonne C. F. Pang, Saelin Bjornson, Susan C. Menzies, Nico van Rooijen, and Laura M. Sly. Malt1 blocks IL-vii  1β production by macrophages in vitro and limits dextran sodium sulfate-induced intestinal inflammation in vivo. J Leukoc Biol. 2018;104(3). Mahdis Monajemi performed experiments, data analysis, and manuscript preparation. Susan C. Menzies maintained the mouse colony, and Susan C. Menzies, Yvonne C. F. Pang, and Saelin Bjornson assisted with blinded histopathological scoring in Figure 2.1, 2.3, and 2.9. Nico van Rooijen prepared and provided clodronate liposomes. Laura M. Sly contributed to experimental design and manuscript preparation. Chapter 3. Figures, 3.1, 3.2, 3.3, and 3.4 of this chapter have been published and were added to the thesis with permission of Copyright Clearance Center Ltd. Mahdis Monajemi, Yvonne C. F. Pang, Saelin Bjornson, Susan C. Menzies, Nico van Rooijen, and Laura M. Sly. Malt1 blocks IL-1β production by macrophages in vitro and limits dextran sodium sulfate-induced intestinal inflammation in vivo. J Leukoc Biol. 2018;104(3). Mahdis Monajemi performed experiments, data analysis, and manuscript preparation. Susan C. Menzies maintained the mouse colony. Yvonne C. F. Pang and Saelin Bjornson assisted with Western blots in Figure 3.1 and 3.6. Laura M. Sly contributed to experimental design and manuscript preparation. Chapter 4. A version of this chapter has been published and was added to the thesis with permission of Copyright Clearance Center Ltd. Mahdis Monajemi, Shera Fisk, Yvonne C. F. Pang, Jessica Leung, Susan C. Menzies, Rym Ben Othman Aniba, Bing Cai, Tobias Kollmann, Jacob Rozmus, and Laura M. Sly. Malt1-deficient mice develop osteoporosis independent of osteoclast-intrinsic effects of Malt1 deficiency. J Leukoc Biol. 2019. Mahdis Monajemi performed experiments, data analysis, and manuscript preparation. Susan C. Menzies maintained the mouse colony. Susan C. Menzies and Shera Fisk assisted with culturing of osteoclasts and Trap staining in Fig 4.6. Yvonne C. F. Pang and Jessica Leung assisted with Western blots and viii  qPCRs in Figure 4.5, and analysis of resorbed area in Fig 4.7. Laura M. Sly contributed to experimental design and manuscript preparation.   ix  Table of Contents Abstract ......................................................................................................................................... iii	Lay Summary .................................................................................................................................v	Preface ........................................................................................................................................... vi	Table of Contents ......................................................................................................................... ix	List of Figures ............................................................................................................................. xiv	List of Symbols and Abbreviations ......................................................................................... xvii	Acknowledgements .................................................................................................................. xxiii	Dedication ...................................................................................................................................xxv	Chapter 1: Introduction ................................................................................................................1	1.1	 Combined immunodeficiency ......................................................................................... 1	1.1.1	 Etiology and pathogenesis of combined immunodeficiency .................................. 1	1.1.2	 The immune response in combined immunodeficiency ......................................... 3	1.1.3	 Clinical presentation of combined immunodeficiency ........................................... 3	1.1.4	 Therapeutic options for combined immunodeficiency ........................................... 4	1.1.4.1	 Hematopoietic stem cell transplantation ............................................................. 4	1.1.4.2	 Gene therapy ....................................................................................................... 5	1.1.4.3	 Immunoglobulin (Ig) treatment ........................................................................... 5	1.1.4.4	 Prophylactic antimicrobials ................................................................................ 6	1.1.5	 Combined immunodeficiency in patients with MALT1 deficiency ....................... 6	1.2	 Inflammatory bowel disease ........................................................................................... 8	1.2.1	 Etiology and pathogenesis of inflammatory bowel disease .................................... 9	1.2.2	 The immune response in inflammatory bowel disease ......................................... 11	x  1.2.3	 Clinical presentation of inflammatory bowel disease ........................................... 13	1.2.4	 Therapeutic options for inflammatory bowel disease ........................................... 14	1.3	 Osteoporosis .................................................................................................................. 16	1.3.1	 Bone structure ....................................................................................................... 16	1.3.2	 Etiology and pathogenesis of osteoporosis ........................................................... 18	1.3.3	 The immune response in osteoporosis .................................................................. 19	1.3.4	 Clinical presentation of osteoporosis .................................................................... 20	1.3.5	 Therapeutic options for osteoporosis .................................................................... 21	1.4	 Myeloid cells ................................................................................................................. 22	1.4.1	 Myeloid cell development ..................................................................................... 22	1.4.2	 Myeloid cell function ............................................................................................ 23	1.4.3	 Macrophage development ..................................................................................... 24	1.4.4	 The role of intestinal macrophages in inflammatory bowel disease ..................... 26	1.4.5	 Osteoclast development ........................................................................................ 28	1.4.6	 The role of osteoclasts in osteoporosis ................................................................. 30	1.5	 MALT1 ......................................................................................................................... 32	1.5.1	 The role of MALT1 in NF-kB activation ............................................................. 32	1.5.2	 The role of NF-kB activation in macrophages and osteoclasts ............................ 35	1.6	 Thesis hypothesis and objectives .................................................................................. 36	1.6.1	 Summary of rationale ............................................................................................ 36	1.6.2	 Hypothesis and objectives ..................................................................................... 38	1.6.3	 Significance ........................................................................................................... 38	xi  Chapter 2: Malt1 blocks IL-1b production by macrophages in vitro and limits dextran sodium sulfate-induced inflammation in vivo ............................................................................40	2.1	 Introduction and rationale ............................................................................................. 40	2.2	 Material and methods .................................................................................................... 42	2.3	 Results ........................................................................................................................... 45	2.3.1	 Malt1-/- mice are more susceptible to DSS-induced colitis than Malt1+/+ mice ... 45	2.3.2	 Concentrations of IL-6 and IL-1b are increased in colon tissues of Malt1-/- mice after DSS treatment ............................................................................................................... 47	2.3.3	 Macrophage depletion reduces DSS-induced colitis in Malt1-/- mice ................... 48	2.3.4	 Macrophage depletion reduces colonic IL-6 and IL-1b concentrations after treatment with DSS in Malt1-/- mice ..................................................................................... 50	2.3.5	 Malt1+/+ and Malt1-/- MCSF-derived bone marrow macrophages produce similar concentrations of pro-inflammatory cytokines when stimulated with LPS, zymosan, or curdlan…. .............................................................................................................................. 51	2.3.6	 Malt1-deficient MCSF-derived bone marrow macrophages produce higher concentrations of IL-1b ......................................................................................................... 52	2.3.7	 Increased IL-1b production by Malt1-/- macrophages is associated with increased transcription of Il1b but not increased inflammasome activation ......................................... 54	2.3.8	 Higher concentrations of IL-1b in colonic tissues of DSS-treated Malt1-/-  mice is associated with increased transcription of Il1b but not increased number of cells with active caspase-1.. ............................................................................................................................. 55	xii  2.3.9	 Treatment with Anakinra, an IL-1 receptor antagonist, reduces DSS-induced colitis in Malt1-/- mice ........................................................................................................... 56	2.3.10	 Treatment with Anakinra does not reduce intestinal inflammatory cytokines concentrations in Malt1-/- mice .............................................................................................. 58	2.4	 Discussion ..................................................................................................................... 59	Chapter 3: Innate immune stimuli increase Malt1 mRNA and protein expression and eliminate Malt1 activity in macrophages in vitro but MALT1/Malt1 mRNA expression is not altered in the inflamed intestine of people with IBD or in mice during DSS-induced colitis ..............................................................................................................................................64	3.1	 Introduction and rationale ............................................................................................. 64	3.2	 Material and methods .................................................................................................... 66	3.3	 Results ........................................................................................................................... 70	3.3.1	 Stimulation of macrophages with LPS, zymosan, or curdlan increases Malt1 protein expression but decreases Bcl10 cleavage ................................................................. 70	3.3.2	 Stimulation of macrophages with LPS, zymosan, or curdlan decreases Malt1 activity…. .............................................................................................................................. 71	3.3.3	 Inhibiting Malt1 activity increases macrophage TNF and IL-6 production ......... 72	3.3.4	 Inhibiting Malt1 activity decreases (LPS+ATP)-induced macrophage IL-1b production ............................................................................................................................. 74	3.3.5	 MALT1, BCL10, and CARD9 expression is not altered significantly in subjects with IBD................................................................................................................................ 75	3.3.6	 Malt1, Bcl10, and Card9 mRNA expression and activity is comparable in mice with or without DSS-induced colitis ..................................................................................... 77	xiii  3.4	 Discussion ..................................................................................................................... 78	Chapter 4: Malt1-deficient mice develop osteoporosis independent of osteoclast-intrinsic effects of Malt1 deficiency ...........................................................................................................84	4.1	 Introduction ................................................................................................................... 84	4.2	 Material and methods .................................................................................................... 86	4.3	 Results ........................................................................................................................... 92	4.3.1	 Malt1-deficient mice are smaller than wild-type littermates at 24 weeks of age . 92	4.3.2	 Trabecular bone volume is lower in 12- and 24-week-old Malt1-deficient mice . 93	4.3.3	 Malt1-/- mice have comparable number of osteoblasts in vivo .............................. 95	4.3.4	 Malt1-/- mice have higher number of osteoclasts in vivo ...................................... 96	4.3.5	 Stimulation of osteoclasts with RANKL increases Malt1 expression and activity…. .............................................................................................................................. 97	4.3.6	 Malt1 deficiency does not impact osteoclastogenesis ........................................ 100	4.3.7	 Osteoclast activity is not affected by Malt1 deficiency ...................................... 101	4.3.8	 Blocking Malt1 activity induces MCSF production and reduces OPG production by macrophages .................................................................................................................. 103	4.3.9	 MCSF increases, and OPG decreases, osteoclast differentiation in vitro, but this is independent of Malt1 deficiency in osteoclasts .................................................................. 104	4.4	 Discussion ................................................................................................................... 105	Chapter 5: Conclusions and future directions ........................................................................111	5.1	 Conclusions ................................................................................................................. 111	5.2	 Future directions ......................................................................................................... 117	Bibliography ...............................................................................................................................123	xiv  List of Figures  Figure 1.1 Role of microbiota-primed myeloid cells in the gut immune response. ...................... 12	Figure 1.2 Structure of long bone (femur). ................................................................................... 18	Figure 1.3 Myelopoiesis. ............................................................................................................... 23	Figure 1.4 Schematic view of osteoclastogenesis. ........................................................................ 30	Figure 1.5 The CARD9/BCL10/MALT1 signalosome complex drives NF-kB activation. ......... 34	Figure 2.1 Malt1-deficient mice have increased susceptibility to DSS-induced colitis. .............. 46	Figure 2.2 Malt1-deficient mice have increased concentrations of colon inflammatory cytokines in their colon tissues after DSS-induced colitis. ........................................................................... 47	Figure 2.3 Macrophage depletion by clodronate liposome reduces susceptibility of Malt1-deficient mice to DSS-induced colitis. .......................................................................................... 49	Figure 2.4 Macrophage depletion by clodronate liposome reduces IL-6 and IL-1b concentrations in colon tissues from Malt1-/- mice colon tissue. ........................................................................... 50	Figure 2.5 Malt1-deficinet macrophages stimulated with LPS, zymosan, or curdlan produce the same concentrations of TNF, IL-6, and IL-12p40 as Malt1+/+ macrophages. .............................. 52	Figure 2.6 Malt1-deficient macrophages produce high concentrations of IL-1b. ........................ 53	Figure 2.7 IL-1b mRNA is higher in MCSF-derived bone marrow macrophages and colon homogenates from Malt1-/- mice compared with Malt1+/+ mice. ................................................. 55	Figure 2.8 IL-1b mRNA is higher in colon homogenates from Malt1-/- mice compared with Malt1+/+ mice. ............................................................................................................................... 56	Figure 2.9 Anakinra treatment ameliorates DSS-induced colitis in Malt1-deficient mice. .......... 58	xv  Figure 2.10 Anakinra does not reduce inflammatory cytokine concentrations in Malt1-/- mice colon homogenates after DSS-induced colitis. ............................................................................. 59	Figure 3.1 Macrophage activation increases Malt1 protein expression but decreases Bcl10 cleavage. ........................................................................................................................................ 71	Figure 3.2 Macrophage activation blocks Malt1 activity. ............................................................ 72	Figure 3.3 The Malt1 inhibitor, mepazine acetate (MPZ), increases macrophage TNF and IL-6 production after stimulation with LPS and curdlan. ..................................................................... 73	Figure 3.4 The Malt1 inhibitor, mepazine acetate (MPZ), decreases macrophage IL-1b production after stimulation with LPS. ......................................................................................... 75	Figure 3.5 Subjects with IBD have comparable MALT1/BCL10/CARD9 mRNA expression compared to control subjects. ....................................................................................................... 76	Figure 3.6 Mice treated with DSS-induced colitis have similar expression of Malt1, Bcl10, and Card9 mRNA, Malt1 and Bcl10 protein, and Malt1 paracaspase activity compared to control mice, which were not treated with DSS. ....................................................................................... 78	Figure 4.1 Malt1-deficient mice are smaller than their Malt1+/+ littermates, but bone length is not reduced. ......................................................................................................................................... 92	Figure 4.2 Malt1-deficient mice have reduced bone volume in trabecular bone. ......................... 94	Figure 4.3 Malt1-deficient mice have reduced trabecular bone and a comparable number of osteoblasts in vivo. ........................................................................................................................ 96	Figure 4.4 Malt1-deficient mice have increased numbers of osteoclasts in vivo. ......................... 97	Figure 4.5 Malt1 is expressed and active in osteoclasts and Malt1 mRNA and protein expression is induced by RANKL. ................................................................................................................. 99	Figure 4.6 Malt1 deficiency does not affect osteoclast differentiation in response to RANKL. 100	xvi  Figure 4.7 Malt1 deficiency does not affect activity of bone marrow-derived osteoclasts. ....... 102	Figure 4.8 Inhibiting Malt1 in macrophages increases MCSF production and decreases OPG production in response to inflammatory stimuli. ........................................................................ 104	Figure 4.9 MCSF enhances and OPG inhibits osteoclast differentiation in vitro, in both Malt1+/+ and Malt1-/- osteoclasts. .............................................................................................................. 105	 xvii  List of Symbols and Abbreviations  β                                  Beta γ                                  Gamma g                                  Gram k                                 kappa  mg                              Milligram μg                               Microgram μm                              Mircometer μM                              Micromolar mL                              Milliliter µL                               Microliter µCT                            Microcomputed tomography ng                                Nanogram °C                                Degrees Celsius  a-MEM                       Minimum essential medium Eagle - Alpha modification  ABC-DLBCL              Activated B cell like diffuse large B cell lymphoma ADA                            Adenosine deaminase ALP                             Alkaline phosphatase  Anova                          Analysis of variance  ATP                             Adenosine triphosphate BCG                            Bacillus Calmette–Guérin xviii  BCL10                        B-cell lymphoma 10 BMD                           Bone mineral density BMDM                       Bone marrow-derived macrophages  BV / TV                      Bone volume / total volume CARD                         Caspase recruitment domain  CBM                           CARD/BCL10/MALT1 CD                               Crohn’s disease CID                             Combined immunodeficiency  Clod-lip                       Clodronate liposomes CLP                             Common lymphoid progenitors CMV                           Cytomegalovirus CMP                            Common myeloid progenitors COL1A1                     Collagen type 1a1 Cort. BMD                  Cortical bone mineral density Cort. Th              Cortical thickness CTX-1                         C-terminal type 1 collagen fragments Cur                              Curdlan DAI                             Disease activity index DMSO                        Dimethyl sulphoxide  DSS                            Dextran sodium sulphate DXA                           Dual-energy x-ray absorptiometry EDTA                         Ethylenediaminetetraacetic acid  EGF                            Epidermal growth factor xix  ELISA                        Enzyme-linked immunosorbent assay  ERa                            Estrogen receptor-a FBS                             Fetal bovine serum FLICA                         Fluorescent labelled inhibitor of caspases FMT                            Fecal microbiota transplant  GAPDH                      Glyceraldehyde 3-phosphate dehydrogenase G-CSF                        Granulocyte colony stimulating factor GMCSF                      Granulocyte macrophage colony stimulatory factor GMP                           Granulocyte/macrophage lineage-restricted progenitor GPCRs                        G protein-coupled receptors GvHD                         Graft versus host disease h                                  Hour H&E                            Hematoxylin and eosin  HLA                            Human leukocyte antigen HSC                            Hematopoietic stem cells HSCT                          Hematopoietic stem cell transplantation HSV                            Herpes simplex virus IBD                             Inflammatory bowel disease IgG                              Immunoglobin G monoclonal antibody IHC                             Immunohistochemistry  IKK                             IkB kinase IL                                Interleukin ILCs                            Innate lymphoid cells xx  IMDM                         Iscove's modified Dulbecco's medium  IP                                 Intraperitoneal injection ITK                             IL-2-inducible Tyrosine kinase  LC                               Langerhans cells LPS                             Lipopolysaccharide MΦ                             Macrophage  MALT1                      Mucosa-associated lymphoid tissue lymphoma translocation gene 1 MAMPs                      Microbe-associated molecular patters MCSF                         Macrophage colony stimulatory molecule MEP                           Megakaryocyte/erythrocyte lineage-restricted progenitor Min                             Minute MMPs                         Matrix metalloproteinases MPZ                            Mepazine  mRNA                         Messenger RNA NFATc1                      Nuclear factor of activated T cells 1 NF-kB                         Nuclear factor kappa-light-chain-enhancer of activated B cells NK cells                      Natural killer cells NOD2                         Nucleotide-binding oligomerization domain containing 2 NSAIDs                      Non-steroidal anti-inflammatory drugs OPG                            Osteoprotegerin OSC                            Osteoclast OSCAR                       Osteoclast-associated receptor P1NP                           Procollagen type 1 N-terminal propeptide xxi  PBMC                         Peripheral blood mononuclear cells  PBS                             Phosphate buffer saline  PD                               Paracaspase dead  PID                              Primary immunodeficiency disease PMA                            Phorbol 12-myristate 13-acetate PSC                             Primary sclerosing cholangitis PTPRC                        Protein tyrosine phosphatase receptor type C qPCR                           Quantitative polymerase chain reaction RAG                           Recombinase activating genes RANK                        Receptor activator factor of nuclear factor kB  RANKL                      Receptor activator factor of nuclear factor kB ligand SCID                           Severe combined immunodeficiency SDS-PAGE                 Sodium dodecyl sulfate polyacrylamide gel electrophoresi SHIP                           SH2 domain-containing inositolpolyphosphate 5ʹ-phosphatase SNPs                           Single nucleotide polymorphisms Tb. BMD                    Trabecular bone mineral density Tb. N                          Trabecular number Tb. Sp                         Trabecular separation Tb. Th                         Trabecular thickness TLR                      Toll-like receptor  TNF                            Tumor necrosis factor  TNFSF11                    TNF receptor superfamily TRAP                         Tartrate resistant acid phosphatase xxii  Treg                            Regulatory T cells UC                              Ulcerative colitis VDR                           Vitamin D receptor WT                             Wild-type ZAP-70                       Zeta-associated protein of 70 kDa Zym                            Zymosan    xxiii  Acknowledgements  This research was funded by Canadian Institutes of Health Research (CIHR) grant. I was also a recipient of BC Children’s Hospital Research Institute (BCCHR) Studentship, CIHR-funded Transplantation Training Program Studentship, and Mitacs-Accelerate Fellowship.  First and foremost, I would like to express my deepest appreciation to my PhD supervisor, Dr. Laura Sly, for giving me the opportunity to persuade my doctoral studies in a supportive and friendly environment in her laboratory. I am grateful for her advice and patience during my PhD journey that made me a better scientist and improved my science communication skills by having the opportunity to present at local and international conferences. I very much appreciate her guidance, kindness, and the time she spent to assist me in writing scientific papers and becoming a better problem solver and leader.  Besides my PhD advisor, I would like to thank the rest of my supervisory committee: Dr. Tobias Kollmann, Dr. Jacob Rozmus, and Dr. Bruce Vallance for their valuable comments and creative questions that helped me expand my knowledge and broaden my research perspectives. I would also like to thank the Centre for High-Throughput Phenogenomics at UBC for access to Micro-CT, and particularly Dr. Guobin Sun who helped me with using the Micro-CT scanner and data analysis. I am also grateful to the staff at BCCHR Histology Core and Animal Care Facility for their assistance during my PhD.  My past and present lab mates, and friends at BCCHR, thank you so much for all your friendship and support throughout my PhD. Thank you to Susan Menzies, Dr. Eyler Ngoh, Dr. Lisa Kozicky, Jean Philippe Sauvé, Young Lo, Peter Dobranowski, Yvonne Pang, Dr. Rym Ben xxiv  Othman Aniba, and Dr. Bing Cai. I am especially thankful for the contribution of the students who I mentored. Thank you to Yvonne Pang, Shera Fisk, Saelin Bjornson, and Jessica Leung.  I would also like to thank my M.Sc. supervisor Dr. Mani Larijani for his continued support towards my progress from the early days I came to Canada.   Last but not least, I would like to thank my family: My husband, Alireza Ensan, for his continued encouragement, love, and support that kept me motivated over these years. Thank you to my parents for always supporting me spiritually and financially. I would never have done this without you.  xxv  Dedication  To my dear Alireza, and mom & dad, for your continued support, encouragement, and love to make this work what it is today. This success is yours, I love you 1  Chapter 1: Introduction  1.1 Combined immunodeficiency  Immunodeficiency disorders occur when the immune system is impaired, leading to higher risk of infection, autoimmunity, aberrant inflammation, and malignancy. There are two main types of immunodeficiency: primary immunodeficiency (PID) caused by genetic defects, which is associated with impaired immune function; and secondary immunodeficiency, the more common type of the disease, which is caused by external factors [1, 2]. The estimated prevalence of PID is between 1:2000 - 1:10000 [1]. PIDs are caused by immune defects. PIDs are often not recognized at birth due to the temporary effect of transferred maternal antibodies, but clinical symptoms can begin to appear within the first few months of life. PIDs can be caused by defects in both the innate and adaptive immune system. Defects in components or activation of innate immunity, such as neutrophils, phagocytes, toll-like receptor signalling, and complement are associated with PIDs [1]. However, PIDs are broadly defined as adaptive immune conditions and the most frequent type of PID is antibody-deficiency disorders associated with impaired humoral immunity [2].  Secondary immunodeficiencies are caused by viral and bacterial infection, malnutrition, acquired metabolic disorders, malignancy, and immunosuppressant drugs [2]. Most secondary immunodeficiencies resolve after the external cause is eliminated.  1.1.1 Etiology and pathogenesis of combined immunodeficiency Combined immunodeficiency disease (CID), a life-threatening genetic defect characterized by lack or defect of the immune response, is a type of PID. CID can be caused by gene defects that lead to loss or malfunction of immune system components. It is very important 2  to know the exact cause of CID to select the most appropriate treatment and to obtain genetic counselling for future pregnancies. Recent advances in molecular biology, human genomics, and immunology obtained through studying genetically determined immunodeficiencies in mutant mice and humans, has had a major impact on the discovery of gene defects that result in CID. Common abnormal genetic defects in patients with CID include defects in genes linked to cytokine receptor, antigen receptor, and immune regulatory molecules. The most common gene defects associated with CID include deficiency in adenosine deaminase (ADA), which is important for immune cell development; IL-2-inducible tyrosine kinase (ITK), which is critical for T cell differentiation and proliferation; the zeta-associated protein of 70 kDa (ZAP-70), which is involved in T cell signalling; recombinase activating genes 1 and 2 (RAG1/2), which are responsible for TCR development; and Janus kinase 3 (JAK3), which mediates signal transduction of cytokine receptors in hematopoietic cells [3]. Altogether, these genetic defects disrupt signal transduction and immune cell effector functions. Although CID is primarily characterized by the absence of a T cell response, there are CID patients with no specific T cell lymphopenia. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is a ubiquitously expressed gene that can be involved in CID. This gene is involved in cellular proliferation and activity of different mammalian cells. Defects in NF-kB signalling result in abnormal immune cell development and responses that cause a failure of the immune system leading to severe immune deficiencies [4]. Since NF-kB signalling is activated in different types of blood cells and tissues, aberrant activation of NF-kB in patients with immune deficiency causes defects in various immune cells and is not restricted to T cells.  3  1.1.2 The immune response in combined immunodeficiency  The “combined” nature of CID primarily refers to defects in T cell development and activity that is sometimes accompanied by poor or absent B cell responses. These diseases are categorized based on absence of T cells with presence or absence of B cells (T-, B+), (T-, B-) [2]. In the severe form of CID (severe combined immunodeficiency or SCID), there is profound T cell deficiency that is diagnosed by counting lymphocytes, as T cells comprise 70% of lymphocytes. Infants with lymphocyte count of less than 2000 cells/µL are highly likely to have SCID [1]. Immunoglobulin concentrations do not help to diagnose CID, especially in early infancy since the IgG concentration is generally normal in infants due to transferred maternal antibodies, and IgA and IgM are usually low during infancy and can be difficult to detect [5].     1.1.3 Clinical presentation of combined immunodeficiency  Clinical presentation is different for each child and appears a few months after birth. Initial diagnosis of CID can be accelerated based on family history or previous, unexplained infant death. Children with CID are more vulnerable to opportunistic infections, which might cause organ damage, morbidity, and mortality if they receive no treatment and/or live in unprotected environments [5]. Children might experience a wide range of symptoms including ongoing and progressive bronchiolitis with chronic cough, persistent respiratory infection, oral and gastrointestinal infection, viral diarrhoea, delayed growth, skin rash due to rapid expansion of lymphocyte clones during infection, and skin sepsis [5]. In addition to these complications, any unusual, persistent, severe, or opportunistic infection by fungi, viruses, and/or bacteria can be manifestations of CID [5]. The most common types of infections that should be further investigated include Pneumocystis carinii pneumonia, cytomegalovirus (CMV), bacillus 4  Calmette–Guérin (BCG), and atypical mycobacterial infections [5]. Fungal infections, such as Pneumocystis carinii, and gastrointestinal viral infections by viruses including rotavirus and adenovirus are the most common types of infections in affected children. Frequency of candidiasis in the gut, skin, and oropharynx is also high in these children [5]. Bacterial infections are less common but transmission of staphylococci, streptococci, or Gram-negative bacteria through broken skin can cause sepsis, which is life-threatening [5]. Acute infection is associated with increased risk of death in children with CID, if they are untreated.  1.1.4 Therapeutic options for combined immunodeficiency  1.1.4.1 Hematopoietic stem cell transplantation Currently, the best treatment available for CID is hematopoietic stem cell transplantation (HSCT) from a related human leukocyte antigen (HLA)-identical donor. For HSCT, patients are injected with whole bone marrow cells including progenitor cells, which repopulate depleted bone marrow, and mature immune cells, which boost immunity [6]. The survival rate was very low in patients with CID prior to the first successful treatment of a 5-month-old infant with HSCT in 1968. HSCT efficiency and methodology have improved significantly and this has increased the survival rate for children with CID [7]. Survival rate following bone marrow transplantation is dependent on multiple factors, including the age of recipients. Railey, M. et al. found that infants who are transplanted before 3.5 months of age have a higher survival rate compared to infants who are transplanted after 3.5 months. The difference in survival rate is caused by higher thymic activity and better immune function following transplantation in younger infants [8]. In addition to age, the use of HLA-matched related donors is associated with decreased rate of graft versus host disease (GvHD) and more successful transplantation outcomes 5  [9]. Although HSCT is the primary treatment considered for patients with CID, difficulties in finding HLA-matched donors, graft-versus-host disease (GvHD), and delayed immunological reactions remain significant complications for this type of therapy.  1.1.4.2 Gene therapy Hematopoietic stem cells (HSCs) are key targets for gene therapy. Approximately 30 years ago, retroviruses were used to transfer genes to reconstitute hematopoietic stem cells [10]. Gene therapy has some advantages over HSCT including eliminating the need for a donor, immunosuppressive therapy, and eliminating the risk of GvHD. Gene therapy can efficiently correct murine models of RAG1/2 and Artemis gene deficiencies [11-14]. Further studies have shown clinical improvement by gene therapy in a few patients with CID, as demonstrated in mice [7]. Although gene therapy is emerging as a potential treatment for patients with CID, there are some challenges associated with this technique that need to be resolved, such as the development of an atypical form of leukemia following gene transfer [15]. Other complications related to gene therapy are designing a safe vector, effective gene editing, as well as preserving cell integrity and viability during the process [7]. Altogether, gene therapy is a progressive new method with great potential to treat complex disease that cannot be resolved using the pharmacological treatment options available.  1.1.4.3 Immunoglobulin (Ig) treatment Therapeutic immunoglobulins (Igs) are polyclonal immunoglobulins (mainly composed of IgG) derived from the plasma of thousands of healthy donors. Igs are used as a therapy in primary and secondary immunodeficiencies. Patients receive 400-600 mg/kg doses of Igs 6  intravenously once a month or subcutaneously once or twice per week [16]. Serum IgG delivers passive immunity through antibody neutralization and opsonization of infectious pathogens. This therapy was first given in 1890 but was replaced with antibiotic therapy due to lower cost and ease of administration [17, 18]. This treatment is administrated to patients with CID as complementary therapy while they are waiting for HSCT. To control potential infections more effectively, patients should receive immunoglobulin therapy early [16]. Since antibody deficiency is not the only complication of patients with CID, Ig treatment should be used as an adjuvant immunotherapy in patients with CID.   1.1.4.4 Prophylactic antimicrobials Live attenuated vaccines are not routinely recommended for CID patients with poor or defective immune responses. Rather, antimicrobial prophylaxis of antibacterials, antifungals, and antivirals against common pathogens is recommended for prevention of infections in these patients. Although pneumocystis is the leading opportunistic infection among patients with CID, other types of viral, bacterial, and fungal infections may also occur in these patients due to their compromised immune system. Patients should receive prophylaxis during the waiting period for HSCT/gene therapy to prevent infection [19]. Prophylactic antimicrobials are effective in some instances but still should be used for a short period of time to avoid additional risks associated with the development of microbial resistance [20].  1.1.5 Combined immunodeficiency in patients with MALT1 deficiency  CID due to mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) deficiency is a rare, genetic disease associated with dramatic inflammation and recurrent 7  infections. Recently, McKinnon et al. diagnosed a patient at BC Children’s Hospital with CID caused by MALT1 deficiency [21]. This patient inherited an autosomal recessive, homozygous missense mutation in the MALT1 gene leading to very low MALT1 expression as well as impaired MALT1 paracaspase activity and scaffolding function. Thus far, three other patients have been identified with loss of function mutations in MALT1. Two of them were siblings, who had inherited a MALT1-associated, autosomal recessive, homozygous missense mutation causing a complete loss of MALT1 protein [22]. Puwani. et al. also reported a patient with a heterozygous mutation for MALT1 (a de novo single nucleotide deletion), which led to abrogated MALT1 expression [23].  All patients with mutations in MALT1 experienced delayed growth, oral lesions, eczematous rashes, and gastrointestinal inflammation. Two of them developed bronchiectasis. Besides inflammatory symptoms, these patients also had concurrent fungal, viral, and bacterial infections by Candida species, CMV, herpes simplex virus (HSV), and S. aureus. McKinnon et al. reported the development of dysmorphic facial features and osteoporosis in the patient. McKinnon et al. also reported B cell lymphopenia with a greater portion of naïve IgD+IgM+CD27− B cells, a small fraction of IgD+IgM+CD27+ marginal zone B cells, and low IgD−IgM−CD27+ memory B cells in the patient. Puwani et al. reported expansion of CD4 and CD8 T cell populations and elevated naïve CD45RA T cells for the patient. Moreover, Punwani et al. reported low immunoglobulin levels accompanied with undetectable levels of IgE in the patient. In contrast, levels of IgM, IgA, and IgG were normal in rest of the patients, while one had abnormally high levels of IgE [23, 24]. The siblings with MALT1 mutations were treated with intravenous immunoglobulins and died by age 13. However, the other two patients, who had HSCT, had significant clinical improvement along with increased weight and height [23, 8  25]. Indeed, HSCT successfully resolved gastrointestinal inflammation and fragile bones in the patient reported by McKinnon et al. To understand how HSCT could resolve these conditions, pathogenic mechanisms of gastrointestinal inflammation and osteoporosis will be described below.  1.2 Inflammatory bowel disease Inflammatory bowel disease (IBD) is a chronic, relapsing, or progressive disease characterized by chronic inflammation along the gastrointestinal tract. There are two main subtypes of IBD: Crohn’s disease (CD) and ulcerative colitis (UC). IBD is a worldwide health issue with a prevalence rate of 4-10 in 10,000. Canada has one of the highest incidence rates of IBD in the world with approximately 1 in 200 Canadians affected [26]. CD and UC can appear at any age, but the median age of diagnosis is 29.5 years for CD and 34.9 years for UC [27]. Generally, sex does not have a significant effect on IBD prevalence; however, there is a modestly higher incidence rate of UC among men [27]. Race and ethnicity have been shown to play an important role in development of IBD, for example Caucasians have higher prevalence of IBD [28, 29].   Though both diseases are under the umbrella of IBD, there are key differences between UC and CD. UC mainly affects the colon and rectum, but some patients with UC also develop inflammation in their terminal ileum [30, 31]. In UC, continuous inflammation is largely limited to the mucosa versus deeper layers, such as muscle [28, 31]. Histologically, UC is characterized by crypt abscess, inflammatory cell infiltration, edema, and goblet cell depletion [28, 31]. UC is being diagnosed by endoscopic and histological assessment [28]. In contrast, CD can occur anywhere along the gastrointestinal tract from mouth to perianal region with higher frequency in 9  the distal ileum [28, 31]. In CD, patches of inflammation are separated by regions of healthy tissue and may be present through all intestinal layers extending to the submucosa [28]. Histologically, CD features include transmural inflammation accompanied by masses of immune cells called granulomas, as well as other clinical features such as fistulas and strictures that may block the intestine [28]. There is currently no definitive diagnostic test for CD and diagnosis is based on patient history and physical examination, supplemented with endoscopy, histology, laboratory, and radiological analysis [32].  1.2.1 Etiology and pathogenesis of inflammatory bowel disease The exact cause of IBD remains unknown. The consensus is that IBD is caused by multiple factors including environmental, genetic, and immunological components. Abundant environmental risk factors have been implicated in the pathogenesis of IBD. These factors include smoking, diet, geographical and social status, stress, non-steroidal anti-inflammatory drugs (NSAIDs), and microorganisms [33, 34]. Risk of developing IBD has increased in developing countries with progressive occupational health services over the last century [35]. This suggests that hygiene plays a role in the manifestation of this condition. The hygiene hypothesis was described in 1989 by Dr. Strachan, who suggested that less exposure to microbes or infection in early childhood could increase susceptibility to allergic disease [36].  An additional environmental factor that has been implicated in IBD is the gut microbiome. The normal gut microbiome has great diversity and consists of 1013 microbes with high numbers of bacteria [37]. Over the past decade, many studies have aimed to improve our understanding of the phenotype and function of gut microbiota. Microbial colonization and composition shape the immune system during the earliest days of life. The gut luminal 10  commensal and pathogenic microbiota are separated from the deeper layers of the gut by the epithelial barrier [38, 39]. The key role of the intestinal epithelial barrier in gut homeostasis is elucidated by studying germ free knockout mouse models, which develop intestinal inflammatory disease following disruption of the epithelial barrier and exposure to commensal bacteria [39]. Development of IBD is significantly mediated by a leaky epithelial barrier leading to microbial invasion that causes an altered gut microbiome, or dysbiosis, with a shift from predominant “symbiont” microbes to potentially harmful microorganisms that initiate a cascade of inflammatory responses [40]. Dysbiosis plays an important role in the pathogenesis of IBD through increased number of organisms such as Pectinatus, Sutterella, Fusobacterium, Mycobacterium paratuberculosis, and Helicobacter hepaticus as well as loss of bacterial diversity including a decline in normal and potentially protective gut microflora including Bacteroides, Eubacterium, and Lactobacillus species [41, 42]. Similar to the effects of gut environment and dysbiosis on abnormal immune response, genetic factors and host immune features also participate in the alternation of the intestinal microbiome. Thus, understanding the interplay between microbiome composition and host genotype may provide insight into the best strategy(ies) for treating people with IBD.  Studying single nucleotide polymorphisms (SNPs) and using transgenic and knock out mouse models, have revealed several genes involved in the pathogenesis of IBD. These genes play a role in IBD through their effect on immunoregulation, mucosal barrier integrity, gastrointestinal homeostasis, and bacterial clearance [43]. Although CD and UC have distinct clinical phenotypes, 110 out of 163 of the IBD-associated loci overlap between CD and UC [44]. Moreover, a significant proportion of IBD genetic markers are associated with other autoimmune and inflammatory diseases suggesting shared mechanistic features [45]. Nucleotide-binding 11  oligomerization domain containing 2 (NOD2) was the first identified gene associated with CD pathogenesis. This gene encodes the microbial associated molecule pattern receptor for muramyl dipeptide that leads to NF-kB activation in monocytes [46]. IRGM and ATG16L1 polymorphisms are also associated with CD and UC through the important role they play in autophagy [47]. TNFSF15 is another IBD susceptibility gene and has various frequencies in diverse ethnic groups [48]. Other genes such as IL-23R and PTPN2 are IBD candidate genes as shown by experimental models, which are also associated with other autoimmune disease like psoriasis and type I diabetes [49-51]. Causes of IBD are highly interrelated and breakdown at several checkpoints increases the risk of developing this disease. Creating models with genetic defects associated with IBD in order to thoroughly analyze these factors is critical.    1.2.2 The immune response in inflammatory bowel disease Both adaptive and innate immune responses have been shown to influence susceptibility to IBD. Defects in intestinal innate immunity consisting of the epithelial barrier and phagocytes within the lamina propria (macrophages, dendritic cells, and neutrophils) increases susceptibility to IBD [52, 53]. Following leakage of luminal contents into the sterile lamina propria during dysbiosis, dendritic cells and macrophages sense microbial antigens by their pattern recognition receptors (PRRs). This causes secretion of pro-inflammatory cytokines, such as IL-1b, IL-18, IL-12, IL-6, TNF, and IFNg that initiates inflammation (Fig. 1.1). Activation of innate immune cells also results in production of chemokines, such as CCL2 and CXCL8, which attract macrophages and neutrophils to the site of inflammation and increase the inflammatory response [54]. Thus, dysregulation of myeloid cell activity contributes to the pathogenesis of IBD. 12   Figure 1.1 Role of microbiota-primed myeloid cells in the gut immune response. (A) During homoeostasis, myeloid cells are involved in immune responses against nonpathogenic microbes, repair mechanisms, and immunotolerance against commensal flora. (B) Disruption of the epithelial barrier during intestinal inflammation allows commensal bacteria to enter the lamina propria. Interactions between infiltrating bacteria and pattern recognition receptors of intestinal macrophages, dendritic cells, and neutrophils initiate production of pro-inflammatory cytokines and chemokines, which play a role in the recruitment of immune cells and IBD-associated chronic inflammation.  Modified and reproduced with permission of Elsevier: Zaki, MH. et al. Trends Immunol. 2011 [54].  In terms of the adaptive immune response, different T cell populations are involved in IBD pathogenesis. Upon pathogen presentation by antigen-presenting cells, a T helper cell type 1 (Th1) immune response is induced in response to IL-12, initiating apoptosis signals or causing differentiation of cytotoxic T cells. Th17 cells are expanded in response to IL-23 and produce pro-inflammatory cytokines such as IL-17 and IL-22 that cause tissue destruction. Upon activation by IL-4, Th2 cells subsequently release cytokines including IL-4, IL-5, IL-13 to promote allergic reaction, induce IgE production, and stimulate eosinophilopoiesis. Moreover, since regulatory T cells (Tregs) play a key role during intestinal homeostasis by suppressing immune responses against commensal antigens, disruption of Treg responses plays a pivotal role in IBD pathogenesis [53]. In summary, various environmental, genetic, and immune mediated 13  factors play a role in development of IBD and understanding the precise mechanism underlying IBD pathogenesis may lead to the development of appropriate therapies.     1.2.3 Clinical presentation of inflammatory bowel disease IBD is associated with both intestinal and extraintestinal manifestations. Intestinal symptoms of IBD can range from mild to severe depending on the level of inflammation and the part of the intestine affected. The clinical manifestation of CD is often subtle, which can be associated with a delay in diagnosis. CD causes pain in the lower right part of the abdomen and is sometimes accompanied by rectal bleeding. A major complication of CD is intestinal blockage caused by edema and gut wall thickening [28]. Presentation of UC is more severe than CD. UC causes pain in the lower left part of the abdomen that extends to the entire abdomen and can be associated with weight loss, diarrhea, rectal bleeding, and fever [28]. Extraintestinal manifestations associated with IBD are common in patients with IBD. These conditions arise in different organs, sometimes prior to diagnosis of the disease, and can be due to systematic inflammation caused by IBD. Inflammatory manifestations of the disease can initially occur in the skin, eyes, joints, liver and biliary tract [55]. A dermatological condition is characterized by skin lesions that occurs in around 10% of IBD patients and psoriasis is a common comorbidity [56]. Problems within the hepatopancreaticobiliary system can develop in one-third of patients with IBD before the onset of gastrointestinal symptoms, and is mainly manifested as primary sclerosing cholangitis (PSC) [55]. Musculoskeletal symptoms are the most common extraintestinal manifestations of IBD and involve articular tissue, periarticular tissue, and muscular tissue as well as increased risk of osteoporosis and arthritis associated joint pain and swelling [55]. Ocular complications include inflammation in episcleral blood vessels, which 14  often occur concurrently with other extraintestinal symptoms [55]. In addition to these complications, IBD can cause growth failure and delayed sexual maturation in children due to chronic malnutrition that is common in both CD and UC [28].   1.2.4 Therapeutic options for inflammatory bowel disease Medical therapy for IBD is focused on attenuating the inflammation and attaining clinical remission and mucosal healing to increase the quality of life of patients. In a step-up approach to therapy, changes in diet and lifestyle are recommended primarily in order to help to maintain remission [57]. If this is not sufficient, drug therapy (antibiotics, steroids, immunomodulators, and biological agents) is administrated followed by surgical intervention, if needed [58]. In terms of pharmacological treatment, the choice of medication is based on disease severity and location. For relieving symptoms of mild to moderate IBD, oral sulfasalazine such as mesalamine, olsalazine, and balsalazide that are especially effective in treating UC are used alone or in combination with other drugs [28]. For treatment of patients with perianal complications and/or infection, antibiotics including metronidazole and ciprofloxacin are considered [28]. In patients with moderate IBD, who failed to respond to antibiotics, corticosteroids, such as prednisone, are recommended for management of symptoms [59]. Immunomodulatory drugs, such as 6-mercaptopurine and azathioprine, are reserved for patients with steroid dependency, unmanageable fistulae, gastroduodenal and perianal disease. However, these drugs are associated with adverse side effects since suppressing immune responses can cause complications such as leukemia, infectious complications, hyper-sensitivity reactions, and gastrointestinal intolerance [28].  15  Recently, biologic agents directed against specific molecules have emerged as novel therapeutic agents to treat patients with moderate to severe IBD. One of the primary complications of IBD pathogenesis is inflammation caused by abnormal production of inflammatory cytokines, including TNF. Biological therapies including infliximab, adalimumab, and certolizumab chimeric monoclonal antibodies that neutralize soluble and membrane bound TNF, have been used successfully to manage IBD symptoms and maintain remission [60]. Recent studies suggest that beginning the therapy with anti-TNF in a “top-down” approach, may improve patient outcomes [61-63]. Although anti-TNF drugs have successful long-term outcomes, some patients are not responsive or become refractory to anti-TNF drugs and other treatments are required. Strategies to target other cytokines and immune cell migration, adhesion, and activation have been under investigation [64]. Ustekinumab is an antibody that targets IL-12/23p40 and has been recently approved for the treatment for CD [65]. Two anti-integrin drugs (natalizumab for CD and vedolizumab for both UC and CD), which are supposed to block migration of immune cells to the site of intestinal inflammation, are currently approved for treatment of IBD [66]. In addition to these drugs, new evidence suggests that fecal microbiota transplant (FMT) from healthy donors to individuals with IBD can restore healthy gut microbes and is under investigation for the treatment of IBD [67]. So far, biological therapies have shown lower risk compared to other medications available for IBD, but further studies are required to evaluate the use of effective biological therapies in a personalized medicine approach to treat disease.   16  1.3 Osteoporosis Osteoporosis is a bone metabolic disease that is associated with reduced bone mass when bone is degraded faster than it is replaced. Osteoporosis is characterized by low bone mass, bone decay, disruption of bone micro-architecture, declined bone strength, and increased risk of fractures [68]. Worldwide, osteoporosis leads to 8.9 million fractures each year [69]. Osteoporosis is mediated by disruption of a process called bone remodelling, the removal and replacement of bone tissue. Bone structure is mainly composed of mineral salts (calcium and phosphorus), organic matrix, and bone cells. There are two main types of bone cells. Osteoclasts remove bone tissue by resorbing bone matrix and dissolving the bone mineral; osteoblasts deposit new bone tissue by laying down layers of bone extracellular matrix [68]. Bone remodelling is controlled by organized signalling mechanisms and imbalances in this process lead to development of different bone diseases. Osteoporosis is represented by a variety of pathophysiological conditions and is classically divided into primary and secondary classes. Primary osteoporosis is triggered by aging as well as hormonal changes in postmenopausal women, whereas secondary osteoporosis is caused by changes in lifestyle, genetic disease, immunodeficiency, digestive and neurological disorders as well as autoimmune diseases [68, 70].  1.3.1 Bone structure Bone is hard and dense connective tissue that provides scaffolding for other body tissues.  Bone also provides an environment for bone marrow and storage of minerals. Bone tissue is mainly composed of collagen that forms soft tissue, and minerals that leads to hardness of bone structure [71]. The structure of the bone mainly consists of 80% cortical and 20% trabecular 17  compartments [72]. Cortical bone is the dense outer surface of the bone that provides strength to longer bone structures, such as the femur (Fig. 1.2) [72]. Trabecular bone, also called cancellous bone, is the spongy tissue found at the end of long bones. The trabecular bone promotes recycling of mineral salts and provides flexible bone structural support [72]. Trabecular bone is composed of an interconnected network of plates and rod structures (Fig. 1.2) [72]. The femur is the longest bone in the body and consists of diaphysis, which runs between the proximal and distal ends of the bone, and contains a hollow region called the medullary cavity that is filled with bone marrow and surrounded by cortical bone [72]. The two wide sections at the ends of the femur are called epiphysis, which is composed of trabecular bone and has spaces filled with fat and bone marrow [72]. Between the epiphyses and diaphysis there is a section called the metaphysis (Fig. 1.2) [72].  During osteoporosis, the plate-rod like structures of the bone become weak and are disrupted [73]. Bone micro-architecture, geometry, and density of trabecular and cortical bone are compromised during osteoporosis. To determine bone strength, trabecular number, thickness, and connectivity as well as cortical bone thickness are assessed. The only possible way to detect micro-architecture and geometry of the bone, which is recommended for diagnosing osteoporosis, is dual-energy X-ray absorptiometry (DXA) or bone densitometry [74]. Since risk of fractures is quite high in the femur, it is one of the preferred measurement sites for DXA [75, 76]. Bone fragility occurs more commonly in this region and examining the changes in metaphyseal trabecular bone is important for determining the risk of fracture [77]. Thus, two layers of cortical bone and trabecular bone function together to maintain bone structure and to protect other internal organs.  18   Figure 1.2 Structure of long bone (femur).  A schematic illustration of basic long bone structure. Diaphysis: long bone main shaft, Epiphysis: the rounded ends of long bones, Metaphysis: the region between the diaphysis and epiphysis at the ends of long bones. Trabecular bone: spongy bone tissue inside epiphysis and metaphysis. Cortical bone: protective bone layer covering marrow cavity. Modified and reproduced with permission of Springer Nature: Morrison, Sean J. et al. Nature. 2014 [78].  1.3.2 Etiology and pathogenesis of osteoporosis  Both environmental and genetic factors are involved in the etiology of bone disease. Life style and environmental factors linked to osteoporosis include inadequate nutrition, low level of physical activity, smoking, air pollution, and stress. Whereas, consuming a well-balanced diet with high intake of dairy products rich in calcium and vitamin D, fish, fruits, vegetables, and whole grain helps promote bone metabolism [79]. Reports show that ethnicity, age, and gender influence the rate of hip fractures and osteoporosis development. For example, Caucasian and Asian women have higher risk of osteoporosis compared to men for people ³ 50 years of age [80]. Furthermore, osteoporosis is a polygenic disorder mediated by different genes, among which vitamin D receptor (VDR), the collagen type 1a1 (COL1A1), and the estrogen receptor-a (ERa) genes are the most commonly implicated. In addition to these candidate genes, 19  polymorphisms in genes encoding the critical immunological mediators such as IL-1, TGF-b, and IL-6 can also contribute to lower bone mass and osteoporosis [81]. It is essential to define critical genes involved in regulation of bone remodelling, and to determine the interaction between these genes and environmental factors.   1.3.3 The immune response in osteoporosis  Recently, numerous studies have reported an interaction between bone health and immune cells, and mechanisms regulating cross-talk between them. The interactions between the immune system and bone metabolism observed during autoimmune and inflammatory disease is termed “osteoimmunology”, which was first described in the year 2000 [82]. The interplay between immune cells and bone cells is mediated by cytokines as well as signal transducers and immunomodulatory molecules. Significant production of pro-inflammatory cytokines by both adaptive and innate immune cells (B cells, T cells, macrophages, and dendritic cells) including TNF, IL-1, IL-7, IL-17, IL-23, IL-6, TGFb, and IFNg lead to osteoporosis downstream of inflammatory disease [83, 84]. Over production of pro-inflammatory cytokines including TNF, IL-6, IL-1, and IFN-g by macrophages and T cells are largely involved in bone resorption. Likewise, immune cells regulate the production of cytokines including macrophage colony stimulatory molecule (MCSF) and receptor activator factor of nuclear factor kB ligand (RANKL), which have a direct role in osteoclastogensis. They also regulate the production of the RANKL decoy receptor osteoprotegerin (OPG), which is a negative regulator of osteoclastogenesis [85-89]. In addition to cytokines, transcription factors are also involved in formation and activity of bone cells. NF-kB is one of these transcription factors that inhibits 20  osteoblast differentiation and function as shown by enhanced bone formation in mice after inhibiting NF-kB signalling in osteoblasts [90]. However, NF-kB positively regulates osteoclast differentiation, which is demonstrated by impaired differentiation of osteoclasts in mice deficient in NF-kB subunits [91]. Overall, during inflammatory disease, aberrant molecular and environmental properties mediate imbalances during bone remodelling leading to either increased or decreased bone mass.  1.3.4 Clinical presentation of osteoporosis  The primary complications of osteoporosis are vertebral fractures caused by mild trauma such as a simple fall. There are also nonvertebral fractures, which are typically undetected as they are often asymptomatic and can only be diagnosed by X-ray. Osteoporosis diagnosis is usually challenging, as it has no clinical manifestations until fractures occur. The prevalence of osteoporosis is rising due to an aging population. In older people, bone mineral density (BMD) decreases, which can be further exacerbated by primary and secondary causes of osteoporosis [92]. Clinical and public health consequences of osteoporosis arise from fractures that cause short- and long-term morbidity associated with pain, limitation of activities, declined quality of life, and increased risk of mortality due to poor health and physical function [93]. The most serious complications of osteoporosis are life threatening hip fractures. One standard deviation decrease in BMD is associated with roughly 1.5-fold increased risk of mortality in patients with osteoporosis [93]. Mortality associated with osteoporosis is often caused by morbidity, limited ability to perform activities and live independently, and poor general health as a result of fractures or fall [93-97]. Hence, preventing fractures and optimal osteoporosis management can reduce morbidity and mortality caused by osteoporosis.  21  1.3.5 Therapeutic options for osteoporosis  Osteoporosis treatment is classified into nonpharmacological and pharmacological groups. Nonpharmacological treatment aims to prevent fracture incidence. This includes calcium and vitamin D intake, avoiding smoking and alcohol consumption, and participating in weight-bearing exercise [98]. Pharmacological treatments for osteoporosis are either antiresorptive or anabolic. Antiresorptive medications including bisphosphonate, hormonal therapy, and denosumab reduce the rate of bone resorption, while anabolic drugs, such as teriparatide, increases the rate of bone formation [98]. Bisphosphonates are remarkably effective against osteoporosis and are generally considered first-line therapy for fragility fractures. These medications inhibit dissolution of mineral crystals and osteoclast bone resorption [99]. Another group of treatments is hormonal therapy, which includes either an estrogen component or an estrogen receptor modulator with similar effects on bone tissue. Estrogen deficiency has an important role during postmenopausal osteoporosis because it increases bone resorption; therefore, hormonal therapy can alleviate estrogen deficiency-induced bone loss in women with postmenopausal osteoporosis [100]. Calcitonin is another hormone used to inhibit bone resorption and reduce the risk of fractures, but it is less effective than bisphosphonate [101, 102]. Another group of treatments are recombinant parathyroid hormone and its N-terminal fragment, teriparatide, both of which are effective anabolic agents approved for treatment of osteoporosis. These drugs exert their effects by increasing bone remodelling thereby mediating increased trabecular and cortical bone formation [103]. Recently, recombinant antibodies have emerged as a novel therapy for treatment of osteoporosis. Denosumab, a human immunoglobin G2 monoclonal antibody (IgG2), acts as a decoy receptor for RANKL (with higher affinity than OPG) to prevent osteoclast differentiation [104]. Despite different therapeutic agents available 22  for osteoporosis, the benefit-risk ratio of these drugs limit their long-term use and it is necessary to design novel medications for treatment of osteoporosis.   1.4 Myeloid cells 1.4.1 Myeloid cell development  Hematopoietic stem cells are multipotent stem cells residing in the bone marrow with the potential to generate both the myeloid and lymphoid lineage of blood cells. The lymphoid lineage, derived from common lymphoid progenitors (CLP), include T, B, and natural killer cells (NK cells). The myeloid lineage arises from common myeloid progenitors (CMP) and consists of granulocytes, monocytes, macrophages, erythrocytes, and megakaryocytes [105-107]. Dendritic cells were classically considered myeloid cells, but recent studies have shown that they can also arise from lymphoid progenitors [108, 109]. CMP give rise to two more distinct progenitors, the granulocyte/macrophage lineage-restricted progenitor (GMP) and the megakaryocyte/erythrocyte lineage-restricted progenitor (MEP). GMP give rise to granulocyte progenitors (GP) and monocyte progenitors (MP) that eventually form differentiated progeny of myeloid cells (Fig. 1.3) [107]. Myeloid cells play significant roles during homoestasis and contribute to acute and chronic inflammation. An understanding of myelopoieses helps to develop novel therapeutic targets for immunodeficiency, malignancy, infection, and other immunomediated disease. 23   Figure 1.3 Myelopoiesis.  Multipotent hematopoietic stem cells differentiate to common myeloid progenitors (CMPs), which give rise to myeloid cells. CMPs then differentiate into megakaryocyte/erythroid progenitor cells (MEPs) or granulocyte/macrophage progenitor cells (GMPs). GMPs differentiate into either granulocyte precursors (GPs), which give rise to granulocytes such as basophils, neutrophils, and eosinophils or monocyte precursors (MPs), which give rise to macrophages. Modified and reproduced with permission of Copyright Clearance Center Ltd: Tidball, J. et al. Development. 2014 [110].  1.4.2 Myeloid cell function  Myeloid cells play a central role in the innate immune system and are the master regulators of adaptive immunity. These cells have unique phenotypes but share features like the ability to adapt to different environments in the body such as in the liver, gut, brain, and bone [111]. Monocytes circulate in the blood stream and can migrate into tissues, where they differentiate into macrophages [112]. Macrophages are widely distributed, phagocytic cells that play an important role in innate immunity and tissue homeostasis [112]. Dendritic cells are antigen-presenting cells and take up antigens to display them on the cell surface and travel to 24  lymph nodes to activate adaptive immune cells [112]. Osteoclasts are bone-resorbing cells that initiate bone remodelling and mediate bone inflammatory disease [113]. Neutrophils are the most abundant granulocyte and fight microorganisms via phagocytosis and releasing microbicidal proteins [112]. Other granulocytes such as basophils and eosinophils release mediators with a key role in immune homeostasis and allergic reactions [112]. Myeloid cells circulate through the blood and are recruited to the affected tissues during inflammation.   Myeloid cells recognize microbes by microbe-associated molecular patters (MAMPs). MAMPs are highly conserved nonspecific molecules, which are shared by most commensal and pathogenic microbes and are not expressed by host cells, helping immune cells to recognize non-self antigens [114]. Upon recognition of microbes, myeloid-dependent-activation of inflammatory responses leads to the release of inflammatory molecules such as reactive oxygen/nitrogen species, antimicrobial peptides/proteins, and secretion of inflammatory cytokines [115]. In addition to immune responses against microorganims, myeloid cells also have regulatory properties and protect the host against excessive immune responses to prevent tissue damage [116]. As a master regulator of immune responses, myeloid cells are key components of development and resolution of immune-mediated diseases through their production of cytokines, induction of inflammatory mediators, and establishment of immunological tolerance [117-119].   1.4.3 Macrophage development Macrophages are resident phagocytic cells located in both lymphoid and non-lymphoid tissues. Macrophages contribute to tissue homoeostasis and immunity through phagocytosis and induction of inflammatory cytokine production [120]. Originally, monocytes were considered to 25  be the only source of macrophages; however, it has become evident that groups of macrophages, such as Langerhans cells (LC) in the skin, microglia in the brain, and alveolar macrophages in the lung are derived from embryonic progenitor cells and renew independently, only occasionally originating from monocytes when depleted [121]. These tissue resident macrophages originate from the embryonic yolk sac and fetal liver prenatally [111]. During injury or inflammation, a large number of bone marrow derived monocytes are recruited to the site of inflammation and differentiate into activated tissue macrophages to replace resident macrophages in some tissues [111]. Some organs such as brain, skin, liver, pancreas, and spleen are comprised of preserved embryonic progenitor-derived macrophages during adulthood, whereas kidney and lung macrophages are derived from both yolk sac and hematopoietic stem cells. However, the gut is distinct from other tissues, perhaps due to predominant exposure to microbiota, which leads to enhanced recruitment of monocytes, and subsequently the gut macrophage population is largely composed of monocyte-derived macrophages [121].  Bone marrow monocyte-derived macrophages support the gut immune system to recognize and fight against pathogens, which breach the epithelial barrier.  Macrophages are remarkably heterogeneous because they arise from different progenitors, exist within distinct tissues (microenvironemnts), and because they are poised to respond to cues in their extracellular milieu. During homeostasis, regulation of resident macrophage differentiation is primarily mediated by colony stimulatory factor-1 (CSF-1 or MCSF), which is a growth factor synthesized by stromal cells. Granulocyte macrophage colony stimulatory factor (GMCSF) is another growth factor important for macrophage development during inflammatory conditions [121]. MCSF monocyte-derived macrophages are largely used as a model for tissue macrophages. GMCSF monocyte-derived macrophages, that mainly express 26  macrophage markers, are sometimes used as a model for DCs due to their shared biomarkers with these cells [122]. MCSF-derived macrophages form the key portion of macrophages in the gut, which is also the site of largest number of macrophages in the body [121, 123]. Overall, MCSF-derived bone marrow macrophages are the best in vitro derived models for gut macrophages.  1.4.4 The role of intestinal macrophages in inflammatory bowel disease  Numerous immune cells including macrophages are located in the gastrointestinal tract. Intestinal macrophages are located in the lamina propria region and mucosa-associated lymphoid tissue of the intestine alongside a vast number of commensal and pathogenic bacteria and antigens [124]. Intestinal macrophages as well as other intestinal phagocytes, including DCs, and specialized epithelial cells called M cells, sense the presence of microbes and act as a first line of defence against microbial invasion [125]. The gut-associated lymphoid tissue (GALT) is one the largest lymph tissues in the body, which is composed of aggregated lymphoid follicles called Peyer’s Patches (PPs) [126]. PPs are located in the mucosal layer of the intestine and play an important role in gut immune responses and tolerance toward antigens and bacteria. Following uptake of antigens by M cells located in PPs, they present antigens to macrophages and DCs [126]. Interactions between immune cells and the microbiota generate a basal level of inflammation that is important for maintaining homeostasis and regulating the inflammatory response [127].  Gut macrophages are anergic and present distinct phenotypic and functional characteristics. Release of inflammatory cytokines by intestinal macrophages is suppressed and their antigen presenting role is less effective compared to DCs; however, they maintain their 27  bactericidal activity [128, 129]. Indeed, gut resident macrophages are hyper phagocytotic and contribute to digestion and absorption of nutrients, water, and electrolytes [111]. In addition to these roles, intestinal macrophages maintain the local clearance of bacteria and antigens that leak through the epithelial layer, signal other immune cells, and contribute to gut homeostasis [130]. Macrophages have dual and well-balanced functions in terms of immune responses including host protection and tissue repair. This effect of macrophages is primarily mediated by cross-talk between macrophages and other immune cells as well as secretion of cytokines. Studies show that interactions between intestinal macrophages and microbiota promote IL-22 production by specialized innate lymphoid cells (ILCs), which induces both antimicrobial activity and tissue repair mechanisms [111, 131]. The engulfment of cell debris by macrophages can also lead to production of anti-inflammatory cytokines such as TGFb and IL-10, which are important for downregulation of the intestinal immune response during homeostasis [111]. Frequent exposure of the gut to microbial flora and food antigens obligates involvement of intestinal macrophages in maintaining gut immune tolerance. Expression of CX3CR1 in gut resident macrophages plays a role in IL-10 production to expand Tregs that provides an additional important layer of gut immune tolerance [132]. In addition, intestinal macrophages are the main source of IL-1b, which promotes intestinal innate lymphoid cells to produce GMCSF that causes myeloid cell-driven expansion of Tregs [133]. Secreting IL-1b following sensing microbial signals by gut resident macrophages also mediates Th17 cell differentiation, which has an important role in the development of many autoimmune and inflammatory diseases [134]. Thus, macrophages are recognized as key players in gut homeostasis and their aberrant function is associated with development of inflammatory disease such as IBD.   28  In patients with IBD, impaired microbial clearance is associated with intestinal inflammation. This can be caused by a reduction in macrophage phagocytic and bactericidal activity, which is associated with detectable levels of serum endotoxin in these patients [135]. In addition to defects in killing bacteria, macrophages also contribute to IBD pathogenesis through aberrant production of inflammatory cytokines. IL-10 plays an important role in maintaining intestinal immune tolerance evidenced by an increased risk of intestinal inflammation in humans and mice with IL-10 deficiencies [136, 137]. It has also been shown that a loss of function mutation in the TGFb receptor is associated with an increased risk of UC in humans, and a lack of macrophage response to TGFb causes delayed resolution of colitic inflammation in mice [138]. Moreover, IL-1b has dual effects in IBD pathogenesis. While production of IL-1b by myeloid cells has a protective role during the earlier stages of disease, it can promote intestinal inflammation in later stages of IBD by increasing the number and activity of key mediators in the cellular immune response [139, 140]. In summary, these studies demonstrate that macrophages play a critical role in IBD pathogenesis through their cytokine production and antibacterial activity.   1.4.5 Osteoclast development Osteoclasts are multinucleated giant cells with the capacity to resorb bone. Osteoclasts can have 2-100 nuclei formed by fusion of mononuclear preosteoclast cells [141, 142]. The unique capacity of osteoclasts to dissolve the bone matrix regulates normal bone growth. Impaired bone resorption mediated by defects in the osteoclast lineage is associated with osteopetrosis, a clinical condition, which is characterized by excessive bone mass [143]. Osteoclasts are a member of the monocyte/macrophage lineage derived from pluripotent 29  hematopoietic stem cells (Fig. 1.4) [144, 145]. Osteoclasts derived from hematopoietic progenitors were discovered when studies showed that transplantation with bone marrow could resolve osteopetrosis in both humans and mice [144, 145]. In addition to differentiation of osteoclasts from the monocyte/macrophage lineage, osteoclasts can be derived from mature tissue macrophages in an appropriate microenvironment [146]. Indeed, osteoclast precursors are mononuclear phagocytes in which the macrophage phenotype is rapidly replaced by an osteoclast phenotype following stimulation with osteoclast inducing factors [147]. Unique phenotypic features of osteoclasts include the expression of tartrate resistant acid phosphatase (TRAP), matrix metalloproteinases (MMPs), cathepsin K, PTH receptors, and calcitonin receptors. These cells also have increased acid phosphatase activity, which causes a high level of acid hydrolysis. Osteoclastogenesis is controlled by interplay between external regulatory factors and intracellular molecules. External factors that enhance osteoclast formation and activity include RANKL, CSFs, systemic hormones such as PTH, as well as calcitriol, prostaglandins, IL-1, TNF, IL-6, IL-11, and oxygen free radicals. On the other hand, external factors such as OPG, IFNg, IL-4, nitric oxide, calcitonin, and sex steroids, like estrogen, negatively regulate osteoclastogenesis and bone resorption [88, 113, 148].   30   Figure 1.4 Schematic view of osteoclastogenesis.  RANKL, MCSF, and OPG control the differentiation of hematopoietic-derived monocytes into osteoclasts. The MCSF/c-fms receptor interaction induces the generation of preosteoclast cells expressing RANK. RANKL triggers fusion of preosteoclast cells and the formation of active bone-resorbing multinucleated giant cells. OPG acts as a decoy receptor for RANKL and downregulates osteoclastogenesis. Modified and reproduced with permission of Oxford University Press: Henriksen, K. et al. Endocrine reviews. 2011 [148].  1.4.6 The role of osteoclasts in osteoporosis Osteoporosis-associated bone resorption is mediated by multinucleated osteoclasts. These cells digest the local matrix by secreting proteolytic enzymes [149]. Following the adhesion of osteoclasts to bone surface via the avb3 integrin, actin polymerisation mediates formation of a clear zone called the actin ring, which isolates the resorption pit [150]. Inside this zone, endosome-like structures form the ruffled border located on the apical side (bone contacting side) of osteoclasts that facilitates bone matrix uptake [150]. Osteoclast resorptive activity includes the concurrent processes of acidification and proteolysis [150]. Conversion of CO2 and H2O into H+ and HCO3- provides protons for acidifying the environment required for proteolysis of the collagen matrix by cysteine proteinase and matrix metalloproteinases (MMPs), which are active at a low pH [150]. Acidification depends on the pumping of protons that transfers the 31  protons against the concentration gradient and also requires passive transport of chloride in order to maintain the electrochemical balance necessary for acidification [142, 148, 149]. In addition to molecules with direct roles in osteoclast resorptive activity, fusion of osteoclast mononuclear cells to form an active multinucleated osteoclast is also critical for bone resorptive activity. This process is mediated by osteoclast cell surface lipids and specific molecules expressed by these cells, such as DC-STAMP, OC-STAMP, and Atp6vo2, all of which have a role in osteoclast fusion through unknown mechanisms [150]. When resorption activity is complete, osteoclasts either undergo apoptosis or roll on the bone surface via actin filamentation to continue their resorption activity [113, 151]. Thus, the main function of osteoclasts is to resorb bone tissue by their proteolytic enzymes. In addition to their role in bone resorption, studies show that there is cross-talk between osteoclasts and other cell types. Some studies show that osteoclast-derived factors can induce osteoblast differentiation and activity, which is important for bone remodelling during osteoporosis [152]. However, the effects of osteoclasts on the osteoblast lineage are controversial. For example, osteoclast function is blocked in patients with mutations in CLCN7 and they have increased numbers of osteoblasts, whereas mice with osteoclast deficiency due to a Csf1 mutation have reduced osteoblast formation [153]. Osteoclasts also have a role in the regulation of T cells as it has been reported that adoptive transfer of osteoclast precursors to a T cell model of rheumatoid arthritis inhibits T cell proliferation and attenuates joint inflammation in mice [154]. Interestingly, osteoclasts can generate autoregulatory negative feedback because they act as antigen presenting cells and can express co-stimulatory molecules as well as MHC class I and class II in humans and MHC class I in mice. T cells primed with osteoclasts have a regulatory phenotype that can suppress osteoclast differentiation [153]. The link between 32  osteoclasts and the immune response has an effect on immune-mediated disease. In addition to osteoporosis and osteopetrosis, abnormalities in osteoclast function are associated with other inflammatory and autoimmune diseases, namely Paget’s disease, bone tumors, breast cancer, and rheumatoid arthritis [155, 156]. Bone manifestations of inflammatory diseases are mediated by either aberrant function of osteoclasts to resorb bone and/or abnormal cross-talk between osteoclasts and other cells.  1.5 MALT1 1.5.1 The role of MALT1 in NF-kB activation The paracaspase MALT1 is a ubiquitously expressed protein that is one of the components critical for activation of NF-kB downstream of specific receptors. NF-kB is a family of inducible transcriptional factors that regulates the expression of a wide variety of genes involved in immune and inflammatory responses [157, 158]. The NF-kB family is also known as the Rel transcription factor family and is composed of members with Rel homology domain including c-Rel, RelA (p65), RelB, NF-kB1 (p50 and its precursor p105), and NF-kB2 (p52 and its precursor p100) [159]. NF-kB1 and NF-kB2 monomers form an NF-kB dimer after proteolytic processing of their precursors triggered by ubiquitination enzymes [159]. NF-kB activation is inhibited by the IkB family including IkB-a, IkB-b, and IkB-e by either sequestering NF-kB in the cytoplasm or suppressing its DNA-binding capability [159]. Stimulation of cell surface receptors leads to the activation of IkB kinase (IKK), which is composed of two catalytic subunits (IKKa and IKKb) and a regulatory subunit (IKKg/NEMO). 33  Degradation of IkB by the ubiquitin-proteasome pathway following phosphorylation by IKK allows NF-kB to enter the nucleus to turn on a large array of target genes [159-161]. Although NF-kB-mediated transcript is triggered by various stimuli through different cascades, activation of NF-kB by MALT1 has been the focus of recent investigations. MALT1 is involved in the activation of NF-kB downstream of immune cell receptors including Toll-like receptors (TLRs), C-type lectin receptors, the TNF receptor superfamily, B and T cell receptors, as well as non-immune cell receptors consisting of G protein-coupled receptors (GPCRs) and the epidermal growth factor (EGF) receptors [162]. In response to cell stimulation, MALT1, together with the adaptor protein B-cell lymphoma 10 (BCL10), associates with a variety of caspase recruitment domain family (CARD) proteins to form a CARD/BCL10/MALT1 (CBM) complex that facilitates downstream signalling pathways leading to NF-kB activation [162]. In this complex, CARD family proteins associate with BCL10 through CARD-CARD interactions, and the C-terminus of BCL10 interacts with N-terminal Ig domains of MALT1. CARD/CARMA is a family of evolutionally conserved proteins and is classified based on their expression in different cell types and tissues. This protein family involves CARD9, CARD10 (CARMA3), CARD11 (CARMA1), and CARD14 (CARMA2) [162, 163]. In T, B, and NK cells, CARD11 is expressed and MALT1 acts through formation of a CARD11/BCL10/MALT1 signalosome complex [164, 165]. In myeloid cells, CARD9 is expressed and transfers signals from ITAM-associated receptors including C-type lectin receptors (Dectin-1, Dectin-2, Mincle) and FcgRs. Upon stimulation of receptors, CARD9 associates with BCL10 and MALT1 to assemble into a CBM complex leading to NF-kB activation (Fig. 1.5) [162, 166].   34   Figure 1.5 The CARD9/BCL10/MALT1 signalosome complex drives NF-kB activation.  Upon stimulation of Dectin-1, SYK becomes activated and mediates cooperation between BCL10 and MALT1 molecules through the adaptor molecule CARD9. Formation of the CARD9/BCL10/MALT1 signalosome complex leads to NF-κB activation and a pro-inflammatory response. Modified and reproduced with permission of John Wiley and Sons: Ruland, J. Annals of the New York Academy of Sciences. 2008 [167].    During NF-kB activation, MALT1 has two different functions. MALT1 acts as a scaffolding molecule, providing a platform for recruitment of other downstream signalling components. Besides its scaffolding function, MALT1 is a member of the cysteine protease family with paracaspase activity [168]. MALT1 protease activity enhances immune cell activation by cleaving different substrates including BCL10, A20, RelB, Regnase-1, Roquin-1, and Roquin-2 [169-173]. However, it has been shown that cleavage of NF-kB subunit (p65), MCPIP1, and HOIL1 by paracaspase MALT1 can inhibit NF-kB activation [174-176]. In summary, MALT1 regulates NF-kB signalling through acting as a scaffolding molecule and a proteolytic enzyme, with a dual role in NF-kB-mediated gene expression.  35  Since MALT1 has a key role in both innate and adaptive immunity, dysregulated MALT1 activation is associated with a broad range of inflammatory diseases. Common chromosomal translocation involving the MALT1 gene results in activated B cell like diffuse large B cell lymphoma (ABC-DLBCL) caused by auto-oligomerization of the apoptosis inhibitor gene AP12 and MALT1 gene, which leads to constitutive activation of MALT1, and allows for abnormal B cell proliferation caused by constitutive NF-kB activation [177]. In contrast to the role of constitutive MALT1 activation in lymphoma, MALT1 deficiency has been identified as a novel cause of primary immunodeficiency [178].  1.5.2 The role of NF-kB activation in macrophages and osteoclasts In macrophages, NF-kB can be activated downstream of TLR4 as well as Dectin-1/2, and Mincle receptors. Stimulation of TLR4, through MYD88/IRAK/TRAF6/TAK1-dependent pathway leads to activation of NF-kB via a BCL10-MALT1 cascade [179]. Dectin-1/2 both bind to fungal, bacterial, and endogenous ligands, whereas the ligands for Mincle receptors remain unknown. Stimulation of macrophage receptors promotes Syk activation that recruits the CBM complex [180]. The activation of NF-kB is important for cell survival through expression of a variety of genes including AP-1, which is composed of c-Fos and c-Jun, proto-oncogene transcription factors [181]. Moreover, NF-kB mediates macrophage inflammatory responses by upregulating production of chemokines, adhesion molecules, and cytokines such as TNF, IL-6, and IL-1b [162, 182]. The role of NF-kB in phagocytosis by macrophages has also been shown in vitro through abrogated Staphylococcus aureus phagocytosis following the blocking of the NF-kB pathway in macrophage cell lines [183].  36  In addition to macrophages, NF-kB is one of the key regulators of osteoclast differentiation, survival, and function. The role of NF-kB in osteoclast differentiation was discovered when studies reported that mice deficient in NF-kB1 and NF-kB2 exhibit modest immunodeficiency. Their immunodeficiency was accompanied by an osteopetrotic phenotype caused by a lack of osteoclasts and could be resolved by hematopoietic stem cell transplantation [184]. In osteoclasts, NF-kB signalling is activated downstream of RANK, one of the members of the TNF receptor superfamily (TNFSF11). In addition to bone resident osteoclasts, RANK is expressed by different cell types including DCs and a large percentage of cancer cells, and is found in different tissues including thymus, mammary glands, prostate, and liver [185, 186]. Upon RANK stimulation by RANKL, NF-kB mediates expression of c-Fos that then stimulates induction of nuclear factor of activated T cells 1 (NFATc1) [184]. NFATc1 is a master regulator of osteoclastogenesis and regulates expression of key osteoclast markers such as TRAP, osteoclast-associated receptor (OSCAR), and cathepsin K [187]. So far, studies have shown that NF-kB can be activated through different cascades in osteoclasts, but the role of MALT1-mediated NF-kB activation in these cells has not been characterized.  1.6 Thesis hypothesis and objectives 1.6.1 Summary of rationale CID due to MALT1 deficiency is a rare genetic condition characterized by various clinical symptoms, including gastrointestinal inflammation and osteoporosis, both of which can be resolved by HSCT [21, 23, 25]. So far, studies have attributed these symptoms to defective adaptive immune responses in MALT1-deficient patients. However, MALT1 is a ubiquitously 37  expressed gene that is not only present in adaptive immune cells. MALT1 plays an important role in innate immune responses, which is predominantly mediated by myeloid cells. Macrophages are a type of myeloid cell derived from hematopoietic precursor cells with key roles in gastrointestinal inflammation [188]. IBD represents a group of disorders characterized by chronic inflammation along the gastrointestinal tract. During IBD development, highly phagocytic macrophages eliminate gut luminal microbes and induce pro-inflammatory responses. One of the key cytokines produced by macrophages that plays a very important role in IBD is IL-1b, which maintains innate and adaptive immune responses [140]. The goal of Chapter 2 was to define the role of Malt1 in the production of inflammatory cytokines, including IL-1b, by macrophages in vitro. I also studied the effect of intestinal macrophage depletion in the protection of Malt1-/- mice against gastrointestinal inflammation, using a dextran sulfate sodium (DSS)-induced colitis mouse model in vivo. I further examined the effect of blocking IL-1b using Anakinra, an IL-1b receptor agonist, on amelioration of DSS-induced colitis in Malt1- deficient mice in vivo.  MALT1 has two different functions, scaffolding and proteolysis. Although the involvement of MALT1 in NF-kB signalling in macrophages has been shown before [179, 180], the role of MALT1 proteolytic activity in these cells has not been assessed. The goal of Chapter 3 was to understand the role of Malt1 proteolytic activity in macrophage inflammatory responses using a Malt1 inhibitor, and to compare the expression and proteolytic activity of Malt1 before and after stimulation of macrophages with PRR ligands in vitro. I also tested whether gastrointestinal inflammation has any effect on Malt1 expression in vivo by measuring Malt1 mRNA expression in inflamed intestinal tissue versus control tissue in humans and mice.  38  Another clinical manifestation of MALT1 deficiency in the patient from BC Children’s Hospital was osteoporosis accompanied by delayed growth, which was also resolved following HSCT [21, 25]. Osteoclasts are a type of hematopoietic-derived myeloid cell with an important role in osteoporosis through their bone resorption activity. Osteoclast differentiation and function is mainly regulated by the NF-kB pathway but the role of MALT1 in this pathway in osteoclasts has not been described. The goal of Chapter 4 was to identify expression and activity of Malt1 in bone marrow-derived osteoclasts. To examine the role of Malt1 in osteoporosis, I measured the bone volume and osteoclast number in Malt1-deficient mice in vivo.  Finally, I investigated whether Malt1 had a cell-intrinsic role in both osteoclast differentiation and activity in vitro.   1.6.2 Hypothesis and objectives I hypothesize that Malt1 deficiency in macrophages and osteoclasts causes intestinal inflammation and osteoporosis, respectively.  Aim 1 (Chapter 2): To determine whether Malt1 deficiency causes intestinal inflammation by increasing macrophage inflammatory responses. Aim 2 (Chapter 3): To determine the level of Malt1 expression in biopsies from subjects with IBD and healthy control subjects, and in mice during DSS-induced colitis. Aim 3 (Chapter 4): To determine whether Malt1 deficiency in osteoclasts contributes to brittle bones by increasing osteoclast numbers and/or activity  1.6.3 Significance This research contributes to our understanding of the role of Malt1 in macrophages and osteoclasts. Moreover, using a pre-clinical mouse model, I examine the potential contributions of 39  those cell types to key features of the pathology associated with CID caused by Malt1 deficiency. My goals were to develop a better understanding of the macrophage and osteoclast cell-intrinsic and cell-extrinsic roles for Malt1 deficiency in intestinal inflammation and osteoporosis, as well as the molecular mechanisms that underlie disease pathogenesis. This research may provide insight into novel treatment options for patients with CID caused by Malt1 deficiency as well as the much broader group of people with immune-mediated diseases accompanied by intestinal inflammation or osteoporosis.    40  Chapter 2: Malt1 blocks IL-1b production by macrophages in vitro and limits dextran sodium sulfate-induced inflammation in vivo  2.1  Introduction and rationale  CID is a potentially fatal disease, which is characterized by immune system defects or dysfunction. CID can be caused by a variety of gene defects [189-191] including numerous defects identified in the NF-κB signalling pathway [192-195]. Recently, mutations in the gene encoding MALT1 have been identified as a novel cause of CID [21-23]. People with MALT1 deficiency have recurrent infections and chronic inflammation including dermatitis, lung inflammation, osteoporosis, and severe inflammation along the entire gastrointestinal tract [24]. Hematopoietic stem cell transplant provides significant clinical improvement for patients including resolution of gastrointestinal disease [23, 25]. During NF-κB activation, MALT1 acts as an adaptor protein bridging CARD11 and BCL10, and has proteolytic activity and is known as “paracaspase” [164, 196]. The role of Malt1's scaffolding function and proteolytic activity in T cells and B cells have been described in vivo comparing Malt1-deficient (Malt1-/-) mice to mice that express a proteolytically inactive form, paracaspase dead (PD), form of Malt1 (Malt1PD/PD) [173, 197, 198]. Malt1-/-  mice are healthy, in the absence of challenge, with no overt immune pathologies [164, 196]. Both Malt1-/- mice and Malt1PD/PD have defects in development of marginal zone B cells, B1 cells, IL-10-producing B cells, T regulatory cells, as well as mature T and B cells [173, 197, 198]. In addition, Malt1PD/PD have reduced T cell IL-2 and TNFα production and defective Th17 differentiation in vitro [198]. The features unique to the Malt1PD/PD mice may contribute to spontaneous inflammatory phenotypes described including a lethal inflammatory syndrome characterized by high levels of IFNγ, [173] autoimmune gastritis, [197] and multiorgan 41  inflammation, [198] the latter two of which are characterized by high levels of IgG1 and IgE. Both Malt1-/- and Malt1PD/PD mice are protected against experimental autoimmune encephalitis, which is consistent with defective T cell, and specifically Th17 cell, development [197-200]. Consistent with that, neither Malt1-/- nor Malt1PD/PD-deficient T cells cause colitis in the T cell transfer model of colitis [198]. Spontaneous ileal or colonic inflammation, like that present in MALT1-deficient patients, has not been described in either Malt1-deficient mouse genotype. Gewies et al. [173] did note that Malt1PD/- mice do significantly worse during dextran sodium sulfate (DSS)-induced colitis than their Malt1+/- counterparts. Of note, DSS-induced colitis is a short-term model of intestinal inflammation that is largely driven by innate immune cells and their responses. Innate immune cells also express MALT1 and may contribute to inflammatory pathology in people with MALT1 deficiency and in Malt1-deficient mice. In macrophages, Malt1 is required for NF-κB activation downstream of TLR4, which recognizes lipopolysaccharide (LPS), and dectin-1, an innate immune receptor critical for antifungal immunity and responses to the antifungal components, zymosan and curdlan [201-203]. Upon receptor ligation, the CBM signalosome induces NF-κB activation by zymosan [204].  Thus far, the role of MALT1 in inflammation in vivo has been attributed to its role in lymphocyte development and activation. However, innate immune cells, and specifically macrophages, contribute to intestinal inflammation during DSS-induced colitis [205, 206]. DSS damages the intestinal epithelial barrier enabling interactions between commensal microbiota and underlying immune cells [207]. Because mice deficient in paracaspase activity did significantly worse than their control counterparts during DSS-induced colitis, we sought to determine whether Malt1-/- mice also do significantly worse in DSS-induced colitis and whether 42  macrophages contribute to that phenotype. We examined the effect of Malt1 deficiency on TLR4- and dectin-1-induced pro-inflammatory cytokine production by macrophages in vitro and ex vivo. Finally, we compared the effect of IL-1 signalling on DSS-induced colitis in Malt1+/+ and Malt1-/- mice.  2.2 Material and methods  Mice. Malt1+/- mice were provided by Dr. Tak W. Mak from the University of Toronto. Mice were generated and backcrossed for more than 10 generations onto C57BL/6 mice [164, 208]. Malt1+/- mice were used to generate Malt1+/+ and Malt1-/- mice that were used between 8 and 12 weeks of age for experiments. Littermates were used for experiments and equal numbers of males and females were used in each group of mice compared in our DSS-induced colitis experiments. Mice were maintained in the Animal Research Center at BC Children's Hospital Research Institute (BCCHR). Protocol numbers are A17-0061 and A17-0071.  DSS-induced colitis. Colitis was induced in mice by adding 2 or 2.5% DSS to their drinking water for 6 or 7 days, as indicated. As controls, groups of mice also received sterile drinking water (no DSS). Mice were monitored daily to measure disease activity index (DAI). DAI was scored on a scale of 0–12 calculated as a sum of the 0–4 score for each of the following parameters: weight loss 0–4, stool consistency 0–4, and rectal bleeding 0–4. A score of 0 = no weight loss, normal stool consistency, no rectal bleeding; 1 = 1–3% weight loss, loose stool, and detectable blood by HEMDETECT paper (Beckman Coulter, Mississauga, Canada); 2 = 3–6% weight loss, very loose stool, and visible blood in stool; 3 = 6–9% weight loss, diarrhea, and large amount of blood in stool; and 4 = more than 9% weight loss, no formed stool, and extensive blood in stool and blood visible at the anus [209]. DAI is reported as the mean score 43  from 9 mice per genotype or group examined in each experiment. After autopsy, colons were excised, and colons lengths were measured. Histological analysis. Colons were harvested from mice and fixed in 10% formalin overnight. Tissue sections were embedded in paraffin, and cross-sections were stained with hematoxylin and eosin (H&E). Histological damage was scored using a 16-point scale by 2 individuals blinded to the experimental conditions. Scoring includes: loss of architecture 0–4; immune cell infiltration 0–4; goblet cell depletion 0–2; ulceration 0–2; edema 0–2; and muscle thickening 0–2 [210]. For macrophage staining, paraffin-embedded tissues were rehydrated and stained with F4/80. F4/80 positive cells were counted at 20× magnification from 6 different fields in 6 tissue sections separated by ≥ 50 μm from each mouse by 2 individuals blinded to experimental condition. For detection of active caspase-1, cells or tissues were stained with YVAD-FLICA and counterstained with Hoechst for nuclei, using the FAM-FLICA Caspase-1 Assay (ImmunoChemistry Technologies, Bloomington, MN, USA), according to manufacturer's instructions. Macrophage depletion. Macrophages were depleted from mice using clodronate liposomes (clod-lip). Mice were treated with 200 μL of clod-lip by intraperitoneal injection (IP), 4 days prior to DSS treatment, again 1 day before treatment, and on days 1, 3, and 5 during treatment. Control mice were injected IP with 200 μL of PBS on the same days before and during treatment with DSS. Anakinra treatment. Mice were injected IP with Anakinra at a dose of 150 mg/kg daily, during DSS treatment. Control mice were injected IP with the same volume of PBS, as an injection control. 44  Macrophage derivation. Bone marrow macrophages were derived from bone marrow progenitor cells isolated from mouse leg bones (femurs and tibias). Bone cavities were flushed using a syringe filled with IMDM supplemented with 10% FBS until the bones appeared to be clear. Bone marrow aspirates were incubated at 37°C in 5% CO2.  After 1h, the mature mesenchymal cells adhered to the flask, and the suspended hematopoietic progenitor cells were harvested. To derive macrophages, following adherence depletion, bone marrow progenitor cells were plated at a concentration of 0.5 × 106 cells/mL for 10 days in IMDM supplemented with 10% FBS, penicillin–streptomycin, and 5 ng/mL MCSF (StemCell Technologies, Vancouver, BC, Canada) at 37°C in 5% CO2 with complete media changes on days 4 and 7. Cells stimulations. Macrophages were cultured in a 96-well plate at a density of 1 × 105 cells/well (1 × 106 cells/mL) and stimulated with 10 ng/mL LPS (Escherichia coli serotype 127:B8; Sigma–Aldrich, St. Louis, MO, USA), 10 μg/mL zymosan (Saccharomyces cerevisiae; Invivogen, San Diego, CA, USA), or 100 μg/mL curdlan (Alcaligenes faecalis; Invivogen, San Diego, CA, USA) for 24 h. The zymosan used at this concentration can activate both dectin-1 and TLR2; the curdlan used at this concentration can activate dectin-1, TLR2, and TLR4. After stimulation, cell supernatants were harvested and clarified by centrifugation for analysis.  Cytokine measurements. Cytokine measurements were performed on clarified colon homogenates and cell supernatants using ELISAs. Mouse IL-1β, IL-6, TNF, and IL-12p40 ELISA kits were purchased from BD Biosciences (Mississauga, ON, Canada). Gene expression analysis. RNA was isolated from mouse colon or macrophages using the RNeasy Plus Mini kit (QIAGEN, Hilden, Germany), followed by reverse transcription using 45  iScript™ Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). For quantitative PCR (qPCR), gene expression was measured using SsoAdvaced™ Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). SYBR Green Supermix was mixed with forward and reverse primers, cDNA template, and nuclease-free water. Il1b and Tnf gene expression were normalized to Gapdh. PrimePCR™ SYBR Green assay primers (Bio-Rad, Hercules, CA, USA) were used. The catalog number for primers is 10025636 and identification numbers were as follows: qMmuCED00027497 (Gapdh), qMmuCID0005641 (Il1b), and qMmuCED0004141 (Tnf). Statistical analyses. Analyses were done using unpaired Student's t-tests, 1-way ANOVAs, and 2-way ANOVAs using Graphpad Prism, as indicated (GraphPad Sofware, San Diego, CA, USA). Differences with P < 0.05 were considered significant.  2.3 Results 2.3.1 Malt1-/- mice are more susceptible to DSS-induced colitis than Malt1+/+ mice Malt1 deficiency in humans causes dramatic inflammation along the gastrointestinal tract [21]. In mice, loss of Malt1 protease activity has been reported to increase inflammation during DSS-induced colitis [173]. However, the role of Malt1 deficiency in DSS-induced colitis in mice has not been investigated. Based on this, we asked whether Malt1 deficiency increases susceptibility of mice to DSS-induced colitis. To examine this, mice were untreated or treated with 2.5% DSS in their drinking water and their DAI including weight loss, rectal bleeding, and stool consistency, was monitored for 7 days. Malt1-/- mice did not display any signs of spontaneous intestinal inflammation. In DSS-treated mice, DAI was significantly higher in Malt1-/- compared with Malt1+/+ mice (Fig. 2.1 A). Colon length decreases during DSS-46  induced colitis and is a measure of disease severity [211]. On day 7, mice were euthanized, and the length of harvested colon tissue was measured. Malt1-/- mice had significantly shorter colons compared to their wild-type (WT) littermates (Fig. 2.1 B). Distal colons were fixed for histological analysis including H&E staining. Histological damage was scored in H&E-stained cross sections by two individuals blinded to experimental condition. Damage scores were based on loss of tissue architecture, immune cell infiltration, loss of goblet cells, ulceration, muscle thickness, and edema. Untreated Malt1-/- mice did not display any abnormal tissue architecture but DSS-treated Malt1-/- mice had significantly higher histological damage scores compared to DSS-treated Malt1+/+ mice (Fig. 2.1 C). Taken together, these data show that DSS induced more severe colitis in Malt1-/- mice than in Malt1+/+ inducing significantly more gross pathology and histological damage.  Figure 2.1 Malt1-deficient mice have increased susceptibility to DSS-induced colitis.  Malt1+/+ and Malt1-/- mice were subjected to either water or 2.5% DSS for 7 days. (A) Disease activity index was measured daily during treatment. (B) On day 7, colons were removed, and their lengths were measured. (C) Colon 47  cross-sections were fixed and stained with H&E and histological damage was quantified. Scale bars, 100 μm. Results are expressed as the mean ± SD; **P < 0.01, ***P < 0.001 for N = 9 mice/genotype from 3 independent experiments using a 2-way ANOVA in (A), and a Student's t-test in (B) and (C).  2.3.2 Concentrations of IL-6 and IL-1b are increased in colon tissues of Malt1-/- mice after DSS treatment To determine if the concentrations of inflammatory cytokines was increased in colon tissue homogenates after DSS treatment, inflammatory cytokines in mice colon tissue homogenates were assayed by ELISA. IL-1β and IL-6 were increased significantly in Malt1-/- mice colon homogenates compared with control littermates (Figs. 2.2 C and 2.2 D), but there was no significant difference in the concentrations of TNF and IL-12p40 between the two genotypes (Figs. 2.2 A and 2.2 B). Taken together, these results show that DSS induced more inflammation in Malt1-/- versus Malt1+/+ mice.  Figure 2.2 Malt1-deficient mice have increased concentrations of colon inflammatory cytokines in their colon tissues after DSS-induced colitis.  Malt1+/+ and Malt1-/- mice were subjected to either water or 2.5% DSS for 7 days. On day 7, colons were removed, and full-thickness colon homogenates were assayed for pro-inflammatory cytokines, (A) TNF, (B) IL-12p40, (C) IL-6, and (D) IL-1b. Results are expressed as the mean ± SD; *P < 0.05, **P < 0.01 for N = 9 mice/genotype from 3 independent experiments using a Student's t-test.  48  2.3.3 Macrophage depletion reduces DSS-induced colitis in Malt1-/- mice Macrophages play an important role during gut inflammation [212]. The role of Malt1 in gastrointestinal inflammation has been largely attributed to its role in lymphocytes but Malt1 is also expressed in macrophages, [204] which play a key role in DSS-induced intestinal inflammation [205, 206]. Based on this, we asked if macrophages contribute to increased susceptibility to DSS-induced colitis in Malt1-/- mice. Mice were subjected to 2% DSS treatment to induce colitis. Mice were injected with clod-lip or PBS, as an injection control, 4 and 1 day prior to treatment with DSS, as well as on days 1, 3, and 5 during treatment. After 6 days of treatment with DSS, mice were euthanized, colons were harvested, and colon lengths were measured. Clod-lip-mediated depletion of macrophages modestly decreased DAI and increased colon length in Malt1-/- mice, whereas depletion of macrophages did not significantly affect DAI or colon length in Malt1+/+ mice (Figs. 2.3 A and 2.3 B). Distal colons were fixed for histological analysis. Histological damage was reduced in H&E-stained colon sections of Malt1-/- mice after macrophage depletion but there was no significant difference in histological damage observed in Malt1+/+ mice (Fig. 2.3 C). Distal colons were fixed for immunohistochemistry and F4/80 staining. Clod-lip injections effectively depleted macrophages in both Malt1+/+ and Malt1-/- mice (Fig. 2.3 D). Taken together, these data demonstrate that macrophage depletion by clod-lip ameliorates DSS-induced colitis in Malt1-/- mice but not in their Malt1+/+ littermates. 49   Figure 2.3 Macrophage depletion by clodronate liposome reduces susceptibility of Malt1-deficient mice to DSS-induced colitis. DSS (2%) was added to the drinking water of Malt1+/+ and Malt1-/- mice for 6 days. Mice were injected with clodronate liposomes (clod-lip) or PBS (as an injection control) 4 and 1 day prior to treatment with DSS, and on days 1, 3, and 5 during treatment. (A) Disease activity index was measured daily during treatment. (B) Malt1+/+ and Malt1-/- mice were euthanized after 6 days of treatment with DSS, and colons were harvested. Colon length was measured in Malt1+/+ and Malt1-/- mice injected with clod-lip and PBS. (C) Colons were fixed and stained with H&E. Scale bars, 100 μm. Histological damage was scored for H&E-stained colon sections from Malt1+/+ and Malt1−/− mice injected with clod-lip and PBS. (D) Colon sections were stained by IHC for mature macrophages (F4/80). Positively stained cells were counted at 20× magnification in 6 different representative fields in 6 sections/mouse separated by ≥50 μm. Scale bars, 100 μm. Results are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 for N = 9 mice/group from 3 independent experiments using a 2-way ANOVA. 50   2.3.4 Macrophage depletion reduces colonic IL-6 and IL-1b concentrations after treatment with DSS in Malt1-/- mice To determine if the concentrations of pro-inflammatory cytokines was decreased in colon tissue homogenates after macrophage depletion, cytokine concentrations were measured in colon homogenates from Malt1+/+ and Malt1-/- mice injected with clod-lip and PBS. There was a significant reduction in the concentrations of IL-6 and IL-1β (Fig. 2.4 C and 2.4 D) in Malt1-/- mouse colon homogenates after macrophage depletion, but there was no difference in the concentrations of these cytokines in Malt1+/+ mouse colon homogenates after macrophage depletion.   Figure 2.4 Macrophage depletion by clodronate liposome reduces IL-6 and IL-1b concentrations in colon tissues from Malt1-/- mice colon tissue.  DSS (2%) was added to Malt1+/+ and Malt1-/- mice drinking water for 6 days. Mice were injected with clod-lip or PBS, as an injection control, 4 and 1 day prior to treatment with DSS, and on days 1, 3, and 5 during treatment. On day 7, colons were harvested and full-thickness colon homogenates from Malt1+/+ and Malt1-/- mice injected with clod-lip or PBS were assayed for pro-inflammatory cytokines, (A) TNF, (B) IL-12p40, (C) IL-6, and (D) IL-1b. Results are expressed as the mean ± SD; *P < 0.05 for N = 9 mice/group from 3 independent experiments using a 2-way ANOVA.  51  2.3.5 Malt1+/+ and Malt1-/- MCSF-derived bone marrow macrophages produce similar concentrations of pro-inflammatory cytokines when stimulated with LPS, zymosan, or curdlan Cytokines play a crucial role during intestinal inflammation. Activated macrophages are the main source of inflammatory cytokines that contribute to gut inflammation during inflammatory disease [213]. To understand the effect of inflammatory cytokines produced by macrophages on Malt1-/- mice susceptibility to DSS, we asked if the concentrations of inflammatory cytokines produced by macrophages is increased in the absence of Malt1. MCSF-derived bone marrow macrophages from Malt1+/+ and Malt1-/- mice were stimulated with a TLR4 agonist or dectin-1 agonists because Malt1-Bcl10 is activated downstream of these innate immune receptors in macrophages activating NF-κB-driven transcription. Macrophages were left unstimulated or stimulated for 24 h with LPS (10 ng/mL) (Fig. 2.5 A), zymosan (Fig. 2.5 B), or curdlan (Fig. 2.5 C) and TNF, IL-6, and IL-12p40 were assayed by ELISA. There were no significant differences in production of these pro-inflammatory cytokines between Malt1+/+ and Malt1-/- macrophages.  52   Figure 2.5 Malt1-deficinet macrophages stimulated with LPS, zymosan, or curdlan produce the same concentrations of TNF, IL-6, and IL-12p40 as Malt1+/+ macrophages.  Malt1+/+ and Malt1-/- MCSF-derived bone marrow macrophages were unstimulated or stimulated with (A) 10 ng/mL LPS, (B) 10 μg/mL zymosan, or (C) 100 μg/mL curdlan for 24 h. Clarified cell supernatants were assayed for TNF, IL-6, and Il-12p40 by ELISA. Data are presented as the mean ± SD for N = 6; macrophages were derived from 1 mouse for each of the 3 independent experiments. No significant differences were observed.   2.3.6 Malt1-deficient MCSF-derived bone marrow macrophages produce higher concentrations of IL-1b Inhibition of Malt1 activity in macrophages has been reported to reduce IL-1β production by macrophages [214]. IL-1β is produced by macrophages in the gut [215] and was elevated in 53  DSS-induced colitis in Malt1-/- mice; and IL-1β has been identified as an important mediator of inflammation in DSS-induced colitis [216]. Thus, to ask whether Malt1 deficiency or inhibition could impact macrophage-mediated IL-1β production in a cell intrinsic manner, Malt1+/+ and Malt1-/- MCSF-derived bone marrow macrophages were left untreated or treated with LPS, zymosan, or curdlan (24 h) to induce IL1b transcription and ATP (5 mM) was added for the final hour to activate the NLRP3 inflammasome and induce IL-1β maturation and secretion. IL-1β production was significantly higher in Malt1-/- bone marrow-derived macrophages compared with Malt1+/+ bone marrow-derived macrophages in response to (10 ng/mL) of LPS (Fig. 2.6 A), (10 μg/mL) of zymosan (Fig. 2.6 B), or (100 μg/mL) of curdlan (Fig. 2.6 C). This is consistent with increased IL-1β concentrations in full thickness tissue homogenates from Malt1-/- mice.  Figure 2.6 Malt1-deficient macrophages produce high concentrations of IL-1b.  Malt1+/+and Malt1-/- MCSF-derived bone marrow macrophages were unstimulated or stimulated with (A) LPS, (B) zymosan, or (C) curdlan for 24 h; with or without ATP (5 mM) for 1 h. Results are expressed as the mean ± SD; macrophages were derived from 1 mouse for each of 6 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 for N = 6, statistical analyses were performed using a 2-way ANOVA.   54  2.3.7 Increased IL-1b production by Malt1-/- macrophages is associated with increased transcription of Il1b but not increased inflammasome activation  Increased IL-1β production can be caused by increased transcription of Il1b or increased processing and secretion of pro-IL-1β by the inflammasome. To determine the cause of increased IL-1β production by Malt1-/- MCSF-derived bone marrow macrophages, Il1b mRNA expressions were assessed in LPS-, zymosan-, or curdlan-stimulated macrophages by Q-PCR; and LPS, zymosan, or curdlan plus ATP stimulated macrophages were stained with YVAD-FLICA for active caspase-1. Malt1-/- macrophages produced significantly more Il1b mRNA in response to LPS, zymosan, or curdlan compared with macrophages derived from Malt1+/+mice (Fig. 2.7 A). As a control, Tnf mRNA expressions were also assessed and were not higher in Malt1-/- macrophages compared with that induced in Malt1+/+ macrophages (Fig. 2.7 A). With regard to inflammasome activation, there is no constitutive activation of inflammasomes in Malt1-/- macrophages because stimulation with ATP is required to activate the inflammasome to produce IL-1β. Thus, to determine whether Malt1-/- macrophages were hyper-responsive to inflammasome activation, macrophages were stimulated with TLR4/dectin-1 ligands plus ATP and stained for active caspase-1 with YVAD-FLICA. There was no significant difference in the number of macrophages that stained positively for active caspase-1 between Malt1+/+ and Malt1-/- macrophages (Fig. 2.7 B).  55   Figure 2.7 IL-1b mRNA is higher in MCSF-derived bone marrow macrophages and colon homogenates from Malt1-/- mice compared with Malt1+/+ mice.  Malt1+/+ and Malt1-/- MCSF-derived bone marrow macrophages were unstimulated or stimulated with LPS, zymosan, or curdlan for 24 h. (A) Q-PCR was performed to measure Il1b (left) and Tnf (right) mRNA, normalized to Gapdh. (B) Macrophages stained with YVAD-FLICA for active caspase-1 and Hoechst, for nuclei. Results are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001; N = 3 using a 2-way ANOVA.  2.3.8 Higher concentrations of IL-1b in colonic tissues of DSS-treated Malt1-/-  mice is associated with increased transcription of Il1b but not increased number of cells with active caspase-1 To determine the cause of increased IL-1β in Malt1-/- colon tissue homogenates, RNA was also isolated from full-thickness colon tissues from DSS-treated Malt1+/+ and Malt1-/- mice. 56  Il1b and Tnf mRNA expressions were assessed by qPCR. Il1b mRNA expressions were higher in colon tissues from Malt1-/- mice compared with Malt1+/+ mice, but Tnf mRNA expressions were not different between genotypes (Fig. 2.8 A). Colon tissue cross-sections from the mice were stained with YVAD-FLICA for active caspase-1 and counterstained with Hoechst. There was no significant difference in the number of cells expressing active caspase-1 comparing tissue from Malt1+/+ to Malt1-/- mice (Fig. 2.8 B).  Figure 2.8 IL-1b mRNA is higher in colon homogenates from Malt1-/- mice compared with Malt1+/+ mice. (A) RNA was prepared from full-thickness colon tissue homogenates of DSS-treated Malt1+/+ and Malt1-/- mice and Q-PCR was performed to measure Il1b (left) and Tnf (right) mRNA, normalized to Gapdh. (B) Colon cross-sections from DSS-treated Malt1+/+ and Malt1-/- mice stained with YVAD-FLICA for active caspase-1 and Hoechst, for nuclei. Scale bars, 100 μm. Results are expressed as the mean ± SD; *P < 0.05; N = 6 using a Student's t-test.  2.3.9 Treatment with Anakinra, an IL-1 receptor antagonist, reduces DSS-induced colitis in Malt1-/- mice  Next, we asked whether increased IL-1β production plays a direct role in exacerbation of intestinal inflammation in Malt1-/- mice. Malt1+/+ and Malt1-/- mice were subjected to 2% DSS 57  treatment to induce colitis and treated with Anakinra, an IL-1 receptor antagonist, or PBS, as an injection control. DAIs were monitored daily and after 7 days of treatment with DSS, mice were euthanized, colons were harvested, and colon lengths were measured. Anakinra treatment decreased DAI and modestly increased colon length in Malt1-/- mice, whereas it did not significantly affect DAI or colon length in Malt1+/+ mice (Figs. 2.9 A and 2.9 B). Distal colons were fixed for histological analysis. Anakinra treatment reduced histological damage evident in H&E-stained colon sections of Malt1-/- mice but there was no significant improvement in histological damage observed by Anakinra treatment in Malt1+/+ mice (Fig. 2.9 C). Taken together, these data demonstrate that antagonizing the IL-1 receptor ameliorates DSS-induced pathology in Malt1-/- mice but not their Malt1+/+ littermates.   58   Figure 2.9 Anakinra treatment ameliorates DSS-induced colitis in Malt1-deficient mice.  2% DSS was added to Malt1+/+ and Malt1−/− mice drinking water for 7 days. Mice were injected IP with Anakinra, an IL-1 receptor antagonist, or PBS, as an injection control, daily during treatment with DSS. (A) Disease activity index was measured daily during treatment. (B) On day 7, colons were removed, and their lengths were measured. (C) Colon cross-sections were fixed and stained with H&E and histological damage was quantified. Scale bars, 100 μm. Results are expressed as the mean ± SD; *P < 0.05 comparing Anakinra treatment to PBS injection controls; N = 6 mice/group from 3 independent experiments. Statistical analyzes were performed using a 2-way ANOVA.  2.3.10 Treatment with Anakinra does not reduce intestinal inflammatory cytokines concentrations in Malt1-/- mice To determine if the concentrations of inflammatory cytokines were reduced in colon tissue homogenates after treatment with Anakinra, pro-inflammatory cytokines were measured in colon homogenates from Malt1+/+ and Malt1-/- mice injected with Anakinra or PBS. Anakinra 59  treatment did not reduce TNF (Fig. 2.10 A), IL-12p40 (Fig. 2.10 B), IL-6 (Fig. 2.10 C), or IL-1β (Fig. 2.10 D) concentrations in mouse colon homogenates from either Malt1+/+ or Malt1-/- mice.  Figure 2.10 Anakinra does not reduce inflammatory cytokine concentrations in Malt1-/- mice colon homogenates after DSS-induced colitis.  DSS (2%) was added to Malt1+/+ and Malt1-/- mice drinking water for 7 days. Mice were injected IP with the IL-1β receptor antagonist, Anakinra, or PBS, as an injection control, daily during treatment with DSS. On day 7, colons were removed, and full-thickness colon homogenates were assayed for inflammatory cytokines, (A) TNF, (B) IL-12p40, (C) IL-6, and (D) IL-1b. Results are expressed as the mean ± SD; *P < 0.05 comparing Malt1+/+ to Malt1-/- mice, N = 6 mice/group from 3 independent experiments. Statistical analyses were performed using a 2-way ANOVA.   2.4 Discussion Herein, we report that higher IL-1β production by macrophages and elevated IL-1 signalling in colon tissue contribute to exacerbated DSS-induced colitis in Malt1-/- mice. Colonic epithelial barrier integrity is compromised in mice by DSS, exposing underlying immune cells to luminal contents, which contain innate immune stimuli. Exacerbated DSS-induced colitis 60  in Malt1-/- mice is characterized by increased disease activity, histological damage, and IL-1β concentrations; and is ameliorated by macrophage depletion or blocking IL-1 signalling. Using the germline knockout mouse, which is deficient in both Malt1 scaffolding and proteolytic activities, data presented here shows that Malt1 deficiency (Malt1-/-) exacerbates DSS-induced inflammation. Moreover, our clod-lip depletion studies demonstrate a critical role for an innate immune/myeloid cell-mediated contribution to that inflammation. Exacerbated DSS-induced colitis in Malt1-/- mice is characterized by high concentrations of the pro-inflammatory cytokine, IL-1β, as well as IL-6. Increased IL-1β concentrations were associated with increased transcription of Il1b but not increased numbers of cells with activated inflammasomes. IL-1β is a key cytokine involved in the development of inflammation during DSS-induced colitis [216]. IL-1β production during inflammation is frequently attributed to myeloid cells, that is, monocytes and macrophages [217], and it can cause autoinflammation, that is, production of more IL-1β as well as other pro-inflammatory cytokines, including IL-6 [215, 218]. Indeed, our Anakinra experiments demonstrate a specific role for IL-1 signalling in exacerbated DSS-induced colitis in Malt1-/- mice. In Malt1+/+ mice, neither macrophage depletion nor Anakinra treatment significantly reduced DSS-induced colitis, though there were modest, albeit insignificant, improvements in DAIs and histological damage scores when macrophages were depleted from Malt1+/+ mice. These data suggest that macrophages and IL-1 signalling play a less important or no role in DSS-induced colitis in WT mice. However, the lack of effect of these interventions may also be due to the lower overall levels of pathology observed in the WT mice making it more difficult to see improvements in disease activity and histological damage. 61  Malt1 expression and proteolytic activity are clearly important in mucosal homeostasis. Inhibition of Malt1 paracaspase activity with paracaspase inhibitors protects mice during DSS-induced colitis [214]. This is caused by decreased NF-κB and NLRP3 activation, decreased IL-1β and IL-18 production by PMA-differentiated THP-1 cells and bone marrow macrophages. This is also consistent with paracaspase activity enhancing Malt1 scaffolding function and augmenting NF-κB-mediated transcription [214]. In contrast, mice deficient in Malt1 paracaspase activity (Malt1PD/-) do significantly worse than their Malt1-sufficient counterparts (Malt1+/-) during DSS-induced colitis. This apparent dichotomy can be rationalized when one considers that this could be due to systemic deficiency in regulatory T cells reported in germline deficient mice Malt1PD/- [173, 197, 198]. In addition, innate immune defects causing bacterial overgrowth or dysbiosis could exacerbate DSS-induced colitis in Malt1PD/- mice [219]. Previous data focusing on T and B cell activation have demonstrated that paracaspase deficiency in the presence of Malt1 scaffolding function exacerbates inflammation. Indeed, paracaspase-deficient mice develop spontaneous inflammatory disease, whereas Malt1-/- mice do not, suggesting a critical role for the Malt1 scaffolding function in driving NF-κB activation, and paracaspase activity in regulating or setting a threshold for that activation, specifically in T and B cells [173, 197, 198].  Malt1 is activated downstream of TLR4 and dectin-1 receptors in macrophages [179, 204] but its role in LPS- and dectin-1-induced pro-inflammatory cytokine production by macrophages has not been thoroughly examined. The microRNA, MiR-26, does reduce IL-6 production by blocking HMGA1 and MALT1 mRNA expression [220]. We found that there is no significant difference in pro-inflammatory cytokine production (TNF, IL-6, or IL-12p40) between Malt1+/+ and Malt1-/- macrophages stimulated via TLR4 or dectin-1 for MCSF-bone 62  marrow macrophages. This is consistent with modest effects of Card9 deficiency on pro-inflammatory cytokine production by bone marrow-derived macrophages [221]. Altogether, this data demonstrates that there is no macrophage cell-intrinsic requirement for Malt1 in the production of TNF, IL-6, or IL-12p40 in response to TLR4 or dectin-1 ligation. Malt1 scaffolding function and proteolytic activity have a role in production of IL-1β by macrophages. IL-1β acts as an alarm cytokine initiating the early innate immune response and so is tightly regulated by a 2-step process. Activation of NF-κB drives Il1b transcription, which is translated to an inactive precursor, pro-IL-1β. A second signal, typically a danger associated molecular pattern, then activates the inflammasome, which catalyzes the processing of IL-1β [217] and its secretion via formation of gasdermin-PI(4,5)P2 pores in the cell membrane [222]. Dectin-1 ligation can also stimulate IL-1β production and secretion via a non-canonical inflammasome, which activates caspase-8 [223]. In both canonical and noncanonical inflammasome activation, TLR4 and dectin-1-induced NF-κB-mediated IL-1β production require the CBM complex. Thus, we would predict that Malt1-/- macrophages would be defective in IL-1β production. In fact, LPS, zymosan, and curdlan-stimulated Malt1-/- MCSF-derived bone marrow macrophages produced more IL-1β than Malt1+/+ macrophages. As in the DSS-treated Malt1-/- mice in vivo, increased IL-1β production was associated with increased transcription of Il1b, but not an increase in the number of macrophages in which the inflammasome was activated.  IL-1β stimulates its own production in vitro and in vivo and IL-1 receptor-driven NF-κB activation is independent of Malt1 [224]. Thus, we posited that a small amount of IL-1β production downstream of TLR4 or dectin-1 or other innate immune stimuli in Malt1-/-macrophages and mice, could lead to autoinflammatory production of IL-1β, which can also lead 63  to IL-6 production in vivo [218]. Indeed, macrophage-derived IL-1β drives spontaneous autoinflammatory ileitis in SHIP-deficient mice, wherein the genetic deficiency in these mice permits elevated NF-κB activation driven by PI3Kp110α activity [215]. Thus, we were somewhat surprised to see that targeting the IL-1 receptor in vivo with Anakinra did not reduce pro-inflammatory cytokine levels (IL-1β and IL-6) in colon homogenates from Malt1-deficient mice. This may be due to DSS-induced colitis being a model of acute intestinal inflammation in which there was not sufficient time for feedback inhibition of IL-1β or IL-6 production by reduced IL-1 signalling. However, this does not preclude the possibility that IL-1 signalling contributes to autoinflammation in DSS-treated Malt1-/- mice. We did not detect any macrophage-intrinsic role for Malt1 in IL-6 signalling. It is possible that our reductionist approach to assessing the role of Malt1 in macrophage cytokine production did not detect differences in IL-6 production. Other cell types or combinations of cells and stimuli may lead to increased IL-6 production by macrophages or other cells in vivo, which could contribute to and exacerbate DSS-induced colitis in Malt1-/- mice. Malt1-independent IL-1 signalling may be critical in normal antifungal responses as IL-β-deficient mice have reduced responses to zymosan-induced peritonitis [225]. In patients, Malt1-deficiency has not been reported to cause defects in antifungal responses specifically [24]. Rather, individuals have increased susceptibility to bacterial infection and debilitating chronic inflammation. Susceptibility to bacterial infection could result from dysregulated and inappropriate innate immune responses to TLR4 in myeloid cells. Importantly, increased IL-1β production associated with Malt1 deficiency in macrophages could cause a systemic autoinflammatory response that contributes to the chronic inflammation experienced by these patients [226]. 64  Chapter 3: Innate immune stimuli increase Malt1 mRNA and protein expression and eliminate Malt1 activity in macrophages in vitro but MALT1/Malt1 mRNA expression is not altered in the inflamed intestine of people with IBD or in mice during DSS-induced colitis   3.1  Introduction and rationale  IBD is the term for a group of gastrointestinal disorders with complex pathogenesis. Two main classes of IBD are CD and UC. CD is characterized by chronic inflammation that can affect any part of the gastrointestinal tract. UC is a severe inflammatory disease that involves acute or chronic inflammation along the colon [28]. Incidence of IBD has been rising in Canada with 1 in 200 people affected [26]. Various genes and genetic loci that increase susceptibility to IBD have been identified [227, 228]. These genetic variants can disrupt different layers of the gut defense mechanism against luminal microbes including the epithelial barrier, systemic innate and adaptive immune cell activation, cytokine secretion, and microbial recognition [227, 228].   Genetically, IBD development can be mediated by unique and rare hereditary mutations rather than common alleles found in a population [228]. However, genome-wide associated studies (GWAS) have found common susceptibility gene candidates that are associated with an increased risk of developing disease [227]. One of these genes is CARD9 that is associated with UC and CD, as well as other inflammatory diseases including primary sclerosing cholangitis (PSC) [227]. CARD9 acts as an adaptor molecule that binds to Malt1 (and indirectly to Bcl-10) to form the CBM complex, which leads to activation of NF-κB signalling in myeloid cells. In this complex, Malt1 interacts with CARD9 and BCL10 and acts as a scaffolding molecule for recruitment of downstream signalling compartments required for NF-kB activation [162]. 65  However, Malt1 also has proteolytic activity and cleaves a number of substrates, including BCL10, which can lead to activation or antagonism of CBM-mediated NF-κB activation [169-172, 174]. Activation of the NF-κB pathway is very important during inflammation because it regulates immune cell development and inflammatory cytokine production [157, 158]. Accordingly, in order to combat inflammatory disease, it is critical to understand how Malt1 protein expression and enzymatic activity are regulated and how they, in turn, regulate pro-inflammatory cytokine production. In T or B cells, Malt1 protein expression is not modified, but Malt1 proteolytic activity is induced after stimulation of these cells with, for example, PMA/Ionomycin [21, 173, 229]. In Chapter 2 of this thesis, I demonstrated that Malt1 deficiency increases susceptibility of mice to DSS-induced colitis and that effect was mediated by macrophage-mediated pro-inflammatory cytokine production. However, Malt1 protein expression and enzymatic activity have not been studied in macrophages in response to innate immune stimuli. Based on this, I examined Malt1 mRNA and protein expression and activity in murine macrophages. To examine the impact of Malt1 activity on macrophage pro-inflammatory cytokine production, I used mepazine, a phenothiazine derivative, which has been reported to inhibit proteolytic activity of Malt1 and treat autoimmune disease and an aggressive type of B cell lymphoma, activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) [214, 230, 231].    Immunodeficiency with severe gastrointestinal inflammation has been shown for all patients with MALT1 deficiency [21-23]. However, MALT1 gene expression has not been investigated in people with IBD. I demonstrated that Malt1 mRNA is up-regulated in murine macrophages in response to innate immune stimuli (this chapter). Because intestinal macrophages are potentially exposed to innate immune ligands in vivo in the inflamed intestine, I 66  hypothesized that MALT1 mRNA expression is up-regulated in biopsies from subjects with IBD compared to those from healthy control subjects, which could contribute to inflammation. I also examined Malt1 mRNA in mice during DSS-induced colitis compared to a non-DSS-treated control group.   3.2 Material and methods  Mice. Malt1+/- mice were provided by Dr. Tak W. Mak from the University of Toronto. Mice were generated and backcrossed for more than 10 generations onto C57BL/6 mice [164, 208]. Malt1+/- mice were used to generate Malt1+/+ and Malt1-/- mice that were used between 8 and 12 weeks of age for experiments. Littermates were used for experiments and equal numbers of males and females were used in each group of mice compared in the DSS-induced colitis experiments. Mice were maintained in the Animal Research Center at BC Children's Hospital Research Institute (BCCHR). Protocol numbers are A17-0061 and A17-0071. Biopsies of individuals with IBD and control individuals. Human studies were approved by the University of British Columbia (UBC)-affiliated research ethics board (REB) and were performed according to their guidelines. Study subjects were not previously diagnosed or treated for IBD and were recruited from BC Children’s Hospital, Division of Gastroenterology. Clinical diagnosis of CD was performed using ileal biopsies based on pathological assessment and ileocolonoscopy. Inflamed or uninflamed biopsies were taken from the same segment of ileum in individuals with IBD (N = 5) or non-IBD controls (N = 9). Biopsies were snap frozen in liquid nitrogen and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at -80°C. Protocol number is H09-01826. 67  Macrophage derivation. Bone marrow macrophages were derived from bone marrow progenitor cells isolated from mouse leg bones (femurs and tibias). Bone cavities were flushed using a syringe filled with bone marrow IMDM supplemented with 10% FBS until the bones appeared to be clear. Bone marrow aspirates were incubated at 37°C in 5% CO2.  After 1h, the mature mesenchymal cells were adhered to the flask, and suspended hematopoietic progenitor cells were harvested. To derive macrophages, bone marrow progenitors were plated at a concentration of 0.5 × 106 cells/mL for 10 days in IMDM supplemented with 10% FBS, penicillin–streptomycin, and 5 ng/mL MCSF or GM-CSF (StemCell Technologies, Vancouver, BC, Canada) at 37°C in 5% CO2 with complete media changes on days 4 and 7. Macrophage stimulation. Macrophages were cultured in a 96-well plates at a density of 1 × 105 cells/well (1 × 106cells/mL) and stimulated with 10 ng/mL LPS (Escherichia coli serotype 127:B8; Sigma–Aldrich, St. Louis, MO, USA), 10 μg/mL zymosan (Saccharomyces cerevisiae; Invivogen, San Diego, CA, USA), or 100 μg/mL curdlan (Alcaligenes faecalis; Invivogen, San Diego, CA, USA) for 24 h. After stimulation, cell supernatants were harvested and clarified by centrifugation for analysis. For inhibitor studies, mepazine acetate (MPZ) (ChemBridge, San Diego, CA, USA) was added 6 h before stimulation of cells at a final concentration of 5–25 μM dissolved in 0.1% DMSO, or cells were simulated with a comparable amount of 0.1% DMSO, as a vehicle control. Gene expression analysis. Human RNA was isolated from ileal biopsies using the NucleoSpin@ RNA II Kit (Macherey-Nagel, Bethlehem, PA, USA). Mouse RNA was isolated from macrophages or mouse colons using the RNeasy Plus Mini kit (QIAGEN, Hilden, Germany). Following reverse transcription using iScript™ Reverse Transcription Supermix (Bio-Rad, 68  Hercules, CA, USA), gene expression was measured by qPCR using SsoAdvanced™ Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). SYBR Green supermix was mixed with forward and reverse primers, cDNA template, and nuclease-free water. For human studies, MALT1, BCL10, and CARD9 gene expression were normalized to PTPRC or GUSB. For mouse studies, Malt1, Bcl10, and Card9 gene expression were normalized to Gapdh. PrimePCR™ SYBR Green Assay primers qPCR (BioRad, Hercules, CA, USA) were used for both human and mice studies. The catalogue number for primers is 10025636 and identification numbers are as follows: qHsaCID0015826 (MALT1), qHsaCID0009788 (BCL10), qHsaCID0021765 (CARD9), qHsaCID0011706 (GUSB), qHsaCED0038908 (PTPRC), qMmuCID0016973 (Malt1), qMmuCED0047589 (Bcl10), qMmuCID0013767 (Card9), qMmuCED0027497 (Gapdh). SDS-PAGE and Western blotting. Bone marrow-derived macrophages were cultured at a concentration of 2.5 × 106 cells/mL and were stimulated with 10 ng/mL LPS (E. coli serotype 127:B8; Sigma–Aldrich, St. Louis, MO, USA), 10 μg/mL zymosan (S. cerevisiae; Invivogen, San Diego, CA, USA), or 100 μg/mL curdlan (A. faecalis; Invivogen, San Diego, CA, USA) for 24 h. Following rinsing twice with cold PBS, cells were lysed on ice in 2 × Laemmli's digestion mix, DNA was sheered by passing through a 26-gauge needle, and samples were heated at 100°C for 1 min. Cell lysates were separated on a 10% polyacrylamide gel and Western blotting was performed. Antibodies used for Western blotting were: anti-Malt1 (sc515389; Santa Cruz Biotechnology, Mississauga, ON, Canada), anti-Bcl10 (4237; Cell Signalling Technology, Whitby, ON, Canada), and anti-Gapdh (10R-G109A; Fitzgerald Industries International, Acton, MA, USA). 69  MALT1 activity assay. Malt1 endogenous protease activity was performed as follow: Macrophages (2.5 × 106) were stimulated with 10 or 100 ng/mL LPS (E. coli serotype 127:B8; Sigma–Aldrich, St. Louis, MO, USA), 1 or 10 μg/mL zymosan (S. cerevisiae; Invivogen, San Diego, CA, USA), or 10 or 100 μg/mL curdlan (A. faecalis; Invivogen, San Diego, CA, USA) for 24 h. Cells were lysed followed by immunoprecipitation of Malt1 with the anti-Malt1 antibody (sc-28246; Santa Cruz Biotechnology, Dallas, TX, USA), and protein A magnetic beads (Cell Signalling Technology, Danvers, MA, USA). 20 μM of acetyl-leucine-arginine-serine-arginine-alphamethylcoumarin (Ac-LRSR-AMC) (Peptides International, Louisville, KY, USA) was added to the beads and incubated for 30 min at 30°C to measure the release of AMC caused by Malt1 protease activity. Fluorescence was measured using an excitation wavelength of 360 nm and emission at 460 nm for 90 min in corning 384-well black flat bottom plate (Greiner bio-one, Kremsmünster, Austria), using a POLARstar Omega plate reader (BMG Labtech, Ortengberg, Germany). Cytokine measurements. Cytokine measurements were performed on cell supernatants using ELISAs. Mouse IL-1β, IL-6, TNF, and IL-12p40 ELISA kits were purchased from BD Biosciences (Mississauga, ON, Canada).                        DSS-induced colitis. Colitis was induced in mice by adding 2.5% DSS to their drinking water for 7 days. As controls, groups of mice also received sterile drinking water (no DSS).  Statistical analyses. Analyses were done using unpaired Student's t-tests, 1-way ANOVAs using Graphpad Prism, as indicated (GraphPad Software, San Diego, CA, USA). Differences with P < 0.05 were considered significant. 70  3.3 Results 3.3.1 Stimulation of macrophages with LPS, zymosan, or curdlan increases Malt1 protein expression but decreases Bcl10 cleavage In Chapter 2, I found that Malt1 deficiency increases susceptibility of mice to DSS-induced colitis, which is a murine model that mimics elements of intestinal inflammation seen in people with IBD. I showed that this effect is mediated by macrophage-derived IL-1b production. In order to better understand the mechanisms underlying the role of macrophages in Malt1 deficiency-mediated intestinal inflammation, it is critical to recognise how Malt1 protein expression and enzymatic activity are regulated in these cells. To determine the impact of innate immune stimuli on Malt1 protein expression in murine macrophages, MCSF-derived bone marrow macrophages were unstimulated or stimulated with LPS (10 or 100 ng/mL), zymosan (1 or 10 μg/mL), and curdlan (10 or 100 μg/mL) for 24 h (Fig. 3.1). Whole cell lysates were analyzed by Western blot, and probed for Malt1 and Gapdh, as a loading control. LPS (Fig. 3.1 A), zymosan (Fig. 3.1 B), and curdlan (Fig. 3.1 C), dramatically increased Malt1 protein expression in a dose-dependent manner. I probed the same membranes for Bcl10. Interestingly, despite an increase in Malt1 protein expression, Bcl10 cleavage was reduced when macrophages were stimulated with LPS (Fig. 3.1 A), zymosan (Fig. 3.1 B), or curdlan (Fig. 3.1 C). Reduced Malt1 activity caused by innate immune stimuli was also dose dependent and more evident at the higher concentrations of the ligands used. This demonstrates that TLR4 and dectin-1 ligation induces Malt1 protein expression but reduces Malt1 proteolytic activity.  71   Figure 3.1 Macrophage activation increases Malt1 protein expression but decreases Bcl10 cleavage. MCSF-derived bone marrow macrophages were unstimulated or stimulated with (A) 10 or 100 ng/mL of LPS, (B) 1 or 10 μg/mL of zymosan, and (C) 10 or 100 μg/mL of curdlan for 24 h. Whole cell lysates (2.5 × 10 6 cells/lane) were analyzed by Western blot for Malt1, Bcl10 (black arrow) and its cleavage product (white arrow), and Gapdh as a loading control.    3.3.2 Stimulation of macrophages with LPS, zymosan, or curdlan decreases Malt1 activity  To confirm that Malt1 paracaspase activity was reduced in macrophages by treatment with LPS, zymosan, or curdlan; I used a biochemical approach to measure Malt1 activity in vitro after stimulating macrophages. First, I monitored the activity of Malt1 in MCSF-derived bone marrow macrophages by examining the cleavage of the fluorogenic Malt1 substrate (Ac-LRSR-AMC), a peptide sequence that is based on the Bcl10 cleavage site [170]. Malt1 was immunoprecipitated from whole cell lysates from 0, 0.5, 1, or 2 × 106 MCSF-derived bone marrow macrophages. Ac-LRSR-AMC cleavage was observed in immunoprecipitates from macrophages and cleavage activity was proportional to input cell number (Fig. 3.2 A). Next, I treated bone marrow-derived macrophages with the Malt1 inhibitor, mepazine (MPZ) and assessed its effect on cleavage of the Malt1 substrate. Upon treatment of the cells with MPZ for 6 h, Malt1 cleavage activity was blocked, demonstrating that MPZ successfully inhibited Malt1 activity (Fig. 3.2 B). Using this in vitro cleavage assay, I asked whether LPS (10 or 100 ng/mL), zymosan (1 or 10 μg/mL), and curdlan (10 or 100 μg/mL) affected Malt1 activity (Fig. 3.2 C). 72  Consistent with my Western blot results for Bcl10 cleavage, Malt1 proteolytic activity was reduced when macrophages were stimulated with either LPS, zymosan, or curdlan.  Figure 3.2 Macrophage activation blocks Malt1 activity.  Whole cell lysates were prepared from macrophages and Malt1 was immunoprecipitated to assay its activity. Intrinsic capacity of Malt1 to cleave the fluorogenic substrate, Ac-LRSR-MCA, was assessed over 90 min. (A) Dose response for Malt1 activity in bone marrow-derived macrophages (0.5–2 × 106 macrophages); control, C, is no lysis buffer. (B) MPZ blocks macrophage Malt1 activity. (C) Malt1 activity from macrophages treated for 24 h with 10 or 100 ng/mL of LPS, 1 or 10 μg/mL of zymosan, and 10 or 100 μg/mL of curdlan.    3.3.3 Inhibiting Malt1 activity increases macrophage TNF and IL-6 production To understand how the opposing effects of Malt1 expression and activity could translate into cytokine production by macrophages, I asked whether specifically inhibiting Malt1 proteolytic activity by MPZ could impact macrophage inflammatory cytokine production. MCSF-derived bone marrow macrophages (WT) were left unstimulated or stimulated with LPS 73  (10 ng/mL) for 24 h after treatment with different doses of MPZ (0–25 μM) for 6 h (Fig. 3.3 A). MPZ treatment increased LPS-induced TNF production in a dose-dependent manner. MPZ treatment also modestly increased LPS-induced IL-6 production but did not affect IL-12p40 production (Fig. 3.3 A). The effect of Malt1 inhibition on macrophage pro-inflammatory cytokine production was also assessed after stimulating macrophages with zymosan (10 μg/mL) or curdlan (100 μg/mL) for 24 h (Figs. 3.3 B and 3.3 C). MPZ did not impact pro-inflammatory cytokine production in response to zymosan (Fig. 3.3 B) and only modestly increased TNF production in response to curdlan (Fig. 3.3 C).  Figure 3.3 The Malt1 inhibitor, mepazine acetate (MPZ), increases macrophage TNF and IL-6 production after stimulation with LPS and curdlan.  MCSF-derived bone marrow macrophages were treated with 5–25 μM MPZ or DMSO, as a vehicle control (V), for 6 h followed by stimulation with (A) 10 ng/mL LPS, (B) 10 μg/mL zymosan, or (C) 100 μg/mL curdlan for 24 h. Clarified cell supernatants were assayed for TNF, IL-6, and IL-12p40 by ELISA. Data is presented as the mean ± SD for N = 3; macrophages were derived from 1 mouse for each of 3 independent experiments. *P < 0.05, **P < 0.01, statistical analyses were performed by using a 1-way ANOVA.  74  3.3.4 Inhibiting Malt1 activity decreases (LPS+ATP)-induced macrophage IL-1b production  Inhibition of Malt1 activity has been shown to protect mice against DSS-induced colitis through impaired IL-1β production in response to LPS [214]. However, I showed that increased production of IL-1β by Malt1-deficient macrophages exacerbates DSS-induced colitis in Malt1-/- mice (Chapter 2). Moreover, I found that blocking Malt1 proteolytic activity actually increases IL-6 and TNF production by macrophages (this Chapter). To understand the precise effect of blocking Malt1 proteolytic activity on macrophage-mediated IL-1β production in my model, I measured IL-1β produced by MCSF-derived bone marrow macrophages in response to LPS, zymosan, or curdlan, while inhibiting Malt1 activity with MPZ. Bone marrow-derived macrophages were treated with MPZ (0–25 μM) for 6 h followed by stimulation with LPS (10 ng/mL) (Fig. 3.4 A), zymosan (10 μg/mL) (Fig. 3.4 B), or curdlan (100 μg/mL) (Fig. 3.4 C) for 24 h and ATP (5 mM) was added for the final hour. There was no significant change in the amount of IL-1β produced after inhibition of Malt1 activity in bone marrow-derived macrophages stimulated with zymosan or curdlan plus ATP (Figs. 3.4 B and 3.4 C), but IL-1β production was decreased after inhibition of Malt1 activity in bone marrow-derived macrophages stimulated with LPS plus ATP (Fig. 3.4 A). In summary, inhibiting Malt1 proteolytic activity decreased LPS-induced IL-1β production by MCSF-derived bone marrow	macrophages.  75   Figure 3.4 The Malt1 inhibitor, mepazine acetate (MPZ), decreases macrophage IL-1b production after stimulation with LPS.  MCSF-derived bone marrow macrophages were untreated or treated with MPZ followed by stimulation with (A) LPS, (B) zymosan, or (C) curdlan with or without ATP. Results are expressed as the mean ± SD for N = 3; macrophages were derived from 1 mouse for each of 3 independent experiments. *P < 0.05 statistical analyses were performed using a 1-way ANOVA.  3.3.5 MALT1, BCL10, and CARD9 expression is not altered significantly in subjects with IBD I found that Malt1 expression is increased in activated murine MCSF-derived bone marrow macrophages, which play a critical role during intestinal inflammation [212]. Based on this, I asked whether MALT1 gene expression is affected in intestinal tissue of subjects with IBD compared to control subjects without IBD. Furthermore, I examined the gene expression of BCL10 and CARD9 in biopsies of individuals with IBD and in control subjects to determine if intestinal inflammation is associated with varied expression of other members of the CBM complex. Ileal tissue biopsies were taken during colonoscopies from (N = 14) treatment-naïve subjects as part of their diagnosis for IBD. Subjects were diagnosed with CD (N = 5) or not (N = 9). The expression of MALT1, BCL10, and CARD9 versus reference genes, including GUSB (a ubiquitously expressed gene) and PTPRC (CD45, a leukocyte marker) were measured. There was no significant difference between MALT1, BCL10, or CARD9 gene expression, relative to GUSB, in the ileum of subjects with CD compared to control subjects (Fig. 3.5 A). I next asked if 76  there is any difference in MALT1, BCL10, and CARD9 gene expression relative to PTPRC, a leukocyte marker, as MALT1 is expressed ubiquitously in both immune and non-immune cells of the intestine, but immune cells are key mediators of intestinal inflammation. Thus, in order to examine if expression of MALT1 is altered in immune cells during intestinal inflammation, MALT1 gene expression relative to PTPRC was assessed. The expression of MALT1, BCL10, and CARD9 gene expression, relative to PTPRC, in the ileum of subjects with CD was similar to control subjects (Fig. 3.5 B).  Figure 3.5 Subjects with IBD have comparable MALT1/BCL10/CARD9 mRNA expression compared to control subjects.  (A) MALT1/BCL10/CARD9 expression in biopsies from ileum, relative to GUSB (a ubiquitously expressed gene). (B) MALT1/BCL10/CARD9 expression in biopsies from the ileum, relative to PTPRC (a leukocyte marker). No significant differences in gene expression were found comparing people with IBD to people with no IBD. 77  3.3.6 Malt1, Bcl10, and Card9 mRNA expression and activity is comparable in mice with or without DSS-induced colitis  I expected to see differences in MALT1 mRNA expression in subjects with IBD compared to control subjects because Malt1 mRNA is up-regulated in response to innate immune stimuli in murine macrophages. Thus, to distinguish whether Malt1 was not up-regulated during intestinal inflammation in vivo or Malt1 mRNA induction was a mouse-specific phenomenon, I measured Malt1, Bcl10, and Card9 mRNA and protein expression in mice colon tissue homogenates after induction of colitis by DSS. Mice were treated with 2.5% DSS in their drinking water. On day 7, mice were euthanized, and their colon tissue was harvested. Mouse colon tissue homogenates were analyzed by qPCR for Malt1, Bcl10, and Card9 gene expression relative to Gapdh. Expression of Malt1, Bcl10, and Card9 genes were not significantly different in mouse colon tissue homogenates treated with DSS compared to mice that were not treated with DSS (Fig. 3.6 A).  I next examined whether Malt1 protein expression is affected in mice colon tissue in response to DSS in vivo as Malt1 protein expression was up-regulated in activated murine MCSF-derived bone marrow macrophages in vitro and could occur independently of the increased gene expression that I observed. Colon tissue homogenates from mice treated with or without DSS were analyzed by Western blot and probed for Malt1 and Gapdh, as a loading control. Malt1 protein expression was comparable in mice with DSS-induced colitis and control mice (Fig. 3.6 B). Finally, I examined whether cleavage of Bcl10, a substrate for Malt1 protease activity, was altered in mouse colon tissue after induction of colitis using DSS. I probed Western blot membranes described above for Bcl10 and its cleavage product, a smaller band visible below Bcl10 (Fig 3.6 B). There was no difference in either Malt1 protein expression or activity 78  (Bcl10 cleavage) in colonic tissue from mice treated with DSS-induced colitis versus control mice (Fig. 3.6 B).   Figure 3.6 Mice treated with DSS-induced colitis have similar expression of Malt1, Bcl10, and Card9 mRNA, Malt1 and Bcl10 protein, and Malt1 paracaspase activity compared to control mice, which were not treated with DSS.  (A) Malt1, Bcl10, and Card9 mRNA expression in mouse colon tissue, relative to Gapdh. (B) Mouse colon tissue homogenates were analyzed by Western blot for Malt1, Bcl10 (black arrow) and its cleavage product (white arrow), and Gapdh as a loading control.  3.4 Discussion I assessed Malt1 protein expression and activity in macrophages along with the role of Malt1 activity in macrophage inflammatory responses in vitro. I also examined a potential role of MALT1 gene expression in IBD pathogenesis in humans and investigated this in mice in vivo during DSS-induced colitis. My data demonstrates that innate immune stimuli that trigger TLR4 and dectin-1 up-regulate Malt1 protein expression but eliminate Malt1 proteolytic activity. Furthermore, blocking Malt1 activity by mepazine decreases IL-1b production by 79  macrophages in response to innate immune stimuli. However, in people with IBD; despite exposure of intestinal immune cells to innate immune ligands in vivo, MALT1 mRNA expression was not altered in intestinal biopsies from participants with IBD.  Similarly, BCL-10 and CARD9 mRNA expression were not significantly different comparing biopsy tissues from people with IBD to unaffected healthy control tissues. To determine whether this is also the case in mice, I examined Malt1, Bcl10, and Card9 mRNA expression in in colon tissue homogenates in mice after DSS-induced colitis. As in people with IBD, there was no difference in mRNA for Malt1, Bcl10, or Card9 in colon tissue homogenates from mice subjected to DSS-induced colitis. In chapter 2, I demonstrated that Malt1-deficient mice are more susceptible to DSS-induced colitis. To identify the association between Malt1 protein expression and the intestinal inflammation, I tested Malt1 protein expression in macrophages in vitro as they play a key role in the immune responses during IBD [212]. Indeed, Malt1 protein expression was dramatically induced by each of the innate immune stimuli (LPS, zymosan, and curdlan) in macrophages. This may occur in response to other innate immune receptors as Malt1 mRNA is among the highest transcripts induced by TLR7 ligation and is required for NF-κB activation and protection against influenza A virus [232]. In contrast, Malt1 paracaspase activity was dramatically reduced by the same innate immune stimuli, which was apparent by examining Bcl10 cleavage by Western blot. In activity assays performed after 24 h of stimulation, Malt1 paracaspase activity was ablated by LPS or zymosan, and also profoundly reduced by curdlan. In T or B cells, Malt1 protein is not induced downstream of antigen receptor ligation [173, 229]. Macrophage Malt1 paracaspase activity is also distinct from activation reported in T or B cells, as in T or B cells, paracaspase activity is only induced when cells are stimulated with CD3/CD28 or PMA/Ionomycin [21, 173, 229]; whereas Malt1 paracaspase is constitutively active in 80  macrophages and reduced upon receptor ligation. In T cells, Malt1 must be associated with the CBM complex to be active [233], which suggests that Malt1 may exist within complexes (associated with Bcl10) constitutively in macrophages. After activation of macrophages, paracaspase activity of Malt1 may not be feasible due to increase in Malt1 protein expression and/or recruitment of other regulatory molecules that bind to Malt1 and block its active site in order to down-regulate Malt1 paracaspase activity in macrophages.  With regard to Malt1's paracaspase activity, a previous report demonstrated that MPZ does not affect TLR4 (LPS)-induced TNF production [234], whereas another report demonstrates that MI-2 inhibits TLR4 (LPS)-induced TNF production by human monocyte-derived macrophages [235]. In contrast, I found that inhibiting Malt1 protease activity with the allosteric inhibitor MPZ [231], actually increased LPS-induced TNF production in a dose-dependent manner, and modestly increased LPS-induced IL-6 production and curdlan-induced TNF production, suggesting that the dominant effect of paracaspase activity in macrophages is to limit NF-κB-driven pro-inflammatory responses. Differences in the effects of inhibitors may reflect differences in inhibitors as it has been previously shown that MI-2 has off target effects on other proteases including cathepsins [236]. Moreover, compared to my study, a similar range of MPZ concentrations (0-100 µM) were used for these assays, but the cells were only incubated with inhibitors for 1 h before stimulation with LPS [234]. This is different from the 6 h incubation done in my study, which has previously been shown to effectively inhibit Malt1 activity and suppress its dependent gene expression in the ABC-DLBCL cell line [231]. In addition to the effects of inhibitors, these differences may also reflect differences in cell source (bone marrow versus blood monocytes) and differences in assay conditions. My data next shows that Malt1 proteolytic activity is critical for the production of IL-1β by macrophages. This is consistent with 81  a previous report that shows blocking Malt1 activity by MPZ decreases IL-1β production by macrophages after stimulation with LPS [214]. Activation of NF-κB drives an inactive precursor form of IL-1β (pro-IL-1β), which is catalyzed to the active form of IL-1β after processing by inflammasomes [217]. It has been suggested that decreased IL-1β production by macrophages is caused by impaired inflammasome activation [214]. In this study, I did not examine the effect of MPZ on inflammasome activation in macrophages, but my data supports their findings and shows that blocking Malt1 activity by MPZ decreases IL-1β production after stimulation with innate immune stimuli. In contrast to IL-1β, my data shows that inhibiting Malt1 activity increases production of TNF and IL-6 by macrophages after stimulation with TLR4 and dectin-1 ligands. The involvement of inflammasomes in IL-1β production may result in the opposing trend that I observed in production of TNF and IL-6 versus IL-1β by macrophages after stimulation by innate immune stimuli following inhibiting Malt1 activity by MPZ. To further assess the effect of blocking Malt1 activity on inflammasomes, IL-1β production can be examined in inflammasome-deficient macrophages following treatment with MPZ in the future. It has been shown previously that recovery is impaired after DSS-induced colitis in mice deficient for Card9, which acts as an adaptor molecule in the CBM complex [162]. Similar to Malt1-/-, Card9-/- mice do not develop spontaneous colitis [237], but the Card9-deficient mice have significant weight loss and a higher histological damage score with shorter colon length on day 12 compared to WT mice, after adding 3% DSS to their drinking water for 7 days followed by an additional 5 days with no DSS [237]. This is consistent with my findings in chapter 2, showing that Malt1-deficient mice have higher weight loss and histological damage scores as well as shorter colon length compared to their WT littermates after adding 2.5% DSS to their drinking water for 7 days. However, unlike Malt1-deficient mice, the susceptibility to DSS-82  induced colitis in Card9-deficient mice is similar to WT mice on day 7 of DSS-induced colitis [237]. The less profound effect of Card9 deficiency on DSS-induced colitis in mice is consistent with delayed and mild clinical features, which is mainly characterized by susceptibility to fungal infections, observed in patients with CARD9 deficiency compared to severe inflammatory phenotype of patients deficient for MALT1 [238]. In addition to the immunodeficiency caused by lack of the CARD9 gene, single nucleotide polymorphisms (SNPs) in the CARD9 gene, which are associated with increased CARD9 mRNA, can increase the risk of developing IBD [44, 239-241]. This is consistent with up-regulated Malt1 protein expression in murine MCSF-derived bone marrow macrophages in response to innate immune stimuli in vitro. Based on this, I asked whether MALT1 gene expression is increased in subjects with IBD. My results actually showed that there is no difference in gene expression of MALT1 or other CBM complex members (BCL10 or CARD9) in biopsies from subjects with IBD compared with those from healthy control subjects. The fact that I did not see any significant difference in gene expression could be because Malt1 induction in response to innate immune stimuli is a mouse-specific phenomenon. To investigate this, I used the DSS-induced colitis model to examine Malt1 gene expression during the induction of colitis in mice. My data shows that DSS-induced colitis does not have any effect on Malt, Bcl10, or Card9 gene expression in mouse colon tissue. It may be that Malt1 is not induced in murine macrophages in vivo during DSS-induced colitis and/or that constitutive Malt1 expression in other cells/tissues overshadows its induction in macrophages as MALT1 is expressed ubiquitously and intestine is composed of a variety of cells including immune and epithelial cells.   In summary, my data demonstrates up-regulation of Malt1 protein expression in macrophages in vitro during inflammatory responses. However, MALT1 gene or protein 83  expression is not changed in human and mice intestinal tissue during inflammation. This indicates that up-regulation of MALT1 expression could be specific to macrophages, which normally express low levels of MALT1. Indeed, MALT1 gene mutations in humans are very rare and the patients with MALT1 deficiency display very unique and dramatic inflammatory phenotypes in various organs and tissues caused by significant immune dysregulation [21-23]. This may suggest that expression of MALT1 is not changed in inflamed intestinal tissue due to its critical role in immune and non-immune cell development and function.      84  Chapter 4: Malt1-deficient mice develop osteoporosis independent of osteoclast-intrinsic effects of Malt1 deficiency   4.1  Introduction  Osteoporosis is a common bone disease characterized by bone loss, change in the bone structure, and increased risk of bone fractures. In homeostasis, bone is constantly remodeled at a microscopic level; damaged or ineffete bone is resorbed by osteoclasts and new bone is laid down by osteoblasts [242]. Osteoblasts are derived from mesenchymal cells whereas osteoclasts are hematopoietic in origin [242].  In osteoporosis, dysregulation of osteoblast or osteoclast number or activity causes bone resorption to overtake bone formation and net bone mass decreases.  Osteoclasts are multinucleated cells that are generated by the fusion of myeloid precursors in the bone marrow. Two hematopoietic cytokines, MCSF and RANKL, are both necessary and sufficient for differentiation of myeloid progenitors into osteoclasts. Osteopetrotic mice (op/op) lack MCSF, are deficient in osteoclasts, and have been used to demonstrate that MCSF is required for osteoclast precursor proliferation and survival [85]. RANKL acts as a master regulator of myeloid cell commitment to the osteoclast lineage and increases the resorptive activity of mature osteoclasts [86]. As such, mice deficient in RANKL are osteopetrotic because they lack osteoclasts [87]. In addition, osteoclastogenesis and activity are regulated by OPG, a soluble decoy receptor that binds RANKL, and blocks osteoclast formation, differentiation, activation, and survival [88]. The RANK/RANKL/OPG signalling pathway is a key regulator of osteoclast number and function and its dysregulation has been implicated in osteoporosis [89]. 85  People with MALT1 deficiency-associated CID have recurrent infections as well as severe, chronic inflammation of the skin, lungs, and gastrointestinal tract [21-23]. Our laboratory participated in the description of a 15-year-old patient with CID at BC Children’s Hospital that was caused by non-ablative MALT1 deficiency [21]. In addition to immune-mediated and inflammatory pathology, the patient was small in stature and had low bone mineral density that led to multiple low-impact fractures [21]. The patient showed significant clinical improvement after HSCT, which resolved the patient’s dermatitis and gut inflammation [25]. Moreover, HSCT increased the patient’s growth velocity from 1.9 to 7.0 cm/year (1 year pre- and post-transplant) [25]. Malt1-deficient mice are viable and fertile with no overt signs of inflammatory pathology [164, 196]. Mice deficient in Malt1 paracaspase activity (paracaspase dead, Malt1PD/PD) are also viable and fertile. However, these mice have low body weight and develop an inflammatory phenotype characterized by high levels of IFNγ, inflammation in multiple organs, and autoimmune gastritis [173, 197, 198]. T cell defects have been demonstrated in both mice strains because T cells from these mice do not cause inflammation in the T cell transfer model of colitis [198]; and both mouse genotypes, as well as inhibition of paracaspase activity, are protective in an experimental mouse model of autoimmune encephalitis, which is driven by the development of pathological Th17 cells [199, 200, 230]. Gewies et al., have reported that mice deficient in paracaspase activity are more susceptible to DSS-induced colitis [173]. In chapter 2, we demonstrated that Malt1-/- mice are also more susceptible to DSS-induced colitis and their increased inflammation and pathology is driven by macrophage-derived IL-1β production. This work suggests that innate immune (and myeloid) cells are poised to respond more robustly to activating stimuli in Malt1-deficient mice. 86  In summary, the patient with MALT1 deficiency had growth inhibition and severe osteoporosis, both of which improved significantly post-HSCT [21, 25]. We have found that Malt1 deficiency leads to hyper-activation of macrophages in Malt1-/- mice (Chapter 2). Osteoclasts, like macrophages, are derived from myeloid precursors and could contribute to the HSCT-corrected bone phenotype observed in the MALT1-deficient patient. Thus, we asked whether Malt1-deficient mice are osteoporotic and whether this phenotype is attributable to cell intrinsic effects of Malt1 deficiency in osteoclasts. We compared growth and bone density in Malt1+/+ and Malt1-/- mice. In vitro, we measured Malt1 expression, activity, and induction in osteoclasts, and examined the effect of Malt1 deficiency on osteoclast differentiation, and activity.  In vivo, we examined osteoclast number and concentrations of cytokines that drive osteoclastogenesis and activity. Finally, we examined the effect of inflammatory stimuli on the production of regulators of osteoclast generation and activity by macrophages and their impact on the differentiation of Malt1+/+ and Malt1-/- osteoclasts in vitro.   4.2 Material and methods  Mice. Malt1+/- mice were provided by Dr. Tak Mak from the University of Toronto.  Mice were generated by breading for more than 10 generations onto C57BL/6 mice [164, 208]. Heterozygous mice (Malt1+/-) were bred to generate Malt1+/+ and Malt1-/- littermates for experiments. Mice were maintained in the Animal Research Center at BC Children’s Hospital Research Institute. Experiments were carried out with approval and according to institutional and Canadian Council on Animal Care guidelines. Protocol numbers are A17-0061 and A17-0071.  87  Microcomputed tomography (μCT) analyses. Ex vivo μCT scanning of the femur was performed on bone specimens using the Scanco μCT 100 (Scanco Medical, Bruttisellen, Switzerland). Bones were scanned with a 0.5 mm Al filter in batches of three at a nominal resolution of 7.4 μm. The X-ray source setting was at 70 kVp and 114 uA, 8w at 100 ms integration time. Ellipsoid contours outline were selected in trabecular and cortical regions. Bone volume / total volume (BV / TV) was determined in 100 continuous slices in the distal femur metaphysis 1.5 mm from the growth plate. Cortical thickness (Cort. Th) and cortical bone mineral density (Cort. BMD) were assessed in 100 continuous slices in the femur midshaft 50 slices above and 50 slices below the midpoint. Trabecular parameters including trabecular bone mineral density (Tb. BMD), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp) were determined in 100 continuous slices in the distal femur metaphysis 1.5 mm from the growth plate.                                                                                                                                                        Serum markers of bone formation and resorption. Blood samples from mice were clotted at room temperature for 15 min, followed by centrifugation at 2000 ´g for 10 min at 4°C. Alkaline phosphatase (ALP), procollagen type 1 N-terminal propeptide (P1NP) (bone formation markers) and C-terminal type 1 collagen fragments (CTX-1) (bone resorption marker) were measured in Malt1+/+ and Malt1-/- mouse serum by ELISA as per manufacturers’ instructions. ELISA kits were from Cusabio for ALP (TX, USA) and FineTest for P1NP and CTX-1 (Hubei, China). In other experiments, sera from mice were assayed for murine MCSF, RANKL, and OPG by ELISA. ELISA kits were from Abcam (Cambridge, United Kingdom). Histological analyses. Femurs were dissected and fixed in 10% formalin overnight.  Bones were decalcified by incubation in 14% EDTA (pH 7.4) for 10 days with gentle shaking. Longitudinal tissue sections extending from the end of the bones and beyond the diaphysis region of femurs 88  were embedded in paraffin, and longitudinal sections were then deparaffinized, rehydrated, and stained with H&E, TRAP, or alkaline phosphatase. TRAP staining was performed using a commercially available TRAP staining kit (Sigma-Aldrich, St. Louis, MO, USA). ALP was stained using an Alkaline phosphatase staining kit (Sigma-Aldrich, St. Louis, MO, USA). TRAP+ cells with ≥ 3 nuclei were counted as osteoclasts and ALP+ cells were counted as osteoblasts; numbers were normalized to mm2 of bone area. Macrophage and osteoclast derivation and culture. Bone marrow progenitors were isolated from mouse femurs and tibias. Bone cavities were flushed using a syringe filled with IMDM supplemented with 10% FBS until the bones appeared to be clear. Bone marrow aspirates were incubated at 37°C in 5% CO2.  After 1h, the mature mesenchymal cells were adhered to the flask, and suspended hematopoietic progenitor cells were harvested. To derive macrophages, bone marrow progenitor cells were plated at a concentration of 0.5 ´ 106 cells/mL for 10 days in IMDM supplemented with 10% FBS, penicillin-streptomycin, and 5 ng/mL MCSF (STEMCELL Technologies, Vancouver, BC, Canada) at 37°C in 5% CO2 with complete media changes on days 4 and 7. Osteoclasts were derived as previously described [243]. Briefly, bone marrow progenitors (1 ´ 106) were plated in 6-well tissue culture plates in 3 mL a-MEM supplemented with 10% FBS, penicillin-streptomycin, 20 ng/mL MCSF (STEMCELL Technologies, Vancouver, BC, Canada), and 40 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) at 37°C in 5% CO2 with complete media changes on days 4 and 6. In some experiments, the concentration of RANKL used to generate osteoclasts was titrated from 0-80 ng/mL; the concentration of MCSF used to derive osteoclasts was titrated from 0-20 ng/mL; or OPG (0-100 ng/mL) was added to osteoclast cultures during derivation. Fixed osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) using a commercially available TRAP staining kit 89  (Sigma-Aldrich, St. Louis, MO, USA). Cells that were TRAP+ and which contained ≥ 3 nuclei were counted as osteoclasts.                       SDS-PAGE and western blotting. Bone marrow-derived macrophages or osteoclasts were rinsed twice with cold PBS, 2.5 ´ 106 cells were lysed on ice in 2´ Laemmli’s digestion mix, DNA was sheered by passing through a 26-gauge needle, and samples were heated at 100°C for 1 min. Cell lysates were separated on a 10% polyacrylamide gel and western blotting was performed. Antibodies used for western blotting were: anti-Malt1 (sc-515389, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Bcl10 (4237, Cell Signalling Technology, Danvers, MA, USA), and anti-Gapdh (10R-G109A, Fitzgerald Industries International, Acton, MA, USA). For dose response studies, osteoclast precursors were untreated or treated with 5, 25, 50, and 100 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) in a-MEM supplemented with 10% FBS, penicillin-streptomycin, and 20 ng/mL MCSF (STEMCELL Technologies, Vancouver, BC, Canada) for 7 days. For time course studies, osteoclast precursors were untreated or treated with 40 ng/mL RANKL for 2, 4, 8, or 24 h in a-MEM supplemented with 10% FBS, penicillin-streptomycin, and 20 ng/mL MCSF.  Malt1 activity assay. Endogenous Malt1 protease activity was measured as follow; mature osteoclasts (2.5 ´ 106) were lysed followed by precipitation of Malt1 with the anti-Malt1 antibody (sc-28246, Santa Cruz Biotechnology, Dallas, TX, USA), and protein A magnetic beads (Cell Signalling Technology, Danvers, MA, USA). 20 µM Ac-LRSR-AMC (Peptides International, Louisville, Kentucky, USA) was added to the beads and incubated for 30 min at 30°C to measure the release of AMC caused by Malt1 protease activity. Fluorescence was measured using an excitation wavelength of 360 nm and detecting emission at 460 nm for 90 min 90  in Corning 384-well black flat bottom plate (Greiner bio-one, Kremsmünster, Austria), using a POLARstar Omega plate reader (BMG Labtech, Ortengberg, Germany). As a control, cells were not lysed prior to immunoprecipiation. For the inhibitor experiment, osteoclasts were incubated with mepazine acetate (MPZ) (ChemBridge, San Diego, CA, USA) at a final concentration of 25 µM dissolved in DMSO (vehicle control; 0.1% DMSO) for 6 hours prior to lysis.                 Gene Expression Analysis. RNA was isolated from osteoclasts using the RNeasy Plus Mini kit (QIAGEN, Hilden, Germany). Following reverse transcription using iScriptTM Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA), gene expression was measured by qPCR using SsoAdvacedTM Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). SYBR Green supermix was mixed with forward and reverse primers, cDNA template, and nuclease-free water. Malt1 gene expression was normalized to Gapdh.  PrimePCRTM SYBR Green Assay primers for qPCR were purchased from Bio-Rad (Hercules, CA, USA). The catalog number for primers is 10025636 and identification numbers were as follows:  Malt1, qMmuCID0016973; Gapdh, qMmuCED00027497.  Dissolution of hydroxyapatite by Malt1+/+ and Malt1-/- osteoclasts. Osteoclasts were generated from myeloid precursor cells that were isolated as described above. After 7 days of culture, 5 ´ 103 cells were lifted and transferred to hydroxyapatite (Corning Inc., New York, USA) in 96-well plates for 7 days. Cells were lysed using 10% bleach for 5 min at room temperature and wells were washed 3 times with distilled water. Wells were stained with a solution of 1% toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Cleared areas were measured using ImageJ from 4 representative fields for each slice imaged by inverted microscopy. Degradation of bone by Malt1+/+ and Malt1-/- osteoclasts. Mature bone marrow-derived 91  osteoclasts were lifted and plated on bone slices made from cortical bovine femur (Immunodiagnostic Systems, East Boldon, UK) at a concentration of 50,000 cells/well for 6 days. Adherent cells were scraped off gently using cotton swabs and bone slices were washed with distilled water. Resorption pits were stained with toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) and resorbed area was quantitated using ImageJ. Separate bone slices were stained using a TRAP staining kit (Sigma-Aldrich, St. Louis, MO, USA) to quantitate osteoclast numbers. CTX-1 released from bone slices was measured by ELISA (FineTest, Hubei, China). Macrophage stimulation. Bone marrow-derived macrophages were cultured in 96-well plates at a density of 1 ´ 105 cells/well (1 ´ 106 cells/mL) and stimulated with 10 ng/mL LPS (Escherichia coli serotype 127:B8; Sigma-Aldrich, St. Louis, MO, USA), 10 µg/mL zymosan (Saccharomyces cerevisiae; Invivogen, San Diego, CA, USA), or 100 µg/mL curdlan (Alcaligenes faecalis; Invivogen, San Diego, CA, USA) for 24 h. The zymosan used at this concentration can activate both dectin-1 and TLR2; the curdlan used at this concentration can activate dectin-1, TLR2, and TLR4. After stimulation, cell supernatants were harvested and clarified by centrifugation for analysis. For inhibitor studies, MPZ (ChemBridge) was added to cultures for 6 h with no stimulation or prior to stimulation at final concentrations of 5-50 µM dissolved in DMSO, or cells were incubated with a comparable volume of 0.1% DMSO, as a vehicle control. Statistical analyses. Analyses were done using unpaired Student’s-t tests, 1-way ANOVAs, and 2-way ANOVAs in Graphpad Prism (GraphPad Software, San Diego, CA, USA), as indicated. Differences with P < 0.05 were considered significant.  92  4.3 Results  4.3.1 Malt1-deficient mice are smaller than wild-type littermates at 24 weeks of age To determine whether compromised bone growth and density can be modeled in Malt1-deficient mice, we measured body length in Malt1+/+ and Malt1-/- mice at 4, 6, 8, 12, and 24 weeks of age. Malt1-/- mice were considerably smaller than Malt1+/+ mice at 24 weeks of age (Fig. 4.1 A). The body weight of Malt1-/- mice was also reduced at 24 weeks of age (Fig. 4.1 B). We measured body length in mice to determine whether their growth was impaired. There were no significant differences in body length in 4-12-week-old mice, but 24-week-old mice were modestly shorter (Fig. 4.1 C). For an accurate measure of bone growth over time, femurs and tibias length were also measured in Malt1+/+ and Malt1-/- mice from 4-24 weeks of age. Femurs and tibias were modestly shorter in some of the Malt1-deficient mice at 12- and 24-weeks of age, but overall, they were not significantly different from their wild-type littermates (Fig. 4. 1 D and E). Taken together, these results show that bone length is not dramatically affected by Malt1 deficiency in mice.   Figure 4.1 Malt1-deficient mice are smaller than their Malt1+/+ littermates, but bone length is not reduced.  (A) Representative images of male Malt1+/+ (left) and Malt1-/- (right) mice at 24 weeks of age. Body weight (B), body length (C), femur length (D), and tibia length (E) of Malt1+/+ and Malt1-/- mice at 4, 6, 8, 12, and 24 weeks of 93  age. Results are expressed as the mean ± SD; *P < 0.05 for N = 6 mice / group (3 male and 3 female), using a 2-way ANOVA.  4.3.2 Trabecular bone volume is lower in 12- and 24-week-old Malt1-deficient mice  While measuring femur and tibia length, we noted that bones from Malt1-/- mice splintered more easily than bones from Malt1+/+ mice. Thus, to determine whether bones in the mice were compromised, we used a 100 µCT scanner to examine the diaphysis region and trabecular bone in femurs from 12- and 24-week-old mice. In 12-week-old mice, cortical thickness was comparable between wild-type and Malt1-/- mice (Fig. 4.2 A, top), but loss of trabecular bone was evident (Fig. 4.2 A, bottom). Quantitative analysis showed that Malt1-/- mice had significantly reduced trabecular bone volume / total volume (BV / TV) (Fig. 4.2 B). Malt1+/+ and Malt1-/- mice had comparable cortical thickness (Cort. Th) and cortical and trabecular bone mineral density (Cort. BMD and Tb. BMD) (Fig. 4.2 B). However, Malt1-/- mice had reduced trabecular number (Tb. N), and increased space between trabecular bone (Tb. Sp) compared to their wild-type littermates (Fig. 4.2 B). Like cortical thickness, trabecular thickness was unaffected by Malt1 deficiency (Fig. 4.2 B). In 24-week-old mice, cortical thickness was modestly but significantly reduced (Fig. 4.2 C, top and Fig. 4.2 D) and loss of trabecular bone was evident (Fig. 4.2 C, bottom and Fig. 2D). Malt1-/- mice had significantly reduced trabecular BV / TV compared to their wild-type littermates (Fig. 4.2 D). Cortical and trabecular bone mineral density were comparable between genotypes (Fig. 4.2 D). Trabecular number and trabecular thickness were significantly lower in Malt1-/- mice compared to their Malt1+/+ littermates, though no difference was seen in trabecular space in mice at this age (Fig. 4.2 D). In summary, our results show that loss of Malt1 reduces bone volume in Malt1-deficient mice, which is particularly evident in trabecular bone. 94   Figure 4.2 Malt1-deficient mice have reduced bone volume in trabecular bone.  Cross-sectional µCT images of cortical bone in the diaphysis region (top) and trabecular bone near the metaphysis region (bottom) from the femur of male Malt1+/+ and Malt1-/- littermates at 12 weeks of age (A) and 24 weeks of age (C). Quantitation of bone volume per total volume (BV / TV), cortical thickness (Cort. Th), cortical bone mineral density (Cort. BMD), trabecular bone mineral density (Tb. BMD), trabecular number (Tb. N), trabecular thickness (Tb. Th), and trabecular spaces (Tb. Sp) from the distal femur of male Malt1+/+ and Malt1-/- littermates at 12 weeks of age (B) and 24 weeks of age (D).  Results are expressed as the mean ± SD; *P < 0.05, **P < 0.01 for N = 3 mice per genotype (2 male and 1 female littermate pairs) using a Student’s t-test.    95  4.3.3 Malt1-/- mice have comparable number of osteoblasts in vivo We next asked whether there were differences in Malt1+/+ and Malt1-/- mice bone histomorphology. Femurs were harvested from 12- and 24-week-old mice, fixed, and decalcified with 14% EDTA for 10 days for bone histomorphometric analysis. H&E-stained longitudinal sections of distal femurs from Malt1-/- mice had less trabecular bone compared to their Malt1+/+ counterparts (Fig. 4.3 A).  We next asked whether there were differences in bone formation markers in sera from Malt1+/+ and Malt1-/- mice. There were not significant decreases in serum bone formation markers, ALP or P1NP (Fig. 4.3 B). Histomorphological analyses were used to measure the number of ALP+ cells in bone. Femurs were harvested from 12- and 24-week-old mice, fixed, and decalcified with 14% EDTA for 10 days for ALP staining. Our data demonstrated comparable levels of osteoblasts in Malt1+/+ and Malt1-/- mice (Fig. 4.3 C). Together, my data suggest that bone formation is comparable in Malt1+/+ and Malt1-/- mice. 96   Figure 4.3 Malt1-deficient mice have reduced trabecular bone and a comparable number of osteoblasts in vivo. (A) Representative images of H&E-stained paraffin sections of distal femur from Malt1+/+ and Malt1-/- mice at 12 and 24 weeks of age. (B) ALP and P1NP concentrations in sera from 12-week-old Malt1+/+ and Malt1-/- mice. (C) Representative images of ALP+ osteoblasts in paraffin-embedded sections of femurs from 24-week-old Malt1+/+ and Malt1-/- mice. Quantitation of ALP+ osteoblasts / mm2 in femurs from 12- and 24-week-old Malt1+/+ and Malt1-/- mice. Results are expressed as the mean ± SD for N = 3 mice / genotype counting 4 representative fields / section. No significant differences were observed.   4.3.4 Malt1-/- mice have higher number of osteoclasts in vivo  We next asked whether there were differences in bone resorption markers in sera from Malt1+/+ and Malt1-/- mice. There were detectable concentrations of the bone degradation marker CTX-1 in Malt1-/- mice only, though the difference between Malt1+/+ and Malt1-/- mice did not reach significance (Fig. 4.4 A).    Histomorphological analyses were used to measure the number of TRAP+ cells in bone. 97  Femurs were harvested from 12- and 24-week-old mice, fixed, and decalcified with 14% EDTA for 10 days for TRAP staining. TRAP staining revealed that there were more osteoclasts in Malt1-/- mice compared to Malt1+/+ mice (Fig. 4.4 B). Collectively, our data suggest that increased osteoclastogenesis may contribute to reduced trabecular bone in Malt1-deficient mice in vivo.    Figure 4.4 Malt1-deficient mice have increased numbers of osteoclasts in vivo.  (A) CTX-1 concentrations in sera from 12-week-old Malt1+/+ and Malt1-/- mice. (B) Representative images of TRAP-stained osteoclasts in paraffin-embedded sections of femurs from 24-week-old Malt1+/+ and Malt1-/- mice. Quantitation of TRAP+ osteoclasts (≥ 3 nuclei) / mm2 in femurs from 12- and 24-week-old Malt1+/+ and Malt1-/- mice. Results are expressed as the mean ± SD for N = 3 mice / genotype counting 4 representative fields / section; *P < 0.05 comparing Malt1+/+ and Malt1-/- using a Student’s t-test.    4.3.5 Stimulation of osteoclasts with RANKL increases Malt1 expression and activity We next asked whether Malt1 was expressed and active in bone marrow-derived osteoclasts from wild-type mice. Osteoclasts express Malt1 and Malt1 is absent in osteoclasts generated from Malt1-/- mice (Fig. 4.5 A). Malt1 protein expressed in osteoclasts was compared to that seen in macrophages, in which we have demonstrated that Malt1 is expressed and active 98  (Chapter 3). Macrophages and osteoclasts (106 cells/well) were analyzed by western blotting for Malt1, Bcl-10 (a Malt1 substrate), and Gapdh, as a loading control. Osteoclasts express more Malt1 on a per cell basis than macrophages and the presence of the Bcl-10 cleavage product suggests that Malt1 is constitutively active in osteoclasts (Fig. 4.5 A). Malt1 activity in osteoclasts was also verified in a fluorogenic assay [170, 231]; Malt1 activity is detectable in osteoclasts and its specificity was further confirmed using the Malt1 inhibitor, mepazine acetate (MPZ) (Fig. 4.5 B). A key difference in the differentiation protocol for macrophages and osteoclasts is the inclusion of RANKL during osteoclast differentiation. To determine whether RANKL induced Malt1 via inducing Malt1 transcription, we titrated RANKL during osteoclast differentiation for 7 days and performed qPCR on mRNA from osteoclasts. RANKL induced Malt1 mRNA in osteoclasts and induction was dose-dependent (Fig. 4.5 C). Differentiation in higher concentrations of RANKL also induced more Malt1 protein expression and activity (indicated by Bcl10 cleavage) in osteoclasts in a dose-dependent manner (Fig. 4.5 C). We also stimulated preosteoclasts with 40 ng/mL RANKL in a short time course (0-24 hours) to determine whether RANKL up-regulates Malt1 mRNA and protein expression. Malt1 mRNA and protein expression were induced in response to RANKL stimulation (Fig. 4.5 D). Taken together, these data demonstrate that Malt1 is expressed and active in osteoclasts, and that Malt1 expression is up-regulated by RANKL, which is required for osteoclast differentiation.    99   Figure 4.5 Malt1 is expressed and active in osteoclasts and Malt1 mRNA and protein expression is induced by RANKL.  A) Whole cell lysates (2.5´106 cells / lane) of bone marrow-derived osteoclast from Malt1+/+ and Malt1-/- mice were analyzed by western blotting for Malt1 and Gapdh, as a loading control, and whole cell lysates (2.5´106 cells / lane) of bone marrow-derived macrophages (Mϕ) and bone marrow-derived osteoclasts (OSC) were analyzed by western blotting for Malt1, Bcl10, and Gapdh, as a loading control. Densitometry for Malt1 and the Bcl10 cleavage product (lower band), relative to Gapdh, are shown below each panel. (B) Whole cell lysates were prepared from bone marrow-derived osteoclasts (2.5´106 cells) and Malt1 was immunoprecipitated to assay its activity in osteoclasts (OSC, closed circles), no lysis buffer control (C, closed squares), and osteoclasts + the Malt inhibitor, MPZ (25 µM) (OSC+MPZ, open circles). (C) Bone marrow-derived osteoclast precursors were differentiated with 0-100 ng/mL RANKL. Gene expression for Malt1 mRNA normalized to Gapdh was measured by qPCR. Whole cell lysates (2.5´106 cells / lane) were analyzed by western blotting for Malt1, Bcl-10, and Gapdh, as a loading control. Densitometry for Malt1 and the Bcl10 cleavage product, relative to Gapdh, are shown below each panel. (D) Bone marrow osteoclast precursors were stimulated with 40 ng/mL RANKL for 0, 2, 4, 8, and 24 h. Gene expression for Malt1 mRNA normalized to Gapdh was measured by qPCR.  Whole cell lysates (5´106 cells / lane) were analyzed by western blotting for Malt1 and Gapdh, as a loading control. Denistometry for Malt1 relative to Gapdh, is shown below the panel. Results are expressed as the mean ± SD; *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001 100  for N = 4 for (C) and N = 3 for (D) using a 1-way ANOVA.  4.3.6 Malt1 deficiency does not impact osteoclastogenesis  We compared osteoclastogenesis in vitro by differentiating bone marrow aspirates from Malt1+/+ and Malt1-/- mice in the presence of 0-80 ng/mL of RANKL. There were no significant differences in the number of osteoclasts generated from Malt1+/+ and Malt1-/- at any concentration of RANKL even when using sub-optimal concentrations of RANKL (Fig. 4.6 A). These data suggests that there is no cell intrinsic effect of Malt1 deficiency in osteoclasts that leads to increased numbers of osteoclasts in vivo in Malt1-deficient mice. We next enumerated osteoclasts generated in the presence of 40 ng/mL of RANKL from bone marrow precursors from Malt1+/+ and Malt1-/- littermates at 4, 6, 8, 12, and 24 weeks of age and found that there were no significant differences in the number of osteoclasts generated from each genotype at any age (Fig. 4.6 B).    Figure 4.6 Malt1 deficiency does not affect osteoclast differentiation in response to RANKL.  (A) Representative images of Malt1+/+ and Malt1-/- bone marrow-derived osteoclasts generated by differentiation in the presence of 0-80 ng/mL RANKL (0, 2, 10, 20, 40, or 80 ng/mL). Osteoclasts were quantitated for each condition. (B) Representative images of TRAP-stained Malt1+/+ and Malt1-/- bone marrow-derived osteoclasts. Quantitation of osteoclasts derived from 4-, 6-, 8-, 12-, and 24-week-old Malt1+/+ and Malt1-/- mice. Results are 101  expressed as mean ± SD for N = 3 mice / genotype for (A) and N = 6 mice / genotype for (B) by counting 4 different representative fields / condition in each experiment using a 2-way ANOVA. No significant differences were observed.  4.3.7 Osteoclast activity is not affected by Malt1 deficiency  We next measured the resorptive activity of Malt1+/+ and Malt1-/- osteoclasts in vitro. Osteoclasts (5 ´ 103) were lifted and re-plated on hydroxyapatite-coated wells. After 7 days, wells were stained with toluidine blue and cleared areas were quantitated using ImageJ. There was no significant difference in osteoclast activity in this assay comparing Malt1+/+ and Malt1-/- osteoclasts (Fig. 4.7 A). Malt1+/+ and Malt1-/- osteoclast activity was also assessed on bone slices (Fig. 4.7 B). Osteoclasts (5 ´ 104) were lifted and plated on bone slices for 6 days. Resorption pits were stained by toluidine blue and quantitated using ImageJ (Fig. 4.7 B). The bone resporption marker, CTX-1, was assayed in culture supernatants by ELISA. There was no significant difference in resorbed area or CTX-1 released comparing Malt1+/+ to Malt1-/- osteoclasts (Fig. 4.7 C). Taken together, these data suggest that there is no cell-intrinsic role for Malt1 in osteoclast activity.   102   Figure 4.7 Malt1 deficiency does not affect activity of bone marrow-derived osteoclasts.  (A) Representative images of cleared area formed on hydroxyapatite incubated with Malt1+/+ and Malt1-/- bone marrow-derived osteoclasts for 7 days. Quantitation of cleared areas measured after staining with toluidine blue. (B) Representative images of Malt1+/+ and Malt1-/- TRAP-stained osteoclasts on bone slices cultured for 6 days (upper panel) and toluidine blue-stained resorption pits on bone slices (lower panel) at 10x magnification. Quantitation of Malt1+/+ and Malt1-/- osteoclasts derived from 12- and 24-week-old mice on bone slices. Quantitation of resorption pits from bone slices cultured with bone marrow-derived osteoclasts derived from 12- and 24-week-old Malt1+/+ and Malt1-/- mice after cells were removed and bone slices were stained with toluidine blue. (C) CTX-1 assayed from supernatant of bone slices cultured with osteoclasts derived from 12- and 24-week-old Malt1+/+ and Malt1-/- mice. Results are expressed as mean ± SD for N = 6 mice / genotype for (A) and N = 3 mice / genotype for (B) and (C) measuring 4 representative fields / slice and quantified using ImageJ. No significant differences were observed.   103  4.3.8 Blocking Malt1 activity induces MCSF production and reduces OPG production by macrophages  MCSF and RANKL play crucial roles in bone remodelling by driving osteoclast development and function [87, 244-246]. In contrast, OPG acts as a decoy receptor for RANKL and suppresses osteoclast differentiation and activity [88, 244, 247].  Thus, we measured serum concentrations of MCSF, RANKL, and OPG in 12-week-old Malt1+/+ and Malt1-/- mice. MCSF was not detectable in serum, likely because it acts rapidly on abundant CSFR1 receptors; RANKL concentrations were not different between Malt1+/+ and Malt1-/- mice; however, serum OPG concentrations were lower in Malt1-/- mice compare to their Malt1+/+ littermates (Fig. 4.8 A). Macrophages are a source of both MCSF and OPG [248, 249] and we have shown in Chapter 2 that Malt1-deficient macrophages are hyper-responsive to innate immune stimuli. Thus, we stimulated wild-type macrophages with LPS, zymosan (Zym), or curdlan (Cur) ± the Malt1 inhibitor, MPZ, and measured MCSF and OPG production. Inhibition of Malt1 activity caused LPS, zymosan, and curdlan to induce MCSF production by macrophages (Fig. 4.8 B). Inhibition of Malt1 activity also reduced LPS, zymosan, or curdlan-induced OPG production (Fig. 4.8 C). Together, this data suggests that deficiency in Malt1 proteolytic activity in activated macrophages may contribute to higher osteoclast numbers in Malt1 deficiency by producing more MCSF to drive osteoclastogenesis and osteoclast activity and less OPG, the endogenous inhibitor of osteoclastogenesis and activity. 104   Figure 4.8 Inhibiting Malt1 in macrophages increases MCSF production and decreases OPG production in response to inflammatory stimuli.  (A) MCSF, RANKL, and OPG concentrations in sera from 12-week-old Malt1+/+ and Malt1-/- mice. (B) MCSF and (C) OPG production by Malt1+/+ (wild-type) macrophages stimulated with 10 ng/mL LPS (left), 10 µg/mL zymosan (middle), or 100 µg/mL curdlan (right) for 24 h in the presence of 0-50 µM concentrations of the Malt1 inhibitor, MPZ, versus vehicle control (V) or left unstimulated in the presence of 50 µM MPZ to test the effect of MPZ independent of inflammatory stimuli. Results are expressed as the mean ± SD; *P < 0.05, **P < 0.01 for N = 3 independent experiments using 1-way ANOVA and 2-way ANOVA.   4.3.9 MCSF increases, and OPG decreases, osteoclast differentiation in vitro, but this is independent of Malt1 deficiency in osteoclasts Finally, we asked whether MCSF and OPG affect osteoclast differentiation in vitro and whether they impact Malt1-/- osteoclasts more profoundly. Osteoclasts were differentiated from bone marrow progenitors (106) in the presence of RANKL (40 ng/mL) ± MCSF (0.2, 2, 5, 10, or 20 ng/mL). MCSF was required for osteoclast differentiation and the number of osteoclasts 105  generated correlated with MCSF provided (Fig. 4.9 A). This was independent of the Malt1 genotype in osteoclasts. Osteoclasts were also generated in the presence of RANKL (40 ng/mL), MCSF (20 ng/mL) ± OPG (1, 10, 20, 50, or 100 ng/mL). OPG inhibited osteoclast differentiation, and inhibition was also independent of the osteoclast Malt1 genotype (Fig. 4.9 B). This data demonstrates that MCSF and OPG concentrations dramatically influence osteoclast differentiation, but differences observed are not affected by Malt1 deficiency in osteoclasts.  Figure 4.9 MCSF enhances and OPG inhibits osteoclast differentiation in vitro, in both Malt1+/+ and Malt1-/- osteoclasts.  (A) Representative images of Malt1+/+ and Malt1-/- bone marrow-derived osteoclasts generated by differentiation in the presence of 0-20 ng/mL MCSF (0, 0.2, 2, 5, 10, 20 ng/mL). Osteoclasts were quantitated for each condition. (B) Representative images of Malt1+/+ and Malt1-/- bone marrow-derived osteoclasts differentiated in the presence of 0-100 ng/mL OPG. Osteoclasts were quantitated for each condition. Results are expressed as mean ± SD; **P < 0.01, ****P < 0.0001 for N = 3 mice / genotype in 3 independent experiments counting 4 different representative fields / condition in each experiment using a 2-way ANOVA.  4.4 Discussion  It has previously been shown that a patient with CID caused by severe, but not ablative, 106  MALT1 deficiency developed osteoporosis [21]. Along with her immune phenotype, she had pathologic fractures associated with low bone mineral density, both of which improved post-HSCT [25]. Consistent with that, our data shows that Malt1-deficient mice have reduced bone volume and trabecular bone, which is evident at 12 and 24 weeks of age. They have an elevated number of osteoclasts in vivo but Malt1 deficiency does not have any cell-intrinsic effect on osteoclast differentiation or activity. Rather, mice had a modest, but significant, reduction in OPG in their serum. We have previously shown that Malt1-deficient macrophages are hyper-responsive to inflammatory stimuli (Chapter 2). Indeed, blocking Malt1 activity increased macrophage MSCF production and reduced macrophage OPG production in response to inflammatory stimuli, both of which could contribute to increased osteoclast numbers present in Malt1-deficient mice. In vitro, osteoclast differentiation increased in response to MCSF in a dose-dependent manner and was inhibited by OPG. However, Malt1 deficiency in osteoclasts did not impact MCSF-induced or OPG-inhibited osteoclast differentiation. Together, this data demonstrates that Malt1-/- mice develop osteoporosis, which is independent of osteoclast-intrinsic effects of Malt1 deficiency. The patient with MALT1 deficiency developed clinical features of immunodeficiency beginning with an eczematous rash at 2 weeks of age that continued as persistent dermatitis [21]. She had multiple infections, which may have contributed to chronic inflammatory lung disease, and she had inflammation along her gastrointestinal tract that caused multiple and severe complications [21]. Her height and weight were below the 5th percentile by 8 and 9 years of age, respectively [25]. Though Malt1-deficient mice do not develop spontaneous intestinal inflammation, in Chapter 2 we found that they are more susceptible to DSS-induced colitis suggesting an increased inflammatory tone in young mice. Low bone volume was evident in 107  Malt1-deficient mice by 12 weeks of age. Bone age in mice is different from their reproductive adulthood. For bone age, 5-month-old mice are young to adult, 12-month-old mice are middle-aged, and 22-month-old mice are considered in ‘old age’ [250]; thus, 12- and 24-week-old mice are young to adult mice. Delayed development of the bone phenotype in mice may reflect different stages of bone development in mice and in humans as well as the chronological time required for bone turnover. However, because our data suggest that the bone phenotype associated with Malt1 deficiency is driven by inflammation, the profound inflammatory phenotype of the MALT1-deficient patient likely contributed to her early onset osteoporosis. HSCT has been used to treat two patients with CID caused by Malt1 deficiency, the patient described here and an 18-month-old male [23, 25]. Though this led us to investigate the cell-intrinsic role of Malt1 in myeloid-derived osteoclasts, HSCT not only resolved the low bone mineral density in our patient, but it also corrected the immune defect and severe inflammatory phenotype seen in both patients [23, 25]. Indeed, our macrophage data demonstrate that inhibiting Malt1 paracaspase activity increases induced MCSF production and decreases OPG production, which may contribute to osteoclast differentiation in vivo and be particularly relevant to the patient with non-ablative Malt1 deficiency and inflammation [21]. Inflammatory macrophages and macrophage-derived cytokines and chemokines have been implicated in inflammatory diseases of the bone [155, 251, 252]. In addition, B cells are another major source of OPG [253]. The MALT1-deficient patient had B cell lymphopenia and impaired B cell development, which were corrected by HSCT [21, 25]. Malt1 deficiency in mice is also associated with impaired B cell activating factor-induced B cell survival and [254] and impaired B cell receptor-induced activation [164]. Thus, reduced OPG concentrations in Malt1-/- mouse serum may result from both B cell deficiency and the inflammatory status of macrophages. 108  Finally, Malt1 is ubiquitously expressed and may play a role in osteoblast activity, including the production of RANKL and OPG. RANKL was not up-regulated in serum from Malt1-/- mice but we cannot rule out the possibility that Malt1 deficiency in osteoblasts causes or contributes to increased concentrations of RANKL, or decreased production of OPG, in the bone microenvironment driving osteoclast differentiation. However, in the MALT1-deficient patient, osteoblasts would not be impacted by bone marrow transplantation. Rather, our data are consistent with a model in which immune cell-mediated inflammation associated with MALT1 deficiency drives osteoclastogenesis in vivo resulting in poor bone density and increased risk of fractures.   Malt1 is expressed and constitutively active in osteoclasts, which is evident by western blotting of Bcl10 (and its Malt1 cleavage product), as well as mepazine-inhibitable Malt1 activity measured in vitro. Malt1 paracaspase is also constitutively active in macrophages (Chapter 3) but is only induced in T cells after activation of the T cell receptor with PMA/ionomycin [173]. There are clearly distinct roles for Malt1 scaffolding function and paracaspase activity in inflammation. Paracaspase-deficient mice develop spontaneous inflammatory disease whereas Malt1-/- mice do not [173, 197, 198]. Mice deficient in paracaspase activity do significantly worse during DSS-induced colitis and we showed in Chapter 2 that Malt1-/- mice are also more susceptible to DSS-induced colitis [173], but inhibition of Malt1 paracaspase activity protects mice during DSS-induced colitis [214]. This apparent paradox may be caused by systemic deficiency in regulatory T cells in the germline paracaspase-deficient mice [173, 197, 198] and/or innate immune defects causing bacterial overgrowth or dysbiosis [255]. In T and B cells, paracaspase deficiency in the presence of Malt1 scaffolding function exacerbates inflammation, suggesting a critical role for Malt1 scaffolding 109  function in driving NF-κB activation, and paracaspase activity in regulating or setting a threshold for that activation in T and B cells [173, 197, 198]. Here, we have found that the loss of activity but not complete loss of the protein increased inflammation-induced MCSF production and reduced OPG production by macrophages. As in T and B cells, paracaspase activity may regulate Malt1-driven NF-κB activation in macrophages that would contribute to pathological osteoclastogenesis. We noted that Malt1 expression was higher in osteoclasts than in macrophages, on a per cell basis, and we found that RANKL induced Malt1 mRNA expression and protein concentrations in a dose-dependent manner. By contrast, in macrophages, LPS, zymosan, or curdlan induce protein concentrations but eliminate Malt1 paracaspase activity (Chapter 3). Thus far, the role of increased Malt1 expression in myeloid cells remains enigmatic. In osteoclasts, induction of Malt1 increased Bcl10 cleavage, suggesting that induction can up-regulate Malt1 paracaspase activity in this cell type, in which Malt1 paracaspase is constitutively active. The creation of cell-specific Malt1 knockout mice and paracaspase-deficient mice will be useful to determine the cell-intrinsic roles of Malt1 concentrations in inflammatory processes and osteoclastogenesis. Systemic osteoporosis and increased fracture rates have been associated with many chronic inflammatory diseases, including rheumatoid arthritis, spondyloarthtritis, and systemic lupus erythromatous. It was recently demonstrated that the Malt1 paracaspase inhibitor, MI-2, protects mice against collagen-induced arthritis [235].  Moreover, the authors demonstrated that MI-2 inhibited the differentiation of PBMC-derived preosteoclasts to osteoclasts in vitro [235]. In contrast, our data showed no difference in bone marrow-derived osteoclast differentiation comparing wild-type to Malt1-deficient cells. This may reflect differences in cell source (bone marrow versus blood monocytes), deficiency of both Malt1 scaffolding function and paracaspase 110  activity in our study versus paracaspase activity alone, or off-target effects of the MI-2 inhibitor that are independent of Malt1. Indeed, the effects of MI-2 were not assessed in Malt1-deficient cells and MI-2 has recently been shown to have non-specific effects [236]. Meloni et al. (2018) recently reported that MPZ, a structurally distinct Malt1 inhibitor, also inhibits RANKL-induced osteoclastogenesis but its activity was independent of its inhibitory effect on Malt1 because it performed equally well in Malt1-deficient cells [256]. These results are consistent with our data, which demonstrate that there is no cell intrinsic role for Malt1 in osteoclastogenesis. Identification of the additional MPZ target(s) that inhibit osteoclastogenesis may provide insight into RANKL-induced osteoclast differentiation and novel therapeutic targets to inhibit osteoclast development during disease.      In summary, our data suggests that Malt1 deficiency does not have a direct effect on osteoclast differentiation or activity in Malt1-/- mice. Rather, inflammation causes an up-regulation of osteoclastogenesis in vivo that leads to reduced bone density in mice. Thus, the extreme bone fragility in the patient with MALT1 deficiency may have resulted from her dramatic and life-long inflammatory pathologies.       111  Chapter 5: Conclusions and future directions  5.1 Conclusions  CID can be a fatal condition characterized by increased susceptibility to opportunistic infections. In humans, CID is associated with aberrant expression of genes necessary for immune system development and function. MALT1 deficiency is a novel cause of CID with severe clinical symptoms including susceptibility to infection, dermatitis, inflammation of the respiratory and gastrointestinal tract as well as osteoporosis [21]. Dysfunction of the adaptive immune response is considered a primary cause of Malt1 deficiency-associated CID. However, Malt1 is ubiquitously expressed and is present in both adaptive and innate immune cells. Despite being expressed by myeloid cells, the roles of Malt1-deficient macrophages and osteoclasts in CID have not been investigated. This thesis addresses three questions: (1) Does Malt1 play a role in macrophage inflammatory responses that can exacerbate DSS-induced colitis in Malt1-deficient mice? (2) Does aberrant Malt1 gene expression contribute to intestinal inflammation in people with IBD? (3) Does Malt1 play a role in osteoclast proliferation and/or activity that contributes to osteoporosis in Malt1-deficient mice? In contrast to humans, Malt1-deficient mice do not develop any clinical phenotype at a young age [164, 196]. However, I demonstrate that Malt1-/- mice have an increased inflammatory tone that leads to increased susceptibility to DSS-induced colitis and Malt1-/- mice develop osteoporosis. Moreover, I have attributed both of these phenotypes to increased macrophage activation associated with Malt1 deficiency. In Chapter 2, the role of macrophage IL-1β release and IL-1 signalling in susceptibility to DSS-induced colitis in Malt1-/- mice has been investigated. I found that IL-1β concentrations are increased in Malt1-/- mice with exacerbated 112  DSS-induced colitis, which is accompanied by increased disease activity and histological damage. By depleting macrophages or blocking IL-1 signalling, DSS-induced colitis in Malt1-/- mice is ameliorated. Furthermore, I found that increased IL-1β is caused by increased transcription of Il1b and not actually by increased activation of inflammasomes. In Chapter 3, I investigated MALT1 mRNA expression in individuals with IBD and then in vivo in mice during DSS-induced colitis. These questions arose because I found that macrophages activated with innate immune stimuli in vitro up-regulate Malt1 mRNA and protein expression but reduce Malt1 activity. I also found that blocking Malt1 activity by mepazine increases pro-inflammatory cytokine production by macrophages. Despite those interesting in vitro observations, I found that expression of MALT1 mRNA is similar in the intestine of subjects with IBD compared to healthy control subjects. Malt1 expression was also not changed in mice during DSS-induced colitis compared to mice that did not receive DSS.  In Chapter 4, I found that 12- and 24-week-old Malt1-/- mice have reduced bone volume and trabecular bone. Malt1-/- mice have more osteoclasts in vivo but Malt1 deficiency does not have any cell-intrinsic effect on osteoclast differentiation or resorption activity in vitro. Rather, Malt1-/- mice have a lower serum concentration of OPG, a negative regulator of osteoclast differentiation. In Chapter 2, I showed that Malt1-deficient macrophages are hyper-responsive to innate immune stimuli, and here I demonstrate that blocking Malt1 activity increases macrophage MCSF production and reduces macrophage OPG production in response to inflammatory stimuli. Together, this data suggests that increased MCSF and reduced OPG production by activated inflammatory macrophages may contribute to increased osteoclastogenesis in vivo and osteoporosis in Malt1-/- mice. 113  Altogether, my data indicates that, similar to patients deficient in MALT1, mice deficient in Malt1 have inflammation and develop some features of CID. Unlike MALT1-deficient patients with severe inflammation in the GI tract, gastrointestinal inflammation does not develop spontaneously in Malt1-/- mice, but my data shows that DSS-induced colitis is exacerbated in these mice. Furthermore, I found that osteoporosis develops in adult Malt1-/- mice, which is consistent with delayed growth and low bone density of the patient at BC Children’s Hospital, who was diagnosed with MALT1 deficiency. My data suggest that osteoporosis detected in adult Malt1-deficient mice is caused by inflammation. Osteoporosis is one of the most common features of inflammation associated with IBD and more than half of individuals with IBD show significant reduction of bone density [257]. Interestingly, it has been shown that development of osteoporosis during IBD is associated with alterations in the RANKL/OPG system, which is mediated by colonic macrophages and DCs [258]. This is consistent with my data showing the role of macrophages in regulation of OPG in Malt1-/- mice may contribute to development of osteoporosis in these mice. Life expectancy in patients with MALT1 deficiency-associated CID is increased following HSCT. HSCT is used to treat conditions such as leukemia, different types of lymphoma, and immunodeficiencies [259, 260]. The first HSCT was performed a half century ago to treat a 5-month old patient with primary immunodeficiency [259]. HSCT procedures include infusion of collected stem cells from donor bone marrow or peripheral blood into the recipient. Subsequently, the healthy transplanted cells can boost the recipient patient’s immune response by providing immune cells that effectively express specific molecular components that are absent in the recipient. HSCT reconstitutes both myeloid and lymphoid progenitors in the recipient. However, the phenotype and proliferative capacity of different subsets of HSC is 114  variable [261]. Recovery of innate immune cells occur in the first few months post-HSCT, starting with monocytes, followed by granulocytes and NK cells, while recovery of adaptive immune cells requires more than 1-2 years [262-264]. Indeed, the rash associated with the severe inflammatory phenotypes in the 18-month old patient with MALT1 deficiency was resolved within a month after transplant, suggesting a key role for innate immune cells in protection from systemic effects [23]. Moreover, bone density was improved in the patient form BC Children’s Hospital with MALT1-deficient-associated CID 6 months post-HSCT [25]. These observations demonstrate the critical role innate immune cells play in the improved clinical phenotype with regard to inflammation and bone health in patients with MALT1 deficiency-associated CID following HSCT.  Here, I show that innate immune cells such as macrophages and osteoclasts play an important role in Malt1-deficient mice susceptibility to DSS-induced colitis and osteoporosis. I found that Malt1-deficient macrophages are hyper-responsive and can contribute to exacerbated DSS-induced colitis in Malt1-deficient mice. Furthermore, increased in vivo osteoclast numbers may play a role in the osteoporotic phenotype of adult Malt1-deficient mice, though Malt1 does not have any direct effect on osteoclast differentiation or function. Indeed, blocking Malt1 activity increased production of MCSF while decreasing OPG production by macrophages after incubation with innate immune stimuli. I confirmed that increasing concentrations of MCSF and lower doses of OPG can both contribute to osteoclast proliferation. However, Malt1 does not play any cell intrinsic role in osteoclast proliferation in response to these factors. Thus, increased MCSF and decreased OPG production induced by blocking Malt1 activity with mepazine in macrophages may contribute to increased osteoclast numbers and osteoporosis that occurs in adult Malt1-deficient mice.  115  Mepazine belongs to a family of chemical compounds called phenothiazines. These components are characterized by a shared tricyclic phenothiazine ring but varied substitutions in side chains that regulate inhibitory effects of different derivatives. As phenothiazines have modulatory effects on dopamine, serotonin, and histamine receptors, they are primarily used to treat psychotic disorders and allergies [265, 266]. It has also been shown that mepazine blocks Malt1 activity and it has been recommended for use in the treatment of autoimmune diseases and B cell mediated ABC-DLBCL [199, 231]. However, recent reports suggest that mepazine does not have optimal potency and selectivity and may not be the best candidate to block Malt1 activity [236, 256]. MI-2 is another inhibitor that binds to the Malt1 active site, which has been shown to protect mice against rheumatoid arthritis and colitis [235, 267], but there is a study that reports MI-2 is a non-selective inhibitor of Malt1 because it is also effective against other proteases such as cathepsins and adenain [236]. A small molecule that is used as a Malt1 inhibitor is Z-VRPR-fmk, which is a peptide substrate analog derived from plants [170]. Z-VRPR-fmk also has off target effects and requires a high dose for efficacy, limiting its application in therapeutic approaches [236]. Recently, other inhibitors (MLT-748 and MLT-747) have been generated that bind to the MALT1 Trp580 residue [268], a critical residue which is mutated to a serine (W580S) in a MALT1-deficient patient, leading to loss of MALT1 protein expression [21]. MLT-748 and MLT-747 inhibit wild-type MALT1 protease activity and restore MALT1 protein expression in MALT1-deficient B cells and primary T cells by acting as a bridge to connect the two domains of MALT1 protein in these cells [268]. However, the effects of these molecules in animal models in vivo (or clinical trials) have not been assessed. Malt1 is a ubiquitously expressed gene and my data shows that loss of Malt1 has diverse effects on proliferation and activity of different cell types. In addition, Malt1 acts both as a scaffolding 116  molecule and a proteolytic enzyme, with complementary or opposing effects in immune cell responses [176]. The side effects of Malt1 inhibitors may be due to the distinct cell specific roles of Malt1 scaffolding function and proteolytic activity, the involvement of Malt1 in various signal transduction pathways, and opposing effects of Malt1 on the activation of different cell types. Ideally, novel inhibitors would be designed that could specifically target Malt1 activity in a single cell type. My data shows that blocking Malt1 activity with mepazine down-regulates IL-1b production by MCSF-derived bone marrow macrophages but release of IL-1b is up-regulated in Malt1-deficient MCSF-derived bone marrow macrophages. It has been shown that decreased production of IL-1β by MCSF-derived bone marrow macrophages is caused by impaired inflammasome activation [214], while here I demonstrate that increased production of IL-1b by MCSF-derived bone marrow macrophages is due to augmented transcription of IL1b. This is consistent with increased levels of IL1b mRNA observed in Malt1-deficient mouse colon homogenates during DSS-induced colitis. IL-1b is an effective mediator of inflammation and its effect is blocked by Anakinra, an IL-1 receptor antagonist. My results show that DSS-induced colitis is ameliorated in Malt1-deficient mice after treatment with Anakinra. Anakinra is a pharmacological drug approved by the US Food and Drug Administration (FDA) for treatment of rheumatoid arthritis in humans [269]. Our group has previously shown that Anakinra can prevent CD-like intestinal inflammation in SHIP-deficient mice by reducing up-regulation of IL1b mRNA in ileal tissues [215]. These findings suggest that Anakinra could also be used to delay or ameliorate intestinal inflammation in humans with IBD.  In addition to Anakinra, other drugs are also available to block IL-1b signalling pathways. These IL-1b targeting drugs that are approved for clinical use include rilonacept, 117  canakinumab, and gevokizumab. Rilonacept is a soluble decoy receptor for IL-1 (cytokine trap) and can bind to extracellular residues of IL-1 for long-term treatment [270]. Canakinumab and gevokizumab are well-tolerated, highly specific, human monoclonal antibodies that neutralize IL-1b but are not cross-reactive with other cytokines [271]. Among all IL-1b blockade treatments, Anakinra is considered the preferred drug in IL-1 therapeutics due to its impeccable safety record in long-term treatment and its short half-life in vivo [272]. These drugs are currently being used in both pre-clinical research and clinical trials for treatment of autoimmune and inflammatory disease in which IL-1b plays a critical role. The mechanism of action of all these drugs is dependent on blocking IL-1b signaling rather than preventing production of IL-1b. Inhibition of IL-1b production may be a more effective approach for preventing IL-1b-mediated disease because it targets an earlier stage of inflammation relative to blocking IL-1b receptors. Current research is focussing on designing a new type of drug targeting production of IL-1b. The histone deacetylase inhibitor (ITF2357) reduces production of inflammatory cytokines in vitro and has anti-inflammatory effects in vivo [273]. However, this inhibitor is not specific for IL-1b and blocks production of other inflammatory cytokines as well; so, may not be an appropriate candidate for conditions mediated primarily by IL-1b. A future strategy to block IL-1b production could be to silence Il1b gene synthesis using gene therapy, which has made tremendous strides over the last two decades.    5.2 Future directions  The goal of this research was to understand the role of Malt1-deficient myeloid cells in pathogenesis of CID and to recognize whether this condition is associated with innate immune dysfunction. I found that Malt1-deficient macrophages are hyper-responsive that leads to 118  increased susceptibility of mice to DSS-induced colitis. However, Malt1 deficiency does not have any cell intrinsic effect on osteoclasts activity and differentiation in mice. Rather, inflammation is a pathway to osteoporosis in Malt1-deficient mice, through up-regulation of osteoclastogenesis in vivo. This suggests that development of osteoporosis in MALT1-deficient patients may be caused by their chronic and life-long inflammatory condition, which affects different organs, including bones. For people with CID and other chronic inflammatory disease, this can be further complicated by treating inflammation with glucorcorticoids, the leading cause of secondary osteoporosis [274]. Preferred treatment for bone-related pathologies that accompany chronic inflammatory diseases would ideally include effective and specific anti-inflammatory therapy (glucocorticoid-sparing) complemented with drugs targeting RANKL. Treatment with OPG or a synthetic analogue could be a great candidate for inhibiting osteoporosis in these patients. Recombinant fc-OPG protein has been used to block RANKL in postmenopausal women with osteoporosis and can effectively block bone resorption in these patients [275].  However, a new antibody that specifically targets RANKL, Denosumab, has recently been developed and approved for the treatment of osteoporosis [276, 277]. Denosumab acts similarly to OPG and profoundly blocks bone resorption, increases bone mineral density, and reduces fracture risk [276, 277]. Studies show that Denosumab is superior to Fc-OPG as it is more potent in decreasing bone turnover markers at lower doses and is effective for longer period of time [278]. Thus, Denosumab or similar strategies targeting the RANK/RANKL/OPG axis could be effectively used to treat osteoporosis in people with CID and/or chronic inflammation. In future studies, Fc-OPG or Denosumab could be provided to Malt1-deficient mice and their bone phenotype could be assessed as I have done in Chapter 4.  This would provide a pre-clinical model to test the potential efficacy of these drugs for treating osteoporosis 119  in people with CID caused by Malt1 deficiency and more generally, for people with osteoporosis secondary to chronic inflammatory conditions, like IBD.  To assess the role of Malt1-deficient macrophages in susceptibility of mice to DSS-induced colitis, my approach in this study was to deplete macrophages using clodronate liposomes. My data show that depleting macrophages can ameliorate DSS-induced colitis in Malt1-deficient mice, which I predict would ameliorate long-term effects on bone health in the mice as well. Clodronate liposomes belongs to a family of bisphosphonates that are widely used to treat various bone related disease. In addition to depleting macrophages, clodronate liposomes can induce osteoclast apoptosis and inhibit osteoclast resorption in vitro [279]. This has interesting potential for future research as clodronate liposomes that reduce macrophage-mediated inflammation are also predicted to have direct beneficial effects on osteoporosis by inhibiting osteoclasts. My data suggests an important role for hyper-responsive macrophages in increased number of osteoclasts detected in osteoporotic Malt1-deficient mice. Interestingly, in humans, depleting leukocytes or adsorption of granulocytes/monocytes in individuals with IBD have been shown to be effective in reducing inflammation [279-281]. Further studies are required to assess the effect of macrophage and osteoclast depletion in patients with CID as well as targeting each cell type individually to examine its potential role in reducing osteoporosis associated with this disease. In future studies, Malt1-deficient mice can be injected with clodronate liposome, as I have done in Chapter 2, to test its effect on osteoclast depletion and assess the possible indirect effect of macrophages on bone phenotype in Malt1-/- mice. This would provide insight into the potential efficacy of this sort of therapeutic approach for people with CID caused by Malt1 deficiency or inflammation-mediated osteoporosis.  120  In addition to treatment of IBD, depleting macrophages has other clinical applications. It has been shown that depleting macrophages can also be used as mobilizing agent before HSCT, as bone marrow macrophages sustain HSCs in the bone niche [282]. Currently, granulocyte colony stimulating factor (G-CSF) is the most commonly used mobilizing agent to force HSC into the blood. However, use of G-CSF has side effects including inducing osteoblast apoptosis and suppressing bone formation [283]. Considering that low bone density is one of the features of MALT1 deficiency in patients, it would be beneficial to use mobilizing agents that do not further compromise bone integrity. Depleting macrophages could effectively mobilize HSC into the blood stream while preventing loss of osteoblasts [282]. Macrophage depletion could be used prior to HSCT in patients with MALT1 deficiency as both a therapeutic treatment and a “bone protective” mobilizing agent. In future studies, clodronate liposomes can be injected into Malt1-deficient recipient mice as I have done in Chapter 2, followed by transplantation of HSCs into irradiated Malt1-/- recipients, to assess the mobilization of donor reconstituted HSCs after transplantation. This would provide a pre-clinical model to test the possibility of using macrophage depletion as a mobilizing agent prior to HSCT in people with CID caused by Malt1 deficiency. The long-term goal of this study was to understand how to apply this knowledge in therapeutic approaches. Nevertheless, some limitations associated with this study regarding the intrinsic role of Malt1-deficient myeloid cells in mice should be addressed prior to translating this work to patients. One caveat in treatment of patients with CID is the role of multiple immune system components in their pathology. Therefore, it would be challenging to study the role of one factor in their immune response exclusively. Consistent with that, my data do not rule out the possibility that Malt1 deficiency in other cell types has effects on the inflammatory tone of 121  Malt1-deficient mice as Malt1 is expressed ubiquitously. To better recognize the intrinsic role of Malt1 in myeloid cells, mice with Malt1 gene deletion only in myeloid cells should be generated. The Cre/loxP system is used for site-specific genetic manipulation in mice. One can develop cell or tissue specific knockout mice with conditional deletion of the target gene using Cre recombinase in floxed mice. Indeed, this method is particularly useful for genetic modulation in the monocyte/macrophage lineage [284]. Basically, the target gene is inserted between the two loxP sites and the promoter. Crossing of loxP mice with Cre mice leads to deletion of the target gene in Cre/loxP conditional KO mice progeny [285, 286]. Thus, in future studies, it would be interesting to generate conditional Malt1-/- mice using the Cre/loxP system to thoroughly assess the specific role of Malt1 deficiency in myeloid cells, independent of other immune factors. Furthermore, as Malt1 deficiency affects multiple organs, generation of mice with site-specific deletion of the Malt1 gene in tissues with severe inflammation in patients with MALT1 deficiency would also be helpful. In future studies, Tie2-Cre mice (for mapping hemopoietic cells), LysM-Cre mice (for mapping the myeloid cell lineage), CD11b-Cre mice (for mapping BMDMs and osteoclasts), and Csf1r-Cre mice (for mapping tissue macrophages, for example, intestinal macrophages) can be purchased from Jackson Laboratories [287]. These transgenic mice could be crossed with Malt1fl/fl mice, generated by flanking the Malt1 gene with loxP. All mice with cell-specific Malt1 gene deletion could be compared with each other, as well as with their Cre-negative floxed mice littermates. This would provide experimental models to test cell specific roles of Malt1 in hematopoietic cells, myeloid cells, BMDMs and osteoclasts, as well as tissue macrophages in DSS-induced colitis and osteoporosis using the same procedures that I have done in Chapters 2 and 4. 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