THE ROLE OF TUMOR NECROSIS FACTOR IN ACUTE AND CHRONIC COLITIS AND COLITIS ASSOCIATED COLON CANCER By Yava L. Jones A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Pathology 2011 ABSTRACT THE ROLE OF TUMOR NECROSIS FACTOR IN ACUTE AND CHRONIC COLITIS AND COLITIS ASSOCIATED COLON CANCER By Yava L. Jones Inflammatory bowel disease (IBD) consists of two disease entities, Crohn’s Disease (CD) and Ulcerative Colitis (UC). Both diseases manifest as chronic, relapsing and remitting bouts of gastrointestinal inflammation. The etiology of these disease, although not completely understood, consists of a complex interplay between genetic alterations rendering increased susceptibility, the gastrointestinal flora, and hyper-responsiveness of the immune system. Tumor necrosis factor (TNF) is a multifunctional cytokine that has been shown to be upregulated in the colon of and systemically in patients with inflammatory bowel disease. Additionally, anti-TNF therapy has proven effective for the treatment of CD. However, TNF has been shown to have divergent roles in the pathogenesis of disease, depending upon the cellular source of secretion, the length of time of production, the local and systemic concentration of the cytokine and the receptor signaling pathway activated. Here we demonstrate that TNF required for protection against acute colitis and enterocytes derived TNF is the determinant for conferring this protection. In addition, the increased inflammation seen in mice with complete Tnf deficiency and enterocyte derived Tnf deficient mice is accompanied by changes in the microbioal flora which could be contributory components of the disease. In contrast, as colitis progresses from acute to chronic, the protective role of TNF is lost and with chronicity, TNF promotes atypical glandular hyperplasia, inflammation, and cancer. Evaluation of the role of TNF from specific cellular sources in the context of colitis associated colon cancer (CAC) revealed disparate roles for TNF in cancer development depending on the cell type secreting the cytokine. Here we show that enterocyte derived TNF promotes CAC, whereas TNF from T cells protects against CAC. These findings highlight the potentially beneficial versus pathogenic effects of TNF in colitis and CAC and underscore the need more evaluation of the current therapeutic practices for IBD patients. ACKNOWLEDGEMENTS I would like to thank everyone who had supported me and this dissertation project. I would like to extend special appreciation to my Principal Investigator, Giorgio Trinchieri, director of the Cancer and Inflammation Program at the National Cancer Institute who has been extremely supportive and gracious with his time, wisdom and resources. I would also like to thank my collaborators Zsofia Gyulai, Rosalba Salcedo, Amiran Dzutsev, Sergei Nedospasov and Caroline Salter. I am extremely grateful for the guidance and assistance provided by the rest of the Trinchieri laboratory with special thanks to: Ren-Ming Dai, Loretta Scheetz, Robin Winkler-Pickett, Anna Mason, Lyudmila Lyakh and Marco Cardone. I would also like to thank my committee members: R. Mark Simpson, Matti Kiupel, Alison Bauer, Vilma YuzbasiyanGurkan, and Behzad Yamini. I’d like to extend a special thanks to my friends and family, especially Cherié Butts, for her scholarly and personal advice; Esmeralda Dickson for her spiritual encouragement and friendship; and the members of the Comparative Molecular Pathology Unit who’ve helped make this a wonderful experience for me. I am eternally grateful to the staff of the Upward Bound program at Talladega College, who had the forethought to invest in the dreams of a young, first generation college student; as well as my mentors, Willie Reed and Olajide Kasali, who have supported and guided me since I was a veterinary student at Tuskegee University. Most importantly I would like to thank my mother for her support of my personal and professional endeavors throughout my life. Her examples, patience, and love are the reasons I have achieved this and all other goals I have set. The people mentioned, my faith and iv hard work have guided me through this evolution and have allowed me to reach this point in my career. This work was performed for the U.S. Government. Copyright protection is not available in the United States for any work of the U.S. Government under Title 17 U.S.C. § 105. Research funding support was provided through the Intramural Research Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD. I was supported on a Cancer Research Training Award as a Molecular Pathology Graduate Fellow in the Comparative Biomedical Scientist Training Program, a component of the NIH Graduate Partnership Program, in partnership with Michigan State University. v TABLE OF CONTENTS LIST OF TABLES.........................................................................................................................vii LIST OF FIGURES......................................................................................................................viii LIST OF ABBREVIATIONS..........................................................................................................x CHAPTER 1: THE ROLE OF TNF IN THE PATHOGENESIS OF HUMAN AND MURINE MODELS OF INFLAMMATORY BOWEL DISEASE……….………………..………..........................................................................1 CHAPTER 2: MATERIALS AND METHODS………….......................................................................66 CHAPTER 3: THE EFFECT OF TUMOR NECROSIS FACTOR IN THE PATHOGENESIS OF COLITIS IS DEPENDENT UPON ON THE SOURCE OF PRODUCTION AND THE CHRONICITY OF DISEASE…………………………………………………………...84 CHAPTER 4: PROMOTING VERSUS PROTECTIVE ROLE OF TUMOR NECROSIS FACTOR IN THE PATHOGENESIS OF COLITIS ASSOCIATED COLON CANCER………………………………………………….............................................128 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS..........................................................154 APPENDIX: APPENDIX …………………………………………………………………………….157 REFERENCES: REFERENCES................................................................................................................164 vi LIST OF TABLES Table 1.1: TNF Targeted Therapeutic Agents…………………………………………………..46 Table 1.2: The Role of TNF in Selected Mouse Models of IBD………………………………..64 Table 2.1: TNF Expression Levels in selected subsets for each strain of mouse……………….68 Table 2.2: Preselected gene set for Nanostring®………………………………………………..73 Table 2.3: Oligonucleotide sequences for RT-PCR analysis of the expression of selected bacterium…………………………………………………………………………………………76 Table 2.4: Histological assessment of colonic inflammation (Colitis Score)…………………...80 Table 2.5: Primary antibodies with dilutions and manufacturers……………………………….81 Table 4.1: Poisson Regression Model comparing group tumor means with the control group tumor mean…………...………………………………………………………………………...143 vii LIST OF FIGURES Figure 1.1: TNF/TNFRI signaling pathway………………………...............................................7 -/- Figure 3.1: TNBS treated Tnf mice have more colitis than TNBS treated WT or SHAM treated mice................................................................................................................................................88 -/- Figure 3.2: TNBS treated E Tnf mice have more severe colitis than TNBS treated Floxed or SHAM treated mice.......................................................................................................................92 Figure 3.3 RNA expression levels of selected genes....................................................................99 Figure 3.4: TNBS colitis induces changes in fecal microbiota composition which are both dependent and independent of TNF…………………….............................................................103 Figure 3.5: TNF from B cells, T cells, and macrophages/neutrophils and B cells had either insignificant or redundant roles in TNBS colitis……….............................................................108 -/- Figure 3.6: TNBS treated Tnf mice have more severe colitis than TNBS treated WT or SHAM treated mice at 5 weeks................................................................................................................111 Figure 3.7: TNBS 5 week colitis induces significantly more fibrosis and mucosal inflammation -/in WT and Tnf mice than observed in acute colitis...................................................................113 Figure 3.8: RNA expression levels of selected genes.................................................................118 Figure 3.9: TNF promotes inflammation at 10 weeks of TNBS colitis……………..................120 min/+ Figure 4.1: APC mice treated for 5 week and harvested at 5 weeks had moderate inflammation and foci of dysplasia, however mice harvested at 15 weeks had neither inflammation nor dysplasia..........................................................................................................137 Figure 4.2: Enterocytes are the major source of TNF constitutively and post AOM/DSS treatment......................................................................................................................................138 Figure 4.3: TNF protects against colon chronic inflammation but depletion of TNF from specific cells neither ameliorates nor augments colitis.............................................................................140 Figure 4.4: Enterocyte derived TNF promotes CAC, while T cell and MN derived TNF protects against CAC.................................................................................................................................142 Figure 4.5: Enterocyte, but not T cell or macrophage/neutrophil derived, TNF mediates the promotion of tumor growth and progression of malignancy.......................................................145 viii Figure 4.6: RNA expression levels of selected genes.................................................................146 Figure 4.7: Divergent Roles of TNF in Tumorigenesis..............................................................152 ix LIST OF ABBREVIATIONS 5-ASA 5-Aminosalicylic Acid ACF Aberrant Crypt Focus ADAM A Disintegrin and Metalloproteinase AID Activation-Induced Cytidine Deaminase AIM2 Absent In Melanoma 2 Ang Angiogenin AOM Azoxymethane APC Adenomatous Polyposis Coli APC Antigen Presenting Cell ARE AU-Rich Elements ATG16L Autophagy Related 16-Like B Tnf -/- B cell specific Tnf -/- BMP Bone Morphogenic Protein CAC Colitis Associated Cancer CD Crohn’s Disease C-FLIP C-FLICE-Inhibitory Protein Ciap-1 Cellular Inhibitor of Apoptosis Protein-1 CL Claudin CRC Colorectal Cancer CRP C-Reactive Protein CS Corticosteroids x DC Dendritic Cell DD Death Domain DF Degree of Freedom DHT Delayed Hypersensitivity DMH 1,2-Dimethylhydrazine DSS Dextran Sodium Sulfate E Tnf -/- Enterocyte specific Tnf -/- EHEC Enterohemorrhagic E. coli ENTERO Enterobacteriaceae ER Endoplasmic Reticulum FADD Fas-Associated Death Domain FAP Familial Adenomatous Polyposis FHC Ferritin Heavy Chain GALT Gut Associated Lymphoid Tissue GI Gastrointestinal GM-CSF Granulocyte Monocyte-Colony Stimulating Factor GWA Genome Wide Association H&E Hematoxylin & Eosin stain HLA Human Leukocyte Antigen IBD Inflammatory Bowel Disease ICC Interstitial Cells of Cajal ICD Intracellular Domain Icos Inducible T-cell Co-Stimulator xi IEC Intestinal Epithelial Cells IFN Interferon IHC Immunohistochemical IKK Iκb Kinase IL Interleukin IP Intraperitoneal IR Intra-Rectal IRGM Immunity Related GTPases ISEMF Intestinal Subepithelial Myofibroblasts Iκb Inhibitor of Nuclear Factor –Κb JNK Jun Kinase JPS Juvenile Polyposis Syndrome KC Keratinocyte-Derived Chemokine LOH Loss of Heterogeneity LPS Lipopolysaccharide LT Lymphotoxin LTβR Ltβ Receptor MN Tnf -/- Macrophage/neutrophil specific Tnf -/- MAP MUTYH-Associated Poliposis MCP-1 Monocyte Chemoattractant Protein-1 MHC Major Histocompatibility Complex MMP Matrix Metalloproteinase MMR Methylation of Mismatch Repair xii MPK MAP Kinases MSI Microsatellite Instability MUC Mucin Myd88 Myeloid Differentiation Primary Response Gene 88 NF-κB Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells NK Natural Killer NLR NOD-Like Receptor NOD Nucleotide-Binding Oligomerization Domain-Containing Protein PAMPS Pattern Associated Molecular Patterns PID Primary Immunodeficiency PJS Peutz-Jeghers Syndrome PPR Pattern Recognition Receptor PR Poisson Regression RELMβ Resistin-Like Molecule Beta RIP Receptor-Interacting Protein RLRs RIG-I-Like Receptors ROS Reactive Oxygen Species SCID Severe Combined Immunodeficiency SFB Segmented Filamentous Bacteria SMAD Mothers Against Decapentaplegic Homolog SOCS Suppressors of Cytokine Signaling SOD Superoxide Dismutase SODD Silencer of Death Domain xiii STAT Signal Transducer and Activator of Transcription STK11 Serine/Threonine Kinase 11 T Tnf -/- T cell specific Tnf -/- TACE TNF Converting Enzyme TCR T cell receptor TGFβ Transforming Growth Factor Beta Th T Helper TIMP Tissue Inhibitor of Matrix Metalloproteinase TLR Toll Like Receptor TNBS Trinitrobenze Sulfonic Acid TNF Tumor Necrosis Factor TNFR TNF Receptor TNP Trinitrophenyl TRADD TNF Receptor1 Associated Death Domain TRAF TNF Receptor-Associated Factor TRAIL TNF-Related Apoptosis-Inducing Ligand Treg Regulatory T Cell UC Ulcerative Colitis WT Wild Type xiv CHAPTER ONE The Role of TNF in the Pathogenesis of Human and Murine Models of Inflammatory Bowel Disease INTRODUCTION The two main subtypes of inflammatory bowel disease (IBD) are Crohn’s disease (CD) and ulcerative colitis (UC), both of which are chronic, relapsing and remitting, inflammatory disorders affecting the gastrointestinal tract (1). Worldwide, the incidence of IBD ranges from 0.5-24.5 cases per 100,000 persons for UC and from 0.1-16 cases per 100,000 persons for CD, with the majority of affected individuals reported to have been disabled at some point in their lives (2-4). The disparity in incidence is due primarily to geographical distributions, with more cases occurring in industrialized areas (4). Most reports indicate that IBD is more common in European Americans than in African Americans, with a higher incidence rate in persons of Jewish descent than other ethnic groups, and the lowest rates in Hispanics and Asians; however these ethnic and racial trends are narrowing (4, 5). The onset of disease in all affected ethnic groups is generally in late adolescence and early adulthood, but the diagnosis may occur at any age (2, 6). The intimate association of the gastrointestinal immune (GI) system with the high concentrations of intraluminal bacteria and the long recognized familial aggregation of the disease syndrome provides the basis for the accepted theory that IBD results from a dysregulation of the immune response to normal commensal microbial antigens in a genetically 1 susceptible host (1, 5, 7, 8). The genetic susceptibility for the development of IBD is further modified by epidemiologically relevant environmental factors (e.g. tobacco use, appendectomy, oral contraceptives, antibiotics) (7). Normally, the intestine is kept in a homeostatic state by maintaining a balance between proinflammatory cytokines and anti-inflammatory cytokines (9). Proinflammatory cytokines proven to be instrumental to the development of IBD include: tumor necrosis factor (TNF), interferon gamma (INF-γ), interleukin (IL)-6, IL-17 and the IL-12 family (9-11). Antiinflammatory cytokines that are considered necessary for controlling excessive proinflammatory responses include: IL-10, transforming growth factor beta (TFGβ), IL-11 and IL-4 (9). Dysregulated production of cytokines has been clearly linked to the pathogenesis of IBD and several reports have established the relationship of mucosal or plasma cytokine levels and the disease activity in patients with IBD (12). Although proinflammatory cytokines appear to be an obvious target for IBD therapy, at the present Infliximab (Remicade®), a humanized monoclonal antibody that binds and neutralizes TNF, is the only approved biological agent for treatment of moderate to severe CD that is unresponsive to conventional therapy (9). Infliximab treatment for UC has shown efficacy in some patients, but is not as effective as in patients with CD (9, 13). Here, we will first review IBD etiopathogenesis and pathology with a focus on the role of TNF in the development, progression and treatment of the disease process. We will highlight some of the murine models used to further our understanding of IBD and discuss the immunopathology and role of TNF in the pathology seen in these animal models, as well as how these models relate to human IBD. TUMOR NECROSIS FACTORS Structure and function 2 TNF, first isolated in 1984, is a cytokine that can have either beneficial or detrimental functions (14, 15). TNF belongs to a superfamily of cytokines (TNF superfamily), of which 19 ligands have presently been identified (15). The mouse Tnf gene is located on chromosome 17, which also contains the major histocompatibility complex (MHC) genes, and is similar to its human counterpart (16). The human TNF gene is located on chromosome 6, also in the region of the MHC genes (17, 18). Control of TNF synthesis largely occurs at the translational level, thus RNA levels may not necessarily correlate with protein quantities in tissues (19). TNF is synthesized as a type II transmembrane protein with a molecular mass of 17 kDa (20, 21). The protein is cleaved from the surface of cells to become active, primarily by the proteinase TNF converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17) (22). TNF is produced by most cells in the body except red blood cells, but is assumed to be primarily made by activated macrophages (23, 24). The effector functions of soluble TNF vary greatly and TNF has been shown to be required for or involved in many processes including, but not limited to: proper lymphoid organ cellular compartmentalization (25); triggering of apoptosis (26); induction and progression of inflammation (23, 27, 28); infection and autoimmune diseases (24, 29); cellular proliferation (24, 30, 31); and tumorigenesis and tumor cytotoxicity (32-35). Acute, systemically high doses of TNF are considered to be pivotal in the pathological sequela associated with septic shock syndrome and tissue injury (14, 36-38). However, exposure to TNF in acute infection can either be beneficial, for example in controlling acute Mycobacterial infection (39) or can promote disease as seen in acute Trypanosoma cruzi infection (40). In addition, the cellular source of production and disease can result in either disease protection or promotion depending on the model. For example, macrophage/neutrophil derived TNF is critical for protection against 3 intracellular Listeria, but promotes autoimmune hepatitis (41). Chronic exposure to the proinflammatory effects of TNF has been clearly linked with the development of cachexia (14) and the induction and/or progression of a wide variety of chronic inflammatory/autoimmune diseases (42, 43) and cancer (44-46). Super-pharmacological doses of TNF can cause tumor necrosis, as the name implies, and therefore be beneficial therapy with controlled local administration (32, 45, 47). The variation in these effects is due to disparate responses to receptor signaling pathways [e.g. TNF receptor 1 (TNFRI) versus TNFRII], TNF concentration, time and course of secretion and source of production (20, 24, 26, 41). Lymphotoxin (LT)α, another key TNF superfamily member, is found on human chromosome 6 and mouse chromosome 17 alongside TNF and is similar in function and binding capacity (16, 48) . Like TNF, LTα is found in both secreted and membrane bound forms (49). Soluble LTα can signal through TNFRI or II, in contrast, membrane bound LTα is tethered to the surface of cells, by forming a heterotrimeric complex with LTβ (LTα1β2) (50) and signals through LTβ receptor (LTβR) (51). Activated lymphocytes, natural killer (NK) cells and a subset of resting B cells express LT. LTβR, however, is mainly expressed on nonhematopoietic and myeloid lineage cells (52). This suggests that the LTα1β2/LTβR signaling pathway functions as a communication link between lymphocytes and stromal cells, but not between T or B cells. Yet, it is clear that both LTα and LTα1β2/LTβR signaling are required for lymphoid organogenesis and the maintenance of lymphoid cellular networks (53, 54). Intestinal lymphoid systems [gut associated lymphoid tissue (GALT)] are covered by specialized epithelia (M cells) which serve to sense luminal bacteria and communicate with the network of the GALT (55). Inhibition of LT signaling results in flattening of Peyer’s patches and reduction in B cell, T cell and dendritic cell (DC) numbers (56, 57). In fact, blocking of LTβR, is protective against Th2 colitis and GALT 4 hyperplasia induced by the colitis (56). Each arm of the LT signaling pathway should be considered when approaching it as a target for therapy. For instance, studies in mice with disruption of the LT gene showed enhanced tumorigenesis and metastasis in the deficient mice due to impaired NK cell function (58). On the other hand, when agonistic monoclonal antibodies were used to trigger the LTβR specifically, an anti-tumor effect was seen (59). These data indicate that the signaling and effector functions of LT are similar to TNF, but also have distinct roles in development and disease. Receptors and signaling pathways The ligands of the TNF superfamily mediate their cellular responses through 29 receptors TNFRs. TNFRs can be divided into 3 broad groups: those containing a death domain (DD), TNF receptor-associated factor (TRAF) binding receptors, and decoy receptors (60, 61). DD containing receptors (such as FAS and TNFRI) are able to induce apoptosis by activation of caspase 8 (Fig. 1.1) (26, 62). However, caspase independent apoptosis by other TNF superfamily members (e.g. through FAS/FAS ligand interaction) can occur as well (63). TRAF binding receptors, such as TNFRII, lack DD domains but instead recruit TRAF proteins which are associated with cellular activation, differentiation, and survival signaling (64). Decoy receptors are nonfunctional but can compete with other receptors for TNF superfamily ligand binding (65). TRAFs are major signal transducers for the TNF superfamily as well as the IL-1 family of proteins. TRAFs have biological roles in cellular metabolism, embryonic development and innate and adaptive immunity (64). Many of the effects of TRAFs are related to their induction of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), which induces expression of pro-inflammatory and anti-apoptotic genes, the details of 5 which are beyond the scope of this review [see reviews: (66-68)]. Briefly, in unstimulated cells inactive NF-κB is sequestered in the cytoplasm by inhibitors of NF-κB (IκB) proteins. Upon stimulation, IκB proteins are ubiquinated and subsequently degraded by IκB kinase complex (IKK) and NF-κB is released from the cytoplasmic complexes, translocates to the nucleus and drives expression of genes, several of which are pro-inflammatory or induce cellular proliferation (67). Overexpression of TRAF2 and 3 can activate of NF-κB expression (69). However, other TRAFs, such as TRAF6 are more instrumental in the TNF and IL-1 signaling pathways. Lack of TRAF6 leads to defective signaling of IL-1 and IL-18 as well as defective responses to lipopolysaccharides (LPS) (70). Alternatively, TRAFs, such as TRAF3 have been shown to recruit to LTβ receptor and induce cell death, as well, it can negatively regulate NF-κB activation (70). DD containing TNFR superfamily members, such as TNFR1, can mediate either apoptosis or activation of NF-κB via the same pathway described above (26). Induction of apoptosis involves TNF binding to TNFR1 with subsequent release of inhibitory protein silencer of death domains (SODD) from the intracellular domain (ICD) of TNFR1. The adaptor protein TRADD recognizes ICD and recruits the adaptor proteins receptor-interacting protein RIP, TRAF2, and FADD. These adaptor proteins then recruit proteins to initiate apoptosis (FADD recruits caspase-8 or 10); inhibit apoptosis (TRAF2 recruits cellular inhibitor of apoptosis protein-1 (cIAP-1) and cIAP-2, which are anti-apoptotic proteins); and/or activate NF-κB (26, 62, 71) (Figure 1.1). It has also been shown that induction of cell killing by another TNF superfamily member, FasL can proceed either dependently or independently of caspase-8 activation; however this cell killing, was ultrastructurally consistent with necrosis and did not exhibit morphological features of apoptosis or DNA fragmentation (72, 73). 6 Figure 1.1 Figure 1.1: TNF/TNFRI signaling pathway [Modified from Karin and Greten (66)] TNF secreted by hematopoietic or non-hematopoietic cells can signal through TNFRI and induce apoptosis or survival. The apoptosis pathway involves recruitment of TNF receptor1 associated death domain (TRADD) to the receptor, which subsequently recruits eceptor-interacting protein (RIP) and TRAF2 (Complex I). Complex I then recruits Complex 2 proteins [Fas-associated death domain (FADD) and Caspase 8] and initiates apoptosis, possibly leading to reduced carcinogensis or anti-tumor resistance. Alternatively, formation of Complex I can lead to activation of the IKK complex, resulting in nuclear translocation of NF-κB or activation of Jun kinase (JNK). Translocation of NF-κB ultimately leads to cell survival and possibly enhanced 7 carcinogenesis by blocking apoptosis and rapidly inhibiting JNK activity. Activation of JNK can lead to activation of the ubiquitin ligase Itch, which leads to c-FLICE-inhibitory protein (c-FLIP) degredation. Apoptosis is inbited by inducing expression of the caspase 8 inhibitor, c-FLIP. JNK activity is blocked by upregulating expression of the antioxidants supersoxide dismutase 2 (SOD2) and ferritin heavy chain (FHC) which inhibit the accumulation of reactive oxygens species (ROS) and prevent activation of MAP kinases (MKP), resulting in termination of JNK activity. For interpretation the references to color in this and all other figures, readers are referred to the electronic version of this dissertation. 8 INFLAMMATORY BOWEL DISEASE Genetic factors Genome wide association (GWA) screening with microsatellite DNA markers have provided information on the relative contributions of various genomic loci common to IBD susceptibility and have been successful in identifying a variety of genes that contribute to disease susceptibility (1, 5, 8). Most GWA studies to date have been performed using cohorts of European ancestry. Results have clearly indicated 75-80% concordance within families, increased concordance in monozygotic twins, and increased frequency of IBD associated with certain genetic syndromes (5, 74-76). Interestingly, 20% of families with IBD have members with CD and members with UC. This finding indicates that some genes which are contributory to the development of IBD are common to both CD and UC (1, 76). GWA studies also revealed that the genetic architecture of CD may not be purely polygenic and that CD result from a collection of single-gene lesions that affect macrophages and display incomplete penetrance at the clinical level, at least in a fraction of patients (77). CD may actually turn out to be a typical primary immunodeficiency (PID) of macrophages, affecting patients who are unable to control their commensal, intestinal microbial flora (77). These studies will be discussed further in the innate immunity section of this review. The first susceptibility gene identified in CD, the nucleotide-binding oligomerization domain-containing protein (NOD) 2 gene (also designated CARD15 and IBD1), was found to be located on chromosome 16 using positional cloning and candidate gene approaches and, surprisingly was expressed in the myeloid rather than the lymphoid lineage cells (1, 78, 79). Subsequent to linkage between IBD and chromosome 16, several other studies have identified more associated loci, including a locus on chromosome 12q (IBD2 locus), 6q (IBD3 locus), 14q 9 (IBD4 locus) and on chromosomes 3p and 5q (5, 78). NOD2 (which encodes nucleotide-binding oligomerization domain protein 2), ATG16L1 (encodes autophagy related 16-like protein), and IRGM (which encodes immunity related GTPases) function in the innate immune system. Single nucleotide polymorphisms in these genes are specific to patients with CD, and are not seen in UC patients (78). Significant associations between UC, but not CD, have been observed with a region on chromosome 12q15, which encompasses the IFN-γ and IL-26 genes (80, 81). Associations between human leukocyte antigens (HLA) and development of IBD have been documented in some subgroups of patients with UC and CD. However, the most significant causal association was observed within the MHCII region near HLA-DRA (alpha chain) (78, 80). NK2 transcription factor related, locus 3 (NKX2-3) and multiple genes in the IL-23 pathway, including IL23R, IL12B, and signal transducers and activators of transcription (STAT) 3 have been associated with both CD and UC (5). Two common polymorphisms in toll like receptor (TLR) 4 in humans have been associated with the development of IBD in Caucasians. These polymorphisms are associated with reduced responsiveness to LPS stimulation (82-86). In addition, it has recently been shown that TLR1 and TLR2 mutations are associated with development of pancolitis in UC patients (82). Autophagy is a catabolic process that is essential for maintaining cell homeostasis and is also required for bacterial clearance and antigen presentation by DCs (87). Defective autophagy has been linked to increased susceptibility to infectious diseases, both in vitro and in vivo (88), whereas excessive autophagy leads to death of host cells. Recent findings from genetic studies including GWA studies of patients with Crohn’s disease have identified two autophagy loci, ATG16L and IRGM1 (immunity-related GTPase M1), that are linked to Crohn’s disease susceptibility (89-91). These findings imply that genetically compromised individuals may have 10 defects in autophagy or handling of enteric bacteria which could contribute to IBD pathogenesis. In fact, it has been shown that interaction between the CD susceptibility loci NOD2 and ATG16L1 results in stimulation of autophagy in response to bacterial sensing (92). DCs ins expressing the Crohn’s disease risk variants NOD2 L1007 300 C (NOD2Fs) or ATG16L1 T A display inefficient bacterial killing and defective antigen presentation due to reduced autophagy following NOD2 stimulation (93). Additionally, Paneth cells deficient in ATG16L1 exhibit impaired luminal secretion of antimicrobial peptides in response to infection, which is proposed to result in bacterial overgrowth and has been linked to CD (94), (95). Mice expressing an hypomorphic variant of ATG16L1 showed defective Paneth cell functions and enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. This enhanced suspecptibility was dependent on both infections by certain strains of murine norovirus and of the presence of commensal flora, suggesting that in the absence of autophagy, mucosal damage induced by the virus alters the physiological interaction with the commensals, leading to inflammation and colitis (96). Microbial Contributions to Mucosal Homeostasis The intestines primary functions are the digestion and absorption of nutrients and water. The gut is composed of two main sections, the small intestine and the colon that present quite different mucosal histology. The colon is the anatomical site most often affected by inflammatory (colitis) and neoplastic pathologies (colorectal cancer; CRC). The regulation of cell proliferation and the characteristics of the gut-associated lymphoid tissues have been the subject of very detailed studies, but relatively less is known about the physiology of the colon. One of the most striking characteristic of the digestive tract is its association with the commensal microbiota (97). Since the time of their birth, all metazoan animals live in mutualistic association 11 with a large number of microorganisms, such as bacteria, fungi, and viruses. It has been estimated that in our body the number of bacterial cells is 10 fold that of our own cells and the number of different bacterial genes 100 fold that of human genes. Although bacteria are present on the entire body surface in direct contact with the external environment, the large majority of commensal bacteria reside in the alimentary tract and the number of bacteria increases dramatically in the more distal portions, the cecum and the colon. The relative composition of the different bacterial class also changes depending of the anatomical position. Thus, in addition to it absorption function, the gut mucosa has the important function of a barrier between the commensal flora and the internal tissues. The colonization of the gut takes place at birth, with the maternal flora being a major determinant of the eventual composition of that of the progeny (98), and then continue for several days with the exposure to the external environment. For centuries scientists have sought a specific etiologic agent as the cause of CD, but none has been discovered, although several agents have gained attention at various points in time (e.g. Mycobacterium, adherent and invasive E. coli, Chlamydia, Helicobacter pylori) (99). However, the accepted hypothesis of the etiopathogenesis of IBD still proposes that dysregulation of the mucosal response to antigens derived from the commensal microbiota significantly contributes to IBD (1, 7, 100-102). In support of this theory, patients with IBD experience improvement in clinical signs after a prolonged course of antibiotic therapy (usually either metronidazole or ciprofloxacin, or both), as well as, IBD does not occur in germ free genetically predisposed animals, in contrast to their naturally colonized littermates (99, 103, 104). Still, the issue of which species of bacteria is significant in the development or suppression of intestinal disease is complex and is may be more integrated in the shift of communities rather than the loss or gain of specific organisms. Although patients with IBD have been shown to 12 benefit from antibiotic therapy, antibiotics have also clearly been shown to result in significant perturbations in the gut microbiota (105). Patients treated with a prolonged course of antibiotics often develop problematic side effects, such as rashes, diarrhea, and peripheral neuropathy (104). In mice, antibiotic therapy related flora changes resulted in increased susceptibility to invasion and colonization by pathogenic organisms (106). In general, patients undergoing antibiotic treatment often develop pathogen-associated or pathogen-independent (functional changes resulting from changes in commensal communities) diarrhea (105). In addition, patients admitted to intensive care and treated with systemic antibiotics were shown to have a higher rate of organ failure and mortality associated with reduction of microbiota diversity and the massive presence of enterococci (107). Trillions of bacteria along with many species of fungi, protozoa, and viruses reside in our intestines (108). There is a large degree of variation in species and compartmentalization of the bacteria present in the intestinal tract. The lowest concentration of bacteria is present in the 11 duodenum and colonization increases to a peak density of 10 12 to 10 cfu/ml in the proximal large intestine (7, 108, 109). For many years our understanding of the bacterial composition of the intestinal tract was limited, however because of the low oxygen tension in the gut, the large majority of intestinal bacteria are anaerobic and thus difficult or impossible to culture (109). Up to 80% of the resident bacteria are non-cultivable (110). The advent of the new sequencing technology, particularly the DNA barcoding and 454 pyrosequencing of the 16S ribosomal RNA from bacteria is allowing investigators a more precise quantification and bacterial identification even for non-cultivable species and has revealed that across vertebrate species, most bacteria present are Gram negative and belong to the Bacteriodes (~64%) and Firmicutes (~28%) phyla (108-110). In fact, a shift in the Bacteriodes/Firmicutes ratio has been linked to several diseases 13 including CD, UC and celiac disease. These conditions are associated with not only inappropriate responses to the commensal flora, but also a decrease in phyla diversity (108). These intestinal microbes have coevolved with humans and contribute many functions to essential human survival (111). The host diet is thought to be one of the key contributory components of the evolution of the microbiota (109). Mammals insufficiently extract the energy content of most plant rich diets, thus the recruitment of bacteria allows for the hydrolyzation of a variety of dietary polysaccharides that would be otherwise indigestible (109). The finding that germ free rodents require approximately 30% more calories to maintain their bodyweight than do conventionally colonized animals and germ free mice are protected against diet induced obesity, emphasizes the importance of the flora for efficient energy consumption (109, 112). Intestinal microbes also provide host protection against pathogenic bacteria by competing for nutrients and stimulating immune responses that are cross-protective against pathogens (109). It seems clear that the primary driving force behind the coevolution of mammals with their enteric flora is enhanced digestive efficiency. However, this coevolution has also led to an integrated effect on the shaping of mucosal immunity (103, 108-110). A growing body of evidence indicates that recognition of commensals via TLR and NOD signaling is required to maintain intestinal homeostasis (80, 99, 103, 111). TLRs are expressed by monocytes, macrophages, DCs and epithelial cells. It has been shown that non-diseased intestinal epithelial cells constitutively express TLR3 (receptor for dsRNA) and TLR5 (receptor for flagellin), but TLR2 (receptor for lipoprotein) and TLR4 (receptor for LPS) are present in much lower amounts (82). In active IBD, epithelial cell expression of TLR3 was downregulated in CD, but not UC. In contrast, expression of TLR4 was strongly upregulated in both CD and UC. TLR2 and TLR5 expression levels did not change in IBD (82). 14 The interaction of the microbiota with any cutaneous/mucosal surface, but in particular in the gut, has a major effect in determining the maturation and the functions of the innate and immune defense systems and also has a major effect on the anatomical development of the gut and in regulating the metabolisms by affecting food breakdown and nutrient absorption (97). The barrier function of the gut is not absolute and in the presence of inflammation but also in physiological conditions a certain level of microbial translocation through the mucosa is observed with bacteria accumulation particularly in the draining lymph nodes. Because of the systemic effects of the inflammatory response to the gut microbiota and also probably because of actual microbial translocation, the physiological and pathological results of the host-commensal flora interaction in the intestine are not confined to the local tissues but can have consequence on distant tissues resulting in DNA damage, autoimmunity, inflammation and cancer (113-115). Particularly important in the regulation of mucosal homeostasis and the interaction with the commensal flora are the inflammatory cells and lymphoid structures adjacent to the intestinal mucosa (GALT). In the small intestine, the GALT is formed by the Peyer’s patches and the isolated lymphoid follicles. The mucosa covering the Peyer’s patches forms a dome without villi and present a specialized flat cell type, the M cell, which facilitates the translocation of antigens to the antigen presenting cell (APC) in the patches. In the colon, there are also large colonic patches, which are somewhat different in structure and are fewer in number than the Peyer’s patches. Lympoid follicles are also present in the colon. In the intestine there are at least three different types of DCs that could favor either the conversion of regulatory T cell (Treg) through retinoic acid and TGF-β or the generation of effector T cell responses, particularly T helper (Th)17, in response to TLR stimulation, e.g. TLR5 by flagellin, and activation of the inflammasome (116). One type of DC, CXCR3+ CD70+ 15 is able to protrude through the epithelial barrier and to come directly in contact with intestinal lumen and its bacterial content (116). The intestinal mucosa, in addition to providing a physical barrier to microbial translocation, needs to avoid excessive inflammatory responses to the commensal flora while remaining able to control the physiological regulation of this symbiosis and response if needed to pathogen invasion. This is obtained by activation of both innate resistance mechanisms as well as adaptive immunity, both humoral, in particular IgA antibodies, and cellular. The innate inflammatory response to the commensal flora is required both for intestinal homeostasis and for the control of microbial translocation and invasion. Myeloid differentiation primary response gene 88 (MyD88) deficient mice that are unable to signal through most TLRs and the IL-1 receptor family have significant defects in the mucosa with an increase in the number of proliferating cells in the crypts that extend much farther from the bottom than in WT animals and also an increase of total number of crypt cells (117). This results in an increased susceptibility to radiation or chemical injury and deficient repair. Signaling through TLR, in particular 4 and 2 by bacterial ligands appears to be required for intestinal homeostasis. MyD88 deficient mice, as well mice in which the commensal flora is decreased by antibiotic treatment, display a similar phenotype and have very low constitutive expression of factors such as IL-6, TNF, CXCL1 and heat shock proteins that are important in preserving intestinal homeostasis (117). Mice lacking TLR signaling components (TLR2, TLR4, or MyD88) or mice treated with antibiotics are also highly susceptible to DSS induced colitis (103). Studies in germ free or antibiotic treated mice have shown that these mice have reduced numbers of CD4+ Th17 cells in the lamina propria and an imbalance in Th1/Th2 T cells as compared to conventionally reared or untreated mice(103, 108). Colonization of germ free mice with Bacteriodes thetaiotaomicron induces epithelial expression of genes involved in several essential 16 intestinal functions (i.e. nutrient absorption and mucosal barrier fortification) and can also recover the Th1/Th2 imbalance, resulting in attenuation of inflammation (103). The polarity of the intestinal epithelial cells also plays a major role in regulating the response of TLR9 to bacterial DNA (118). TLR9 is expressed both on the apical and basolateral membrane in polarized cells but only intracellularly in hematopoietic cells. Whereas stimulation of basolateral TLR9 induces NF-κB activation and a proinflammatory response, stimulation of apical TLR9 induces accumulation but not degradation of ubiquitinated IκBα and thus fails to activate NF-κB but rather induces tolerance to other stimuli (118). Although apical and basolateral activation induces expression of different gene patterns, only apical stimulation results in the production of Wnt ligands that induce the production of anti-bacterial factors and activate an alternative anti-microbial mechanism (118). Thus, activation of apical TLR9 by commensal bacteria contributes to intestinal homeostasis by suppressing inflammation through a mechanism that may be in part due to induction of type I Interferon whereas in the presence of alterations of mucosal integrity and microbial translocation the basolateral TLR9 is activated with a classical inflammatory response. Changes in the composition of the intestinal microbiota or the colonization with specific pathogens may alter intestinal homeostasis and the nature of the mucosal immune response and in some case may result in spontaneous colitis or carcinogenesis. A very recent example is the colonization of mice from certain vendors with segmented filamentous bacteria (SFB) that adhere to epithelial cells in the terminal ileum of mice and it is associate with a predominance of Th17 cells whereas very few of these cells are present in animals that are not colonized by SFB (119). Mice colonized by SFB are better in responding to infections but are also more prone to autoimmunity (119). Interestingly, colonization with SFB is not only responsible for changing the nature of the mucosal immunity but also has systemic 17 effects and is sufficient to enhance Th17 response and induce arthritis in a T cell receptor (TCR) transgenic mouse model (115). Another example is provided by the change in intestinal microbiota composition in T-bet and Rag1-deficient mice that is able to induce colitis and cancer, both in the knock out animals as well as in co-caged littermates (120, 121). Mucosal IgA production has also been shown to be important in maintaining “tolerance” to commensal bacteria. Studies have shown that expansion of anaerobes, such as SFB occurs in mice lacking activation-induced cytidine deaminase (AID), which results in defective class switching and, subsequently, decreased levels of IgA producing plasma cells (122). The intestinal flora also regulates the development of IgA secreting cells as evidenced by the fact that germ free mice and antibiotic treated mice have severe reductions in fecal IgA levels and IgA+ cells in the lamina propria (103). In addition to affecting T helper cells’ development and functions and B cell production of IgA, the commensal flora is instrumental in the induction or suppression of development and functionality of the GALT and many other key immune cells in the intestine, including Tregs, DCs, macrophages, NK cells, and CD8+ cells (108, 123-125). Macroscopic and Microscopic Pathology of Inflammatory Bowel Disease IBD has traditionally been defined by a non-strict combination of clinical symptoms, pathological features, and endoscopic and radiological findings (1, 126). In order to determine the best approach to therapy, patients are assessed by determining the IBD phenotype (CD versus UC), disease extension and distribution, extra intestinal manifestations, disease severity and response to various pharmacologics (8). Despite this comprehensive and cumbersome approach to diagnosis, approximately 10-15% of patients are still diagnosed with indeterminate colitis due to the inability to distinguish between UC and CD (8). 18 The lesions of UC are typically mucosal with inflammation and ulceration affecting the rectum and either the distal or the entire colon. The lesions appear in a continuous and confluent manner, with no intervening normal tissue (1, 8, 127). Symptoms include, but are not limited to, melena, tenesmus, nocturnal defecation and abdominal pain. Complete blood count and chemistry often reveal anemia; inflammation (increased levels of C reactive protein); and malabsorption (low vitamin levels) (126). CD may affect any portion of the gastrointestinal tract, however involvement of the terminal ileum is most common and early mucosal lesions often appear over Peyer’s patches (1, 126). As with UC, laboratory tests reveal evidence of chronic inflammation, anemia and signs of malnutrition/malabsorption. CRP levels correlate better with disease activity in CD patients than in UC patients (126). None of these indices is specific enough to allow differentiation from UC, however. Colonoscopy is the gold standard for diagnostic confirmation of both UC and CD, in addition to assessment of disease activity and drug responsiveness. The most conclusive colonoscopic finding of UC is continuous and confluent colorectal inflammation, evidenced by erythema, mucosal erosions or ulceration, and/or spontaneous bleeding (1, 126, 128, 129). Chronic inflammation may result in mucosal atrophy and formation of pseudopolyps (126). Distinctive features of CD are unaffected or only partially affected rectum; asymmetrically affected regions intervened by healthy mucosa (“skip” lesions); linear and serpiginous lesions; a “cobblestone” mucosal appearance; and the presence of fistulas or stenosis (1, 126, 130). The value of colonoscopy, however, is limited in severe cases due to the increased risk of bowel perforation. Histopathology is a key diagnostic tool for both UC and CD. A diagnosis favoring UC over CD is based on the combination of the presence of basal cells around or below crypts, 19 diffuse hypercellularity of the lamina propria and distortion of the crypts and mucosal architecture. The presence of crypt microabscesses and of villous surface also tends to be a feature of UC rather than CD (1, 126). Histological lesions which characterize CD include: aggregation of macrophages with formation of non-caseating granulomas, focal (segmental or discontinuous) crypt architectural abnormalities; patchy or segmental, often transmural, chronic inflammation; and mucin preservation at sites of active inflammation (1, 126). Biopsy sampling via endoscopy often results in extraction of the superficial layers of the mucosa, however to accurately diagnose CD, a full thickness bowel wall sample displaying evidence of granuloma formation and at least one other of the above mentioned changes is needed (126). Similar to UC, multiple biopsies from various sites improve diagnosis accuracy (126, 131). Innate Immunity and Adaptive Immunity in Inflammatory Bowel Disease It is important to remember that the luminal intestinal microbes are separated from the submucosal antigenic compartment by 2 layers of mucous and a single layer of epithelial cells which serve as a physical barrier (109, 111, 132-134). Local inflammation is persistently present at low levels in the intestine, however and can be regarded as a component of innate defense (132, 135). Innate and adaptive immunity are the two essential elements of remission/treatment and progression of IBD. Innate immunity provides a relatively non-specific, but immediate protection against invading pathogens, but lacks the capability of generating immunologic memory (132). The immune cells of the innate immunity, which includes phagocytic cells (neutrophils, monocytes, macrophages), NK cells, γδ T cells, Paneth cells, intestinal epithelial cells, fibroblasts, myofibroblasts and the intestinal epithelium itself express a vast network of pattern recognition receptors (PPRs) (8, 132, 135-137). The TLR and NOD receptor families of PPRs are essential in mucosal homeostasis and alterations, as seen with NOD mutations in CD, 20 contribute to the pathogenesis of IBD (82). Activation of the innate immune response is essential for the excessive adaptive immune response that characterizes IBD (1). Immunoglobulin (Ig) and antibody secreting B cells and T cells with various helper or effector phenotypes are classically considered the mediators of the adaptive immune response (1, 135, 136). The simplest categorization of IBD is that UC exhibits a Th2 cytokine profile and CD exhibits a Th1/Th17 cytokine profile (1, 8). T cells The adaptive immune system has traditionally been thought to be the primary driving force of the pathogenesis of both UC and CD. Tremendous effort has been placed on characterizing the pathogenic CD4+ Th cell population considered to be instrumental in the production of cytokines responsible for the initiation and propagation of IBD. In addition to elevation of disease specific proinflammatory cytokines (with simultaneous inhibition of antiinflammatory cytokines), IBD patients have also been shown to have a down-regulation of apoptosis of pathogenic T cells. In these cases, decreased apoptosis allows for unregulated expansion of pathogenic T cells and thus aggravation of the condition (138). Although the mechanism of this phenomenon is not clear, the efficiency of anti-TNF therapies is mediated by their ability to induce apoptosis of effector T cells. It is therefore possible that certain therapeutic anti-TNF antibodies, but not others, are able to recognize membrane-bound TNF on T cells thereby inducing their death by apoptosis (138, 139). It should also be noted that patients with IBD undergo bouts of remission throughout the course of the disease. It is thought that the primary mediator if remission is the production of anti-inflammatory cytokines such as IL-10, IL-35 and TGFβ by Tregs. These cells develop from naïve T cells under the regulation of the transcription factor FoxP3, in the presence of TGFβ and retinoic acid (7). 21 Classically, Th cells are phenotyped based on a polarized production of cytokines produced secondary to activation by an antigen presenting cell (138, 140). The Th1 cells predominantly produce the IFN-γ, IL-12 and TNF. Production of these cytokines is regulated by the transcription factor T-bet, induced by a microenviroment rich in Th1-inducing cytokines including IL-12 (7). CD has traditionally been considered to be the result of a shift towards a Th1 mucosal environment. Support for this theory is evident in the fact that tissues from patients with CD have elevated IL-12 and IL-18, as well as TNF and INF-γ (7, 141). Recently the identification of the Th17 cells that produce IL-17, IL-6, IL-22 and granulocyte colony stimulating factor under the control of the transcription factor RORγt further advanced our understanding of IBD. Initially, IL-23 was thought to be the regulator of development of Th17 cells; however it was subsequently discovered that Th17 cells only develop from naïve T cells in the presence of IL-6 and TGFβ, with or without the presence of IL-23 (7, 142). Studies of patients with CD have shown that IL-17 is present in the intestines of affected patients, but not normal controls. Additionally, serum levels of IL-22 are elevated and CD4+ T cells isolated from the intestines of patients with CD (but not normal controls) produce IL-22 (143, 144). Taken together, these findings highlight the role for both Th17 and Th1 cells in the pathogenesis of CD (145). Th2 cells are associated with IL-4, IL-5, IL-13 and IL-25, cytokines which are primarily described as anti-inflammatory. Th2 cells develop from naïve T cells under the regulation of the transcription factor GATA 4 and in the presence of IL-4 and other Th2-inducing cytokines (7). IL-4 is the definitive cytokine of the Th2 response and although a clear link between T cells isolated from UC patients and the production of IL-4 has not been established, it is generally accepted that UC is a Th2 skewed disease and the disease has been associated with elevated T 22 cell production of both IL-13 and IL-5 (146, 147). Additionally, Th2 cells have been shown to more efficiently activate B cells and patients with UC have a higher rate of development of autoantibodies—indirectly implicating Th2 cells in the pathogenesis of UC (148, 149) B cells As alluded to above, alteration of TLR expression has been implicated in IBD. But in addition to alteration on T cells and epithelial cells, alteration of TLRs can also occur on B cells (150, 151). The significance of these possible alterations as they relate to how B cells may affect responses to microbes or B cell function in the context of IBD has not yet been elucidated. Perhaps more significant is the fact that B cells play a very important role in maintaining homeostasis of the mucosa, primarily by production of abundant levels of IgA and IgM (152). Patients with IBD have been shown to have low levels of mucosal IgA, evidenced by strikingly reduced J-chain expression. High levels of J-chain expression are required for prevention of the generation of secretory antibodies, necessary for colonization of the mucosa by microbes and penetration of the mucosa by soluble antigens (152, 153). There is also a shift from IgA2 to IgA1, which is more susceptible to degradation (152) B cells may promote pathology in UC patients by inducing autoimmune reactions (148, 149). CD5+ B1 cells, which are localized to mucosal sectors as well as peritoneal and pleural cavities function in the production of antibodies which, in addition to having broad specificity against many pathogen associated molecules, also initiate a number of autoimmune reactions (154). As many consider IBD to be a disease of altered response to commensal bacteria that may also involve autoimmune responses, these cells were studied in affected individuals. It was found that these cells were decreased in the serum of patients with UC and that dexamethasone therapy 23 (often used to treat IBD patients) was a significant factor in this reduction through the induction of apoptosis (154). Epithelial Cells Intestinal epithelial cells (IECs) are the most abundant intestinal cell and represent the major site of sampling of luminal antigens, which these cells can translocate to the submucosal compartment, suggesting that epithelial cells may act as APCs for submucosal T cells or the intraepithelial lymphocytes (109, 135, 155). IECs likely also play a role in anergy of T cells, as evidenced by the fact that they express MHC class II molecules on their surface, but have no to low levels of costimulatory molecules (155). The mechanisms that underlie an antigen inducing tolerance or immunogenicity are unclear. However, the lack of responsiveness to commensal organisms is thought to be, at least in part, dependent upon 3 factors of the microorganisms (156). The first of which is the fact that commensal bacteria are unable to effectively colonize the intestinal mucosa due to the lack of pathogenic factors such as adhesins and invasins. Secondly, this lack of colonization results in persistent removal of the bacteria and their pattern associated molecular patterns (PAMPS) via peristaltic contractions. Finally, the endotoxic portion of LPS anchored in the outer wall of Gram negative bacteria (the primary component of the commensal flora) is pentacylated in the intestine making it of low endotoxicity. Reports have shown that LPS mutated by pentacylation was unable to: induce TNF production in murine peritoneal macrophages; activate NF-κB in transfected cells expressing murine TLR4; or induce conformational changes or oligomerization of TLR4 to allow for signal transduction. These findings suggest that this mutated form of LPS is nonpathogenic (157). IECs also participate in the induction of tolerance to commensals. Some IECs have been shown to lack expression of TLR4 and cofactors required for LPS recognition. In other cases, location of the TLR may be 24 key, such as seen with TLR5 which recognizes a subunit of bacterial flagellin (155). TLR5 expression is limited to the basolateral surface of the IEC, suggesting that flagellin recognition might require delivery of this ligand to the basal site of the epithelium. Basolateral translocation, however, requires overcoming the barrier of epithelial tight junctions. Finally, some recognition of PAMPs occurs intracellulary. Commensal bacteria, in contrast to pathogenic bacteria are unable to transport PAMPs into epithelial cells (155). Maintenance of the integrity of the intestinal mucosa is of utmost importance to prevent excessive entry of bacteria and other antigens from the intestinal lumen into the circulation (1, 80). The key component for an intact mucosal barrier is the functionality of the tight junctions, which seal the space between adjacent epithelial cells (1, 80, 158). The junctions are dynamically regulated in response to cytokines such as IL-13, TNF, IFNγ, and IL-17, as well as chemokines and the submucosal immune cell network (1, 158). Patients with CD and UC have been shown to have altered permeability related to reduction in tight junction strand number, reduced depth of tight junctional meshwork and the appearance of strand discontinuities (1, 159). These strand discontinuities allow macromolecules like food antigens and bacterial LPS to penetrate the mucosal barrier (159). Expression analysis from IBD biopsies has revealed downregulation of junctional complexes (E-cadherin and β-catenin) and upregulation of apoptosis of epithelial cells (1, 159). An alteration of tight junction architecture in intestinal epithelial cell monolayers, similar to what has been seen in UC patients, was observed after exposure of cells to TNF and IFN-γ (159). TNF was shown to induce an increase in intestinal tight junction permeability mediated by an increase in myosin light chain kinase in an NF-κB dependent manner (160). Treatment with anti-TNF biologics has been shown to stimulate improved mucosal healing and decrease epithelial cell apoptosis in some patients with CD (9, 159). Apical and lateral tight 25 junctions, situated at the membrane between apical and lateral regions of polarized epithelial cells, selectively regulate the passage of molecules and ions via the paracellular pathway and also restrict the lateral movement of molecules in the cell membrane (161-163). Claudins (CL) are one of the most important classes of proteins shown to be important in paracellular maintenance of epithelial tight junctions and comprise a family of at least 24 proteins (164). Studies evaluating the effect of cytokines that are typically elevated in CD (i.e. TNF and INF-γ) on claudin expression revealed that the combination of TNF and INF-γ resulted in decreases in CL2 and 3, the redistribution of CL4 and a marked increase in paracellular permeability. IL-13 also resulted in increased paracellular permeability due to dramatically increased CL2, with little effect on CL3 or 4 (164). These data suggest that, in contrast to IL-13, TNF may act by removing claudins from the tight junction but, not by altering expression of specific claudin isoforms. Thus, presumably, anti-TNF therapy could improve intestinal barrier function also by abrogating the effect of specific claudin loss. Intestinal surfaces harbor other epithelial lineage cells, such as Paneth cells and goblet cells, which also contribute to limiting bacterial penetration of host tissue. Goblet cells secrete large quantities of mucin, composed of glycosylated proteins, which forms a bi-layered, semipermeable protective coat over the mucosal surface, which is also important in helping to accelerate the repair of intestinal damage (109, 156, 165). The outer layer of mucous is loosely adherent and colonized by bacteria. The inner layer is adhered to the epithelial surface and is resistant to bacterial penetration, thus preventing epithelial access by nonpathogenic bacteria which do not posses enzymes that would facilitate penetration of this barrier (156, 165). Both of these layers are composed primarily by the secreted gel forming mucin (MUC), MUC2. In contrast to wild type (WT) mice, Muc2 deficient mice have bacteria in direct contact with the 26 mucosal epithelium and extending down into the crypts (165). These mice are reported to develop colitis by 7 weeks of age and colon cancer by 6-12 months, again highlighting the contribution of the intestinal flora to the development of colitis and colitis associated colon cancer (165, 166). Patients with UC have been shown to have a defective mucous layer and decreased numbers of goblet cells in upper third of the crypts (167, 168). Surprisingly, however, patients with CD have an upregulation of goblet cell differentiation associated with inflammation (167, 168). Also of note, in areas of goblet cell depletion, MUC2 expression has been demonstrated in cytoplasmic granules of flattened, cuboidal, nongoblet-cell-like (precursor) surface cells, possibly representing a nonspecific repair function of the colon cells to compensate for damage to barrier function (169). Goblet cells from patients with UC and CD have been shown to express MUC5AC, which is not expressed by normal colonic tissue (169). The MUC5AC gene product has been identified as a component of the effusions seen in patients with otitis media with effusions and has been shown to be upregulated in response to LPS (169-171). In these cases the TNF has been shown to stimulate MUC5AC production, resulting in hypersecretion of mucin (170, 171). These findings may partially explain the increase in goblet cell differentiation noted in CD patients and/or present a possible role for TNF in attempting to promote intestinal healing. Paneth cells are specialized epithelial cells that normally reside at the base of the crypts in the small intestine, however Paneth cell metaplasia has been observed in cases of colonic infection and in patients with long standing IBD and is considered a pre-neoplastic lesion in the progression to colorectal cancer (7, 111, 172, 173). In addition, humans with either loss of NOD2 or ATG16L1 function or deletion of at least one gene associated with the endoplasmic reticulum (ER) stress response not only have a genetic predisposition for the development of IBD, but also 27 present with abnormalities in Paneth cell structure and function (7). Paneth cells secrete a diverse repertoire of antimicrobial peptides, including α-defensins, lysozyme, and RegIIIγ (7, 109, 111). Paneth cells directly sense gut commensals and maintain homeostasis by controlling mucosal penetration of both commensal and pathogenic bacteria through MyD88 dependent TLR activation (109, 111). Studies utilizing mice with genetic ablation of Paneth cells have shown increased translocation of bacteria to the submucosa and to mesenteric lymph nodes in these deficient strains of mice (111). Traditionally, aberrant Paneth cell structure and function have been considered the primary contribution by these cells to the progression of IBD. However, experiments using Il-10 deficient mice, which spontaneously develop colitis, revealed that these mice expressed high levels of TNF within the granules of the Paneth cells, prior to lesion development (174). The function of TNF derived from Paneth cells is unknown, however this finding led to the hypothesis that TNF production by Paneth cells, possibly triggered by intestinal microbes, may be a key component of the chronic intestinal inflammation seen in IBD (174, 175). In the adult human intestine, TNF expression by Paneth cells appears to be induced when crypts and Paneth cells are damaged rather than constitutively produced (174). These reports suggest that TNF may a role in the reconstitution of the crypt cell population (176). Mesenchymal Cells The intestinal myofibroblast population consists of two cell types: the interstitial cells of Cajal (ICC) and the intestinal subepithelial myofibroblasts (ISEMF) (177, 178). These cells not only provide the structural support of the lamina propria, but also have immunomodulatory effects on epithelial cells and leukocytes, which may play an important role in the pathogenesis of IBD (179). The ICC are located in an intramuscular space in the submucosa and provide pacemaker functions such as regulating peristalsis neuronal transmission (180). The ISEMF 28 exhibit features of both fibroblasts and smooth muscle cells and reside subjacent to the basement membrane (177). One of the potential complications of IBD is the induction of significant fibrosis and recent studies suggest that mesenchymal cells derived from bone marrow stem cells are crucial in intestinal repair and fibrosis (177). The ISEMF and ICC isolated from the intestinal mucosa and submucosa have been shown to be able to undergo in vitro differentiation into various tissue types (e.g. bone, fat, cartilage) as well as trans-differentiation from hepatic stellate cells, skeletal muscle cells, and cells of neural crest origin; induce the differentiation and organization of intestinal epithelial cells; inhibit T-cell proliferation; and increase the levels of anti- inflammatory mediators such as IL-10 (179, 181). These cells are usually activated by IL-6 and TFGβ and have been shown to secrete growth factors such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF); cytokines such as IL-1, IL-6, or IL-8; or chemokines such as RANTES or monocyte chemoattractant protein-1 (MCP-1) (135, 181). Evidence suggests that TNF can promote or inhibit collagen synthesis in a tissue-specific manner (182). In patients with IBD, there is a transmural increase in the number of myofibroblasts and these cells colocalize with sites of type I collagen mRNA expression and collagen deposition (183, 184). In vitro studies utilizing intestinal myofibroblasts indicate that TNF increases collagen accumulation and decreases collagen degradation and matrix metalloproteinase (MMP)-2 activity via signaling through TNFR2 (177, 182). TNF has also been shown to induce myofibroblasts to secrete the growth factors GM-CSF and G-CSF as well as, increase secretion of IL-6. IL-17 enhances this IL-6 secretion induced by TNF by stabilizing IL-6 mRNA (185) . In addition, human colonic myofibroblasts actively secrete “destructive” 29 MMPs, such as MMP-3, but also tissue inhibitor of matrix metalloproteinase (TIMP)-1 in response to IL-17, IL-1β, and TNF (186). Mice carrying a deletion in the TNF AU-rich elements (ARE), resulting in overexpression of TNF (Tnf ΔARE mice) develop spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis and Crohn’s-like IBD with elevated TNF levels (187, 188). Bone marrow grafting experiments demonstrated that development of arthritis is dependent upon TNFR1 expression in the radiation-resistant compartment. It was shown that in these mice, mesenchymal cells are activated before onset of inflammatory disease with upergulation of MMP-3 and downregulation of TIMP-1 (187). Specific deletion of TNFR1 from mesenchymal cells resulted in decreased evidence of arthritis and CD-like IBD, demonstrating that activated mesenchymal cells were sufficient TNF targets to induce the pathology seen in Tnf ΔARE (187). Professional Antigen Presenting Cells Antigen presenting cells represent a major population of cells in the innate immune system. These cells are defined by their ability to express PRRs, and their ability to internalize, process, and subsequently present antigens on their cell surface (7). Surface presentation of antigens in conjunction with MHC Class I and II molecules, when combined with the enhanced expression of costimulatory molecules also induced by microbial stimuli acting through the same or different receptors, enable the APCs to activate adaptive immune responses (189). Professional APCs are distinguished from non-professional APCs by their expression of either MHC class I or II molecules and their ability to prime naïve T cells, however most cells in the body can present antigen to CD8+ T cells via MHC class I molecules and, thus, act as 30 nonprofessional APCs (190). Monocytes, macrophages, and DCs are the primary classes of professional APCs and have a common origin in hematopoietic stem cells (191). Monocytes circulate in the blood, bone marrow and spleen. These cells are equipped with chemokine receptors and adhesion receptors that mediate migration from blood to tissues during infection, but they do not proliferate in a steady state (192). Monocytes are capable of migrating to tissue under inflammatory or non-inflammatory conditions and differentiating into macrophages or DCs (193, 194). In the absence of inflammation, bone marrow monocytes have an immature phenotype and are characterized by low levels of expression of MHC class II and costimulatory molecules (194). Microbial infection induces the recruitment of monocytes to mucosa-associated lymphoid tissues and in acute cases of Salmonella typhimurium or Listeria infections in mice, for example, these cells are the main producers of TNF and iNOS (195). It should be noted that in the absence CD11b+ monocyte mediated recruitment of granulocytes and monocytes, L. monocytogenes replicates abundantly within nonphagocytic cells. Thus, CD11bmediated recruitment of inflammatory cells is essential for bacterial containment and killing during the acute stages of Listeria infection (194). In patients with IBD, it has been demonstrated that monocytes are recruited at a high rate to the inflamed mucosa and these cells are more susceptible to LPS stimulation, which likely results in propagation of inflammation in the diseased intestine (196). Different studies have also shown that monocytes in the IBD intestine produce high levels of TNF, IFN-γ, and IL-6 and that, in vivo, the most potent inhibitors of these proinflammatory cytokines was the combined addition of IL-4 and IL-10 or IL-13 and IL-10 (197). When comparing UC versus CD, it has been shown that blood monocytes from CD patients produce higher levels of TNF and IL-1β than those from UC or normal controls. In addition, the cytokine production from both UC and 31 CD patients is higher from patients with active disease versus those in remission (198). These data indicate the monocytes are important in the enhancement of IBD pathology, but may play a propagating or diminishing role in certain intestinal infectious diseases. Cell-extrinsic activation of the innate immune system is mainly mediated by macrophages and DCs and does not require the cells expressing these receptors to be infected. This type of activation is achieved via signaling through transmembrane receptors (including TLRs and dectins) (199). Cell intrinsic activation generally requires that the cell is infected and is mediated by NOD-like receptors (NLRs) or RIG-I-like receptors (RLRs); these PRRs are broadly expressed because most cells can potentially be infected by pathogens (199). Macrophages and DCs express a wide array of PRRs, are efficient phagocytic cells and produce a myriad of inflammatory cytokines. Both cell types differentiate from the monocyte lineage and are widely dispersed throughout the body in lymphoid and nonlymphoid sites (189, 193). Macrophages and DCs are integral in tissue homeostasis, via the clearance of apoptotic cells, and the production of growth factors (191). DCs are very similar to macrophages in that they are also important in removal of apoptotic cells and are responsive to the same growth factors and express the same surface markers (including CD11c) (193); however, exposure of monocytes to GM-CSF and IL4 induces differentiation of human and mouse monocytes into DCs, whereas exposure to M-CSF induces monocytes to differentiate into macrophages (191). Supplementing M-CSF with IFN-γ or lipopolysaccharide (LPS), produced for example by activated macrophages or DCs, results in the differentiation of “classical” M1-like macrophages; whereas addition of IL-4 induces the differentiation of “alternative” M2-like macrophages (191, 200). M1 macrophages exhibit potent microbicidal properties and promote strong Th1 responses, mediated by IL-12. In contrast, M2 macrophages support Th2 effector responses and may play a role in resolution of 32 inflammation (201). Alternative activation can be of both innate and acquired origin and several cell types can produce the required cytokines (IL-4 and IL-13), including conventional CD4+ Th2 and CD8+ T cells, NKT cells, basophils, mast cells, and eosinophils (201). As alluded to in the genetic section of this review, two studies, ~30 years apart, made the seemingly paradoxical discovery that patients with CD had a defect in granulocyte influx, both systemically and in the gut and that this defect was related to impaired cytokine secretion by macrophages (202, 203). In 2006, Anthony Segal’s group (204) demonstrated that patients with CD had defect in wound healing and neutrophil influx secondary to cutaneous E. coli infection. Combined, these studies suggest a causal link between impaired inflammation and impaired bacterial clearance. Further investigation revealed that these patients’ neutrophils were functionally and morphologically intact, however, their macrophages failed to secrete proinflammatory cytokines and chemokines, including some that attract granulocytes (204). Remarkably, the defect applied to various experimental conditions and like the impaired attraction of granulocytes and bacterial clearance, this phenotype was distinct from healthy controls and was shared by all CD patients tested, regardless of the NOD2 (204). The macrophages were shown to transcribe adequate levels of cytokines; however the cytokines were destroyed in lysosomes inside the cell. This decreased secretion of cytokines resulted in deficient neutrophil influx and subsequent killing of pathogens and eventually a T-cell mediated granulomatous reaction in response to high bacterial loads (204). Despite the attractiveness of the theory that CD results from a primary immunodeficiency of macrophage (77), many studies have shown that activated DCs and macrophages secrete several cytokines that actively regulate the inflammatory response in UC and CD by triggering the differentiation of T cells (205). The primary orchestrating cytokine produced by APCs (and 33 lymphocytes) is TNF and its expression has been noted in both UC and CD patients, demonstrating its central role in both diseases (206, 207). TNF has pleiotropic effects such as increased production of IL-8, IL-12, IL-18, IL-1β and IL-6, expression of adhesion molecules, proliferation of fibroblasts and procoagulant factors, as well as initiation and inhibition of apoptosis (207-210). Studies have shown that in CD or UC high numbers of activated macrophages and lymphocytes were seen in the lamina propria, whereas few were seen in non-disease mucosal samples. In addition, there was an increase in monocyte turnover, suggesting that most of the macrophages present were newly recruited cells from the peripheral blood (211). Although many cell types can produce granulocyte monocyte-colony stimulating factor (GM-CSF) and TNF, a correlation between the increased numbers of macrophages and increased expression of GMCSF and TNF in diseased areas of the colon compared with control mucosa has been postulated (212). GM-CSF (and TNF) has many functions, but in IBD, mucosal production of GM-CSF (and G-CSF) was related to a delay in neutrophil apoptosis in IBD (213). Macrophages are capable of producing a variety of cytokines (e.g. IL-6, IL-1, IL-12, IL-18) with varying effector functions, however, as previously stated, TNF is considered to be a master regulator of effector responses (207). In addition to secretion of TNF, TNF can induce macrophages to secrete a variety of cytokines and in most cases cytokines themselves, which are present in the environmental milieu, such as INFs, modulate the cytokine/chemokine secretion by macrophages in response to TNF (214). Macrophages also release reactive oxygen and nitrogen species and proteases that degrade the extracellular matrix. The combination of cytokine production and matrix degredation suggests that macrophages are key players during resolution of inflammation and repair of the intestinal mucosa that occurs during disease remission (207). 34 DCs are located at mucosal interfaces, are phenotypically heterogeneous and control several aspects of the immune response including type (Th1, Th2, Th3, Th17 or Treg) and homing of antigen specific effector cells (215, 216). DCs also play a key in discriminating between commensal and potentially pathogenic microorganisms, thus maintaining the balance between tolerance and active immunity. Studies of DCs in human IBD and murine models of colitis have shown increased numbers of activated DCs at sites of intestinal inflammation, suggesting play a role in the pathogenesis of IBD (217-219). In addition, Baumgart et al demonstrated (216) that patients with active IBD lacked immature blood plasmacytoid and myeloid DC, suggesting recruitment of these cells to the inflamed colon. This increase in numbers of DCs has been shown to be related to elevated levels of the DC and memory T cell attractant, CCL20, which was dependent upon increased TNF and blocked by anti-TNF (219). A subset of human DC precursors has been described in the peripheral blood recognized by the monoclonal antibody M-DC8. This subset of cells, detected in the inflamed mucosa of CD patients, has been shown to secrete in response to LPS >10 times the amount of TNF as MDC8(-) monocytes, and produce significantly less IL-10 and thus, may contribute to the high level of TNF production seen in CD patients (220). Activation of CD40, was enhanced on lamina propria DC of patients with CD and UC and these produced more IL-6 and IL-12p40, than controls (221). Supporting the role of TNF as an inducer of DC activation, treatment of patients with CD patients with anti-TNF therapy resulted in a reduced expression of CD40 by lamina propria DCs (221). DCs can also be the source of the cytokines that contribute to the T helper profile seen in CD or UC (222). In UC, production of IL-27, was increased in the lamina propria and was associated with an atypical Th2 response mediated by NKT cells producing IL-13, suggesting that in UC, DCs may regulate 35 NKT cell activity through production of IL-27 (222). In CD patients, mesenteric lymph node myeloid DCs produced higher amounts of IL-23 and lower amounts of IL-10 and was associated with a stronger Th1 immune response in mixed leukocyte reactions compared with those from UC and controls (223). The established roles of DCs in promoting effector functions which contribute to the propagation of inflammation in IBD suggest that DCs may present a novel target for regulating IBD. IBD ASSOCIATED COLORECTAL CANCER Cancer of the colon and rectum (CRC) is the third most common malignancy in humans (224). The frequency is higher in males than females and the ratio of colon versus rectal cancer is approximately 2:1. There are wide geographical variations in the incidence of CRC that it is very frequent in North America, Europe and Australia whereas is much less frequent in Africa, Asia, and South America (224). Asian populations that transferred in North America acquire the same risk of the local populations within one generation, suggesting that environmental differences and particularly alimentary customs, likely affecting the commensal microbiota, are responsible of the geographical variation in incidence (225). Indeed obesity, lack of exercise, fat-rich diet and use of alcohol and tobacco represent risk factors for CRC (226). The gut microbiota affects the development and activity of mucosal innate and adaptive immunity, the catabolism of natural mutagens and carcinogens, and, acting through innate receptors, the inflammatory environment in the gut. In addition to the canonical example of Helicobacter pyloris for gastric cancer, enterotoxigenic Bacteriodes fragilis, able to activate the Wnt and NF-κB pathway (227), and attaching/effacing Escherichia coli, able to downregulate genes encoding DNA mismatch mutation repair (MMR) proteins (228), have been suggested to associate with increased risk of CRC. IBD, both CD and UC, represents a major risk factor for CRC (colitis-associated cancer, 36 CAC) with an overall risk (OR) of approximately 3 (229, 230) but the majority of CRCs develop without any obvious pre-existing inflammatory pathology. In the adenoma-carcinoma development, the earliest identifiable lesion both in humans and in experimental animals is the aberrant crypt focus (ACF) represented by a small dysplastic lesion of the crypt opening. Both a top-down model with the transformation initiating from the surface epithelial cells spreading down the crypt and forming new crypts or a bottom-up model with the lesion originating in the deep layer of the crypts have been proposed for CRC (231, 232). Approximately 30 percent of CRCs are familial as determined by kindred and twin studies and 5 percent of the cases are associated with well characterized highly penetrating inherited gene mutations (233). The remaining inherited cases of CRCs are not well understood and may be due to single less penetrating genes or to combinations of them. The most frequent CRC genetic syndrome is the Lynch or hereditary nonpolyposis CRC syndrome characterized by high susceptibility to CRC and endometrium cancer as well as other types of cancer and it is due to autosomal dominant mutations in genes controlling methylation of mismatch repair (MMR), predominantly hMLH1 and hMHS2, with almost complete penetrance, the severity and precocity of the clinical manifestation depending by the gene affected and type of mutation (234). As in all autosomal dominant CRC genetic syndromes, loss of heterogeneity (LOH) with mutation or silencing of the normal allele is responsible for the initiation of the neoplastic transformation. Most CRCs from these patients are characterized by microsatellite instability (MSI) (235). MSI is observed also in many sporadic CRCs, usually associated with a more favorable prognosis, and in these cases an epigenetic bi-allelic silencing of MMR genes rather than mutations is observed (235). The second most common CRC genetic syndrome is the Familial Adenomatous Polyposis (FAP) with different autosomal dominant mutations of the APC genes, thus resulting in activation of the β- 37 Catenin pathway in cells with LOH (233, 236). Depending on the type of adenomatous polyposis coli (APC) mutation, FAP may present in a classic or attenuated form (236). In the most severe cases starting at young age hundreds of adenomas form in the colon and rectum, but also in the higher gastro-intestinal tract and if not treated they invariably progress to CRC in the fourth or fifth decade of life (233). Among the most rare syndromes, of particular interest are the MUTYH-associated poliposis (MAP) due to autosomal recessive mutation of the MUTYH gene controlling the base excision repair pathway important for defending against DNA oxidative damage (237), and two autosomal dominant hamartomatous polyposis syndromes, the PeutzJeghers syndrome (PJS), due to a mutation in the tumor suppressor serine/threonine kinase 11 (STK11) gene (238) and the Juvenile Polyposis Syndrome (JPS) due to mutations of the genes encoding bone morphogenic protein (BMP) receptor 1A or its signaling protein mothers against decapentaplegic homolog (SMAD)4 (239). GWAS studies have identified approximately ten genes significantly associated with sporadic CRC (240) including BMP4 and SMAD7, cadherin1, and colorectal adenomas and carcinoma susceptibility gene 1 (CRAC1) previously identified as responsible for hereditary mixed polyposis syndrome in a large family of Ashkenazi descent (241). However, the effect of each of these mutated genes is responsible only for an OR between 1.10 and 1.25, indicating the low penetrance of each mutation and that the combination of different mutations or other contributing factors are required for CRC development (240). For example, variants in the TGFBR1 gene have been shown to modify CRC frequency in high risk family and this effect has been confirmed in mouse genetic models (242). Thus, the molecular mechanisms involved epithelial self-renewal and controlled differentiation and apoptosis in the gut and those involved in the deregulated growth of CRC are identical and mutations leading to change in their functions, particularly for the genes controlling the Wnt/β-catenin, MMR, and 38 BMP pathways, lead to adenoma and CRC progression (243). Interestingly, one of the genes that was found to be targeted for mutation in CRC with altered MMR is the cytosolic DNA activated inflammasome Absent In Melanoma 2 (AIM2); restoration of AIM2 in CRC cells induce cell cycle arrest but also promote invasion by affecting adhesion to extracellular matrix (244). Various rodent experimental models have been established that mimic the human CRC. These models are based on genetic alteration of the pathways known to be involved in clinical CRC (Wnt/β-Catenin, MMR genes, TGF-β pathways) or to affect immune responses (IL-10, IL2, β2-microglobulin, TCRα, Gαi1) as well as treatment with carcinogens, particularly Azoxymethane (AOM) or its precursor 1,2-dimethylhydrazine (DMH) (245, 246). Both DMH and AOM are procarcinogens with organotropism for the colon and they need to be further 6 metabolized into methylazoxymethanol to able to methylate the 0 position of guanine (246). Different mouse strains are variably susceptible to AOM induction of CRC with A/J being the most susceptible strain and the widely used C57Bl/6 having low but variable susceptibility depending on the sub-line (N, J, and Ha) (246). AOM induction of CRC is linked to its ability to induce mutation in several signal pathways including K-ras, Src/PI3K/Akt, β-catenin, TGFβ and p53 (247). Unlike human CRC in which APC mutations are frequent and β-catenin mutations are present only in a minor subset of patients, in the mouse AOM model the latter predominate (248). The number of adenoma and adenocarcinoma is dramatically increased if the mice after AOM administration are chronically treated with DSS that induces colitis and an inflammatory environment and acts as tumor promoter, mimicking the pathologic process of CAC (249). Among the most widely used genetic model is the APC Min/+ mice (Min, multiple intestinal neoplasia) that are heterozygous for a nonsense mutation at codon 850 of the Apc gene, 39 the murine homolog of the human APC gene (250). This mutation is analogous to one seen in humans with FAP. APC Min/+ mice develop numerous intestinal tumors and in some conditions CRCs, but are also more susceptible to mammary and alveolar neoplasia (250). AOM treatment induces appearance of in the colon of APC Min/+ larger number of polyps mice that are otherwise rare; these polyps are larger and with more infiltrating characteristics than in AOM treated WT mice but unlike in the latter they do not express β-catenin mutations (251). In studies in which APC Min/+ mice were adoptively transferred with proinflammatory T cells to induce intestinal inflammation, these mice had a high rate of intestinal carcinogenesis and (in females) mammary tumor formation. Neutralization of TNF with anti-TNF antibodies abolished tumor formation at both sites and was associated with downregulation of c-Myc. These data indicate TNF has a tumorigenic role in this model (252). In contrast, a recent study in which APC crosses with Tnf -/- Min/+ mice were mice and treated with DSS, the lack of TNF had no effect on the level of inflammation or tumorigenesis (253) suggesting that TNF is important for T cell-mediated inflammation in the adoptive transfer model but not for the DSS induced inflammation secondary to interaction with the commensal flora In addition to using animal models which exploit known human genetic alterations associated with CRC to induce CRC in mice, there are models which attempt to evaluate the relationship between the chronic inflammation seen in IBD patients and the development of CAC. One of the more commonly used models utilizes the colonic genotoxic carcinogen AOM to induce mutations in the context of chronic inflammation induced by the synthetic sulfate polysaccharide, DSS. In a recent paper, Popivanova et al (254) showed that mice deficient for TNFR1 or treated with soluble human TNFR2 (etanercept) dysplayed decreased intestinal 40 inflammation and decreased formation of polyps. These data suggest that TNF has a promoting role in the development of chronic inflammation which leads to intestinal tumor formation. However, TNFR1 and TNFR2 bind both TNF and lymphotoxin-α, making it impossible to attribute the observed effect to either or both members of the TNF family. Recently, Onizawa et al (255) showed that anti-TNF antibodies decreased the formation of polyps but not inflammation in the AOM/DSS model of colitis, suggesting that the role of TNF and other ligands of the TNFRs might be dissociated in the inflammatory mechanisms leading to colitis or carcinogenesis. Both types of animal models have provided much information on the role of inflammatory mediators in the development of CAC and in particular the role of innate and immune cells, cytokines such as TNF, IL-1, IL-6, IL-10, IL-11, IL-17, IL-22, IL-23 and of the axis STAT3 and NF-κB (256). Interestingly the activation of STAT3, which is mostly downstream of IL-6 and IL-11 signaling, plays a central role both in intestinal mucosal regeneration in response to injury as well as in the development of CAC (257-259). THERAPY IN IBD PATIENTS Antibiotics Therapy for IBD is a multifaceted approach. It is widely accepted that the high bacterial content of the distal ileum and colon are permissive to the pathogenesis of both CD and UC (260, 261). In fact, IBD patients have been shown to have increased intestinal Bacteriodes, E. coli and Enterococci with increased adherence of anaerobes such as Bacteriodes, and Enterobacteriaceae to the mucosal surface. As well, they have decreased levels of “healthy” bacteria, such as Bifidobacterium and Lactobacillus. These facts underscore the fact that the balance of beneficial 41 to pathogenic commensal bacteria determines the severity of disease in affected individuals (262). Given the role of the enteric flora in the pathogenesis of IBD, it is no surprise that the use of antibiotics is the foundation of therapy for the treatment of IBD (263, 264). Antibiotics are often used for the management of many of the secondary effects of CD such as localized peritionitis, toxic megacolon, bacterial overgrowth secondary to strictures, abscesses, pouchitis and perianal disease (260, 265). Antibiotics have been shown to decrease luminal bacteria, eliminate certain subsets of bacteria, and decrease bacterial translocation and systemic dissemination (260). However, the rate limiting affect of antibiotics appears to be the bacterial composition itself. In patients with CD, the two most widely used antibiotics are metronidazole and ciprofloxacin and alone or in combination, these two compounds have shown some success in CD patients. Clinical trials utilizing these agents have shown that responders are patients with disease limited to the ileocolonic or colonic regions, but not those with ileal disease (266). It is also important to note that both of these agents have significant immunomodulatory affects in addition to their anti-microbial properties, which may account for much of their therapeutic successes (266, 267). Unfortunately, antibiotic therapy has been relatively unsuccessful as a single agent for the routine treatment of UC, regardless of severity of disease (265). Overall, carefully designed studies to delineate the method and effectiveness of antibiotics are lacking. Primary problems with antibiotic trials include, but are not limited to, high dropout rates, poor study design, and insufficient numbers of study subjects (260). The result is that definite conclusions regarding antibiotics as therapy for primary CD cannot be made and antibiotic therapy for UC is not currently justified (104). Immunosuppressive agents 42 Corticosteroids (CS) are powerful inhibitors of T-cell activation and inhibit secretion of proinflammatory cytokines (268). These compounds are highly effective at reducing remission of IBD, but due to their inability to maintain remission for long periods of time and their high rate of side effects, they are only appropriate for short term use (269). CS therapy shows rapid reducing effects on active disease in CD patients, however by one year most patients are either steroid resistant or steroid dependent. In addition, although CS effectively treats the symptoms of CD, they usually do not promote mucosal healing which is the inciting cause of symptoms (270). Oral 5-aminosalicylic acid (5-ASA) compounds are used for management of mild to moderately active UC. CS therapy is reserved for patients with severe disease or for those that don’t respond to 5-aminosalicylic acid (271). Similar to CD patients, however, about 15% of patients are resistant after their first course of therapy and many are dependent by one year (272, 273). To combat the side effects of systemic therapy, switching patients to topical steroid therapy has been used and has shown good success. In CD patients, topical steroids resulted in a decrease in CS side effects and an increase in plasma cortisol levels (274). However, although studies have shown that CS enemas are effective for treatment of UC, they are inferior to treatment with 5ASA drugs for treatment of active UC (275). Immune modulators A host of pharmaceuticals which alter the immune system in some form have been used to treat IBD which target the pathogenesis of disease from many different angles. Leukocytes infiltration into the intestine is a key feature of UC and CD. Thus several agents have been developed which target molecules key in the process of leukocyte adhesion to endothelia and extravasation of cells from the lumen of vessels to tissue (276, 277). CD is accepted as having a predominant Th1/Th17 cytokine profile. To this end, inhibitors of Th1 polarization, namely anti43 IL-12 and anti-INF-γ antibodies have been employed in CD therapy (276). Anti-IL-12 therapy has shown some encouraging results in clinical trials, but no conclusive results on efficacy and safety have yet been drawn (278). Anti-IFN-γ trials have produced evidence of higher remission and response rates compared to placebo as well as good safety and tolerability (279). These results suggest that further clinical evaluation of both inhibitor of Th1 polarization should be undertaken. NF-κB is a key mediator of transcription of proinflammatory cytokines shown to be instrumental in the pathogenesis of IBD and may be a good therapeutic target for IBD patients. Non-specific inhibition of NF-κB can be achieved by anti-inflammatory aminosalicylates, which have shown to be effective in treating IBD (280). But, while aminosalicylates such as 5-ASA are the mainstay of therapy for UC they are only mildly effective for CD patients (269). Additionally, selective inhibition of NF-κB has also recently shown positive results (e.g. improved clinical, endoscopic and histological parameters) in clinical trials, but further evaluation is needed (265). Pharmaceuticals taking advantage of the anti-inflammatory properties of IL-10 and IL-11 have either failed to show efficacy or have shown mild efficacy with safety concerns, respectively (265). Anti-TNF Therapy Several components of the immune system have been targeted in the attempt to cure or effectively treat IBD. As already stated TNF plays a crucial role in both the initiation and propagation of inflammation in IBD and has been shown to be elevated in the mucosa of both UC and CD patients (281-284). To date, the only approved biological therapy for IBD patients, Infliximab, is an antagonist of TNF (265, 285). All of the compounds in this class, however, area able to bind both soluble and membrane TNF. Their ability to fix complement, induce 44 antibody dependent cytotoxicity, and induce apoptosis of T cells varies within amongst the compounds (Table 1.1) (276, 286). Infliximab (REMICADE®) is a mouse (25%)-human (75%) chimeric IgG1 monoclonal antibody approved for the induction and maintenance of moderate to severe CD in nonresponders to conventional therapy and in fistulizing CD (287). However, human anti-chimeric antibodies (HACAs) may develop in treated patients and is associated with acute infusion reactions, delayed hypersensitivity reactions and loss of response to therapy (286, 288). Patients with active UC and are refractory to conventional treatment have also shown efficacy to Infliximab therapy in 2 large trials (13). The most promising alternative to Infliximab is Adalimumab (D2E7/Humira®), a full humanized monoclonal IgG1 antibody. This compound has the same mechanisms of action but is less immunogenic than Infliximab (265, 289, 290). Undesirable adverse reactions occurred much less frequently than with Infliximab treatment and were often dose dependent. Preliminary studies indicate that there is good efficacy and safety with this compound and that it is effective in patients who are allergic/nonresponsive to Infliximab (265). Controlled trials are needed to determine if this compound will be useful in inducing remission of CD or treating fistulizing CD. 45 Table 1.1: TNF Targeted Therapeutic Agents [Modified from Danese et al (276)] Drug Infliximab Structure Binds both soluble and membr ane TNF Fixes Compl ement Induces antibody dependent cytotoxicit y Induces Disease Results in apopto Indicat Clinical sis in ion Investigat TNF ion express ing T cells Chimeric mousehuman IgG1 mAb Yes Yes Yes CD/UC Effective/ promising Yes Yes Yes CD Effective Pegylated Yes Fab Fragment of humanize d antiTNF antibody No No ? CD Effective in CRP>10 Humaniz ed anti TNF IgG4 No No ? CD/UC Effective in CRP>10/ modest Yes Yes Adalimumab Full humanize d antiTNF IgG1 CDP870 CDP571 Yes 46 Table 1.1 (continued) Human Yes Etanercept soluble p75 fused with Fc domain of human IgG1 Onercept Human Yes soluble p55 fused with Fc domain of human IgG1 No No No CD Ineffective No No No CD Ineffective CDP870 is fab fragment of humanized IgG1 monoclonal antibody attached to two polyethylene glycol molecules. This drug has been evaluated for treatment of CD, but not UC. In a phase II trial patients with high baseline levels of C-reactive protein (CRP) had greater response than those with low CRP levels (291). In a phase III trial, patients treated with certolizumab showed a higher rate of clinical response and remission than placebo treated controls, regardless of CRP levels (292). In the aforementioned phase II trial, adverse affects reported included injection site reactions, infection, and aggravation of CD (291). CDP571 (HUMICADE™) is a murine (5%)-human (95%) IgG4 monoclonal antibody, which was expected to be less immunogenic than Infliximab. However, due to limited therapeutic effects and limited steroid-sparing capabilities (e.g. no disease flare and no longer requiring corticosteroid therapy), this compound is no longer in clinical development (293-295). Soluble versions of the TNF receptors p75 and p55, Etanercept (ENBREL®) and Onercept, respectively, were also developed, but discontinued due to limited efficacy or limited conclusive data (265). 47 Antagonists of TNF are the most recent additions to the milieu of IBD therapeutic agents. Infliximab has shown good results in induction and maintenance of CD, however Adalimumab may be a better alternative due to its decreased immunogenic potential. Currently most therapy has been aimed at and/or is most successful at treating CD, rather than UC. Given the multifaceted pathogeneses of UC and CD, it is most likely that a combinational regime will be more effective than a single agent and the agents effective at treating CD may differ significantly from those effective at treating UC. Despite the beneficial effects of anti-TNF therapy for CD patients, the use of these drugs is not without risk. TNF antagonists are associated with increased morbidity and mortality in patients related to reactivation of latent tuberculosis, fungal infections, fatal sepsis or development of autoimmune disorders (296-299). In addition to these adverse reactions, there has been an increase association of malignancy in patients with rheumatoid arthritis (RA) and CD patients that are treated with TNF antagonists (300-302). CD (adult and child) patients treated with anti-TNF agents have been linked to a variety of malignancies, including: melanoma, malignant thyroid tumors, malignant leukemia, lymphoma, skin cancer, gastrointestinal (including colon) cancers, lung cancer, and breast cancer (302). It has been difficult to explain the mechanism of these findings due to the heterogeneity of the diseases and risks, patients with other diseases (such as RA) that increase the potential for malignancy, and confounding factors associated with combined treatment with other, potentially carcinogenic, compounds (302). Some studies have indicated a potential decrease in malignancy in patients treated with Etanercept versus other anti-TNF compounds, possibly due to the shorter half-life Etenercept (303, 304). The alterations in cytokine secretion may also be important. Polymorphisms in TNF are associated with autoimmune diseases and an increased risk of 48 lymphoma (305). It is therefore possible that inhibition of TNF results in a shift from a predisposition for autoimmunity to one of neoplasia. Gene expression profiling studies have also shown that anti-TNF therapy may contribute to alterations in apoptosis and DNA repair pathways, a potential mechanism for the increases in malignancy and autoimmune diseases reported with therapy (306). It is clear that there are potentially many reasons (some of which could be due to a combination of events) for the adverse side effects reported with anti-TNF therapy. These data suggest that patients with autoimmune disease may have an increased risk for cancer development, either due to the cause/effects of their disease or due to anti-TNF therapy. Despite the benefits patients have received with anti-TNF treatment, the associated toxicities warrant more in-depth studies into the mechanisms of the therapy as well as, continued attempts to improve this therapy. THE ROLE OF TNF IN SELECTED MOUSE MODELS OF IBD ΔARE The Tnf Mouse Model of Colitis As previously stated, TNF is a pleotropic cytokine and its role in the pathology associated with inflammatory bowel disease (as well as RA) is well established and is supported by the beneficial effects of anti-TNF therapy (307, 308). It has been shown previously that AU-rich elements (ARE), mapped to the 3’-untranslated region of transcipts of cytokines, including TNF, regulate of the stability of mRNA (309). Studies have shown that the ARE region of TNF mRNA is responsible for translation repression during unstimulated conditions (19). This repression is thought to be irreversible in stromal cells, but inducible, with appropriate stimulation, in hematopoietic cells (19, 310). Subsequent studies confirmed this theory by deleting ARE 49 function in fibroblasts (a non-hematopoietic cell type), resulting in these cells having increased accumulation of TNF mRNA and readily detectable levels of TNF protein secretion in culture (311). To address the impact of deletion of TNF ARE from the mouse genome, a 69 bp deletion of the TNF gene, encompassing the TNF ARE region was instituted in 129/SvE mice. Tnf ΔARE mice are stunted; have chronic overexpression of TNF with an increased susceptibility to endotoxemia; have hypoplastic thymuses; and develop chronic inflammatory arthritis and inflammatory bowel disease (311). The IBD seen in Tnf ΔARE mice has many features of CD and is primarily localized to the terminal ileum, with some involvement of the proximal colon. Intestinal lesions consisted of: blunting and widening of villi, chronic mucosal and submucosal inflammation (macrophages, lymphocytes and plasma cells with scattered neutrophils). With disease progression there was transmural infiltration of inflammation and development of undeveloped noncaseating granulomas (311, 312). In addition to hyperproduction of TNF, Tnf ΔARE mice have also been shown to have elevated production of the proinflammatory mediators IFN-γ and IL-17, as well as the antiinflammatory cytokine, IL-10 (Table 1.2) (313). Molecular characterization of Tnf has revealed that the development of intestinal pathology in Tnf ΔARE ΔARE mice mice is also dependent upon functional non-γδTCR CD8+ T cells and production of the Th1 cytokines IFN-γ and IL-12. Surprisingly, Tnf ΔARE mice lacking CD4+ T cells exhibited increased mortality and exacerbation of intestinal disease (311). In contrast, the lack of CD8+ T cells resulted in marked attenuation of IBD. Backcrossing of the Tnf ΔARE 50 mice onto IL-12p40 or IFN-γ deficient mice also resulted in attenuation of disease. The histopathological and effector T cell features described in this model make it an intriguing option for the study of CD. However, to date, mutations in TNF ARE have not been described in humans and CD4+ T cells, unlike in the Tnf ΔARE mice, play a prominent role in the pathology of the disease in humans. The IL-10 -/- Mouse Model of Colitis IL-10 is a cytokine produced by the Th-2 subset of CD4+ T cells and is a potent inhibitor of the Th1 dominant cytokines (e.g. INF-γ and IL-12) (314). In addition to inhibiting the Th1 cytokine response, it also enhances the viability of B cells and their expression of MHC class II, while unaffecting B cells proliferation and class switching (315, 316). IL-10 is also an effective suppressor of activated macrophage production of the proinflammatory cytokines IL-1, IL-6 and TNF (314). In addition, IL-10 can decrease serum elevation of TNF (317). Of note, patients with IBD have been shown to have lower production of IL-10 when compared to healthy control subjects (318). -/- These findings led to the development of Il-10 deficient (Il-10 ) mice which were subsequently found to develop an age associated, chronic enterocolitis and anemia with overproduction of TNF, IL-1 and IFN-γ (319). The intestinal flora has also been shown to -/- contribute to the pathogenesis of disease in Il-10 mice. Both narrow and broad spectrum antibiotics are capable of preventing disease in Il-10 deficient mice and germ free Il-10 -/- mice do not develop colitis (320, 321). As well, it has been shown that colonization of the intestine of germ free reared, Il-10 deficient mice with certain populations of bacteria either had no inducing effect of colitis or, as in the case of Enterococcus faecalis, not only induced inflammation, but 51 also dysplasia and adenocarcinoma (322, 323). However, lesions were attenuated in the duodenum, but similar and more severe and localized in the colon of Il-10 deficient mice reared under specific pathogen free (SPF) conditions (319). Histopathology of the small and large intestinal lesions was profound. Lesions consisted of both excessive regeneration and degeneration of intestinal epithelium. In the small intestine, excessive inflammation and regeneration resulted in intestinal thickening and abnormal crypt and villus formation (319). Erosions, mononuclear to granulomatous inflammation and hypoplasia of the gut associated lymphoid tissue (GALT) were also present, but severity decreased from the duodenum to the distal small intestine. -/- In depth analysis of Il-10 revealed normal development of B and T cells as well as a normal immune response to T cell dependent immunization. However, these mice develop a Th1 response to nematode infection, which was not seen in WT mice, thought to be due to the lack of suppression by IL-10 (319). Given the spontaneous elevations of Th1 dependent cytokines in these mice, studies using antibodies to block both Th1 and Th2 cytokines (including, but not limited to: TNF, IFN-γ and IL-12) where undertaken (324, 325). Rennick et al found (324) that -/- blockade of IFN-γ and in 3 week old, but not 3 month old, Il-10 mice resulted in drastic reduction of colitis, suggesting that IFN-γ is instrumental in the inductive phase of the disease in these mice, but is not required for maintenance of the disease. Similar results were found in 3 -/- week old Il-10 mice using neutralizing antibodies against IL-12. Surprisingly, no effect was -/- noted when TNF was neutralized in both 3 week and 3 month old Il-10 mice (Table 1.2) (324). Scheinin et al, however, found (325) that antibody blockade of TNF commencing at 4 weeks of age and administered 3 times/week until mice reached 20 weeks of age markedly ameliorated 52 -/- clinical and histopathological disease in SPF Il-10 mice. In addition, levels of TNF-R p75 and -/- IL-1β were decreased in the stools of anti-TNF treated Il-10 mice, correlating well with the decrease disease scores (325, 326). Additionally, IL-10 therapy has been shown to prevent trinitrobenze sulfonic acid (TNBS) mediated pathology with concomitant decreases in proinflammatory cytokines (327). The findings from this model support the protective role of IL-10 in IBD and the beneficial effects of blocking both IFN-γ and TNF. The CD4+CD45RBHIGH T Cell Transfer Mouse Model of Colitis In both CD and UC, it is largely accepted that disease development and progression are predominantly due to the excessive immune reactions elicited by pathogenic effector CD4+ T cells, disproportionate to the regulatory T cell response (328). Expression of CD45 is necessary for the activation of T cells and in the mouse, high levels of expression of CD45RB on T cells is believed to distinguish naïve T cells from the low expressing activated memory T cells (329, 330). In humans, the CD45 isoforms, CD45RA designates the naïve T cells from the CD45RO memory T cell isoforms (331). ten Hove et al first demonstrated that in the circulation, virtually high all CD4+CD45RB low CD4+CD45RB high (CD45RB ) T cells were of the CD45RA isoform and the low (CD45RB ) cells were of the CD45RO isoforms. They next showed that in patients with IBD, most of the intestinal T cells in both the lamina propria and in lymphoid follicles were CD45RA+, however in normal controls 90% were CD45RO+ (331). Cytokine profiling of these subsets revealed that the CD45RBlow population in IBD patients produced less IL-10 and their CD45RB high T cells produced more TNF than those of the controls (331). 53 One model developed to study the mechanisms of IBD exploits the nature of a pathogenic high subset of CD4+CD45+ T cells (332). This model involves the transfer of CD45RB T cells from immunocompetent mice to immunodeficient mice, resulting in the development of chronic colitis with features reminiscent of both CD and UC (332). Whereas, when CD45RBlow T cells high are co-transferred with the CD45RB cells, disease is prevented or delayed (329). The high histopathological features of the disease in mice receiving CD45RB cells reflected chronicity as evidenced by the predominantly granulomatous inflammation and epithelial hyperplasia. The inflammation was often transmural, similar to that seen in CD, but features reminiscent of UC were also present and included extensive mucin depletion, as well as crypt elongation and branching(332). This model is also dependent upon the microbiota. Disease could be high ameliorated when CD45RB cells were transferred to SCID mice with a reduced intestinal flora or to mice which had been treated with antibiotics (333). high The CD4+ T cells seen in mice transferred with CD45RB cells had a highly polarized Th1 response similar to what has been seen in CD patients (334). In addition, Williams et al showed (335) that in general, myeloid cells infiltrating into diseased tissue predominantly comprised macrophages, neutrophils and DCs. Activated macrophages in the lamina propria had upregulation of both transcription and translation of TNF and these cells clustered in areas of tissue destruction (335). The Th1 profile in diseased mice was evidenced by elevated colonic levels of IFN-γ and TNF, as well as low levels of IL-10 and IL-4 (Table 1.2) (334). The Th1 profile, wasting syndrome, and colitis could be abrogated or significantly reduced by administration of anti-IFN-γ antibody administered soon after T cell transfer or by 54 continuous administration of neutralizing anti-TNF antibody (334). These findings indicate this model sufficiently replicates the pathogenic response elicited by TNF and IFN-γ producing CD4+ activated T cells. Transfer of Tnf +/+ -/- Rag 2 or Tnf -/- high CD45RB cells to Tnf -/- recipients resulted in a complete absence of disease. Surprisingly, transfer of Tnf -/- CD45RBhigh cells to Tnf +/+ -/- Rag 2 recipients resulted in colitis similar to that reported in the original description of the model. This report provides evidence that TNF production by non-T cells plays a nonredundant role in the pathogenesis of this model (336). The histopathological features of this model, however, exhibit features of both UC and CD making it of questionable use to study the specific pathogenesis of one form of IBD versus the other. The DSS Mouse Model of Colitis The DSS model of colitis was originally reported as a reliable model of acute and chronic ulcerative colitis in mice, induced by providing them with drinking water containing 5% DSS (337). It is thought that DSS causes direct toxicity to colonic crypt epithelia, as well as disrupts mucosal integrity (338). One 7 day cycle of DSS followed by 10 days consumption of distilled water results in severe damage, primarily confined to the distal colon (337, 339). Mucosal damage can be achieved in both WT and immunodeficient mice [severe combined -/- immunodeficiency (SCID) and Rag1 ], indicating that innate, rather than acquired immunity is the driving force of this model (338). In addition, the microbial flora is a critical component of this model, evidenced by the fact that treatment with antibiotics proven to be effective in patients with IBD ameliorates the disease (8, 340). Notably, although the role of specific components of the microbiota is unclear in this model, it has been shown that luminal concentration of Bacteroides are increased in both acute and chronic DSS colitis (337). 55 Clinically, DSS treated mice lose weight secondary to diarrhea and are often anemic due to rectal bleeding. DSS causes crypt depletion with more delayed regeneration than other chemical models (e.g. ethanol and acetic acid) (339, 341). Histopathology following one cycle of DSS reveals extensive damage to epithelia with marked inflammation (neutrophils and mononuclear cells), as well as marked edema and often, severe ulceration and crypt abscesses. Subsequent to 5 cycles of DSS, signs of chronicity are evident and include: regeneration of the mucosa with dysplasia and formation of lymphoid follicles in the mucosa (337). DSS colitis has been shown to induce secretion of cytokines of both the Th1 (IL-12, IFNγ, IL-1 and TNF, as well as the Th2 class-activating cytokine IL-4, but not IL-3 (Table 1.2) (342, 343). Cytokine expression is linked to chronicity and dose. In fact, studies have shown a transition from Th2 to Th17 serum cytokine profiles as disease progresses from acute to chronic (344). Studies blocking TNF in mice subjected to acute and chronic DSS colitis concluded opposing roles for TNF dependent upon disease chronicity. Animals with acute colitis which were treated with anti-TNF or anti-IL-1 antibodies or dexamathosone had exacerbated disease as evidenced by increased weight loss, and increased colonic inflammation (345). However, in chronic DSS colitis neutralization of TNF or dexamethasone therapy, but not neutralization of IL-1, resulted in a significant decrease in disease. These results indicate that TNF (and IL-1) is beneficial in acute disease, whereas TNF contributes to perpetuation of pathology in chronic colitis (345). These results underscore the benefits of anti-TNF therapy in patients with chronic IBD, but this therapy could be detrimental for patients combating acute disease. conclusions were made using Tnf -/- Similar mice subjected to acute DSS colitis. Naito et al showed (346) that DSS treated TNF deficient mice had enhanced disease as assessed by shortened colon lengths, increased colonic nitric oxide (NO) production, and elevated histopathology scores 56 when compared to WT mice. Similarly, Noti et al showed (347) that DSS treated Tnf -/- mice had increased weight loss and myeloperoxidase (MPO) activity and decreased colonic glucocorticoids production. The marked ulceration present and edema present has often led to this model being reported as a model for human UC. However, unlike UC, the cytokine response indicates a role for both Th1 and Th2 pathways in this disease, rather than the predominant Th2 effector response. As well, unlike UC, inflammation is patchy in distribution and crypt abscesses are not frequently seen. Additionally, the chronic phase of DSS colitis is more reminiscent of CD in that there is focal inflammation, prominent lymphoid follicles, and fissuring ulcers (348). Finally, the lack of adaptive immune mediation of the model indicates that it is a model of chemical injury rather than immune mediated chronic disease. The TNBS Mouse Model of Colitis TNBS is a contact-sensitizing agent which, when instilled intra-rectally, haptenates enteric compounds with trinitrophenyl (TNP) groups and induces a delayed hypersensitivity (DHT) reaction (349). The DHT reaction persists even after TNP-haptenated proteins have disappeared, perhaps because infiltrating immune cells elicited by TNBS cross react with autologous mucosal antigens, such as bacteria or bacterial components, and thus continue to be stimulated (349). Support for this theory is found in the fact that T cells from mice with TNBScolitis lack normal tolerance to their own flora and will proliferate in response to it (350). Similar to other models of colitis, treatment with probiotic bacteria, such as Lactobacillus and Bifidobacterium longum, has been shown to have a protective effect against the severity of TNBS colitis in mice (351, 352). Additionally, antibiotic treated mice have a significant 57 reduction in TNBS mediated clinical disease, histopathology, and proinflammatory cytokine production (353). Colitis is induced in this model via intra-rectal (IR) instillation of TNBS dissolved in ethanol. Ethanol is required to breach the mucosal barrier, allowing penetration of TNBS into the intestinal mucosa (354). TNBS is a covalently reactive compound and IR administration results in the development of acute necrosis in susceptible strains of mice (355). Histopathological lesions occur within 2-3 days post IR injection and consist of discrete foci of necrosis and inflammation, usually transmural, and cryptitits. Granulomas may develop, but are not a prominent feature. This model induces a Th1/Th17 effector response with elevated levels of TNF, IFN-γ, and IL-12, IL-6 and IL-18, as well as decreased levels of IL-4 (344, 349, 356, 357). Hollenbeck et al showed (358) that TNF, IFNγ, IL-2, IL-12, and IL-18 levels peak 2-3 days post IR TNBS administration (Table 1.2). The levels return to baseline, with the exception of IL-12, by day 5. Both IL-17 and IL-18 have been shown to be critical to the pathology of TNBS colitis. Blockade of IL-18 or signaling of IL-17 through its receptor resulted in significant reduction of colitis and pro-inflammatory cytokines (359-361). Despite elevations of Th1 and Th17 effector cytokines, studies have shown that IFN-γ is dispensable in the TNBS model and disparate role for the two subunits of IL-12 (p35 and p40) (362, 363). The discovery of the cytokine IL-23, which shares the subunit p40 with IL-12, has led to belief that the role of IL-12 in CD and TNBS colitis may well have been overestimated. The subunit p35 is unique to IL-12 and the subunit p19 is unique to IL-23 (364). To better understand the role of p35, mice deficient in p35 or p40 were evaluated in TNBS colitis. In this study, lack of the subunit p40, effectively negating the function of both IL-12 and IL-23, resulted in exacerbation of disease, but p35 deficient mice had decreased 58 pathology (362). These results suggest that IL-12 is more pathogenic than IL-23 in this model. Becker et al, however, showed there is cross regulation of IL-12 and IL-23 by demonstrating that -/- p19 mice were highly susceptible to TNBS colitis, but had elevated IL-12 production. Additionally, blockade of p40 in IL-23 deficient mice ameliorated the lethality of the colitis (365). Elucidation of the role of TNF in the TNBS model has resulted in varied findings, which are likely related to differences in the protocols, susceptibility differences among mouse strains, contributions of the intestinal flora, and utilization of knock-out mice versus anti-TNF antibody. Kinoshota et al (366) showed that Tnf -/- (C57Bl/6 generated and reared in Japan) mice had less colitis scores and decreased MPO activity subsequent to TNBS colitis when compared to WT mice. In this study, mice were given an IR TNBS injection and tissues were harvested 2 days later. Using a similar protocol, Noti et al (347) showed that Tnf -/- (C57Bl/6 generated and reared in Switzerland) mice had a decrease in clinical responses to therapy (e.g. less weight loss and shorter colons), however, and rather than the expected decrease in colonic steroid production, colonic glucocorticoids were increased. Histopathological colitis scores to ensure this discrepancy translated to differences in pathology were not provided in this study. Nakai et al (367) showed that Tnf deficiency (C57Bl/6 Tnf -/- mice reared at Jackson labs) did not result in significant differences in colonic inflammation when compared to WT mice. Mice in this study were harvested 1 week post IR injection. Although there were no differences in the level of inflammation between WT and Tnf -/- mice, rather than healing occurring during the week post IR as expected, the level of inflammation was surprisingly elevated in both WT and Tnf 59 -/- mice (367). In all 3 studies, C57Bl/6 mice were used, but were not presensitized, which is described as necessary for induction of TNBS colitis in this strain (368), which brings into question the confounding cause of the severity of the inflammation seen in the latter study in both WT and Tnf -/- mice. Additionally, the efficiency of the level of T cell contribution is questionable in animals harvested 2 days post IR injection. In a chronic model of TNBS colitis Neurath et al (356) found a pathogenic role of TNF, comparing Tnf -/- (SJL/J mice generated at NCI in Bethesda, MD) mice to WT mice. In this study TNF deficient mice were resistant to colitis. Anti-TNF antibody therapy has also been used in TNBS (and DNB) colitis. These studies indicate that the therapy results in decreased inflammation during acute disease as compared to untreated mice (369, 370). Both studies site apoptosis as a cause for decreased inflammation, but report different results regarding induction of apoptosis. Fries et al showed (369) that both Infliximab and Etanercept (two anti-TNF treatments) effectively prevent TNF-TNFR1 mediated apoptosis of enterocytes. It has been shown that apoptosis of enterocytes occurs early during IBD and results in disruption of epithelial conductivity, and thus, results in impaired function of the mucosal barrier (371). In IBD patients, apoptotic enterocytes found in actively inflamed areas of mucosa have been shown to have marked upregulation of TNF-related apoptosisinducing ligand (TRAIL), indicating that TRAIL may be instrumental in maintaining epithelial barrier function (209). Shen et al showed (370) that anti-TNF therapy induced apoptosis of macrophages and monocytes, the cell type thought to be primarily responsible for the elevated TNF production found in colitic patients. Although these reports indicated different mechanisms the knowledge that CD patients have prolonged survival of lamina propria inflammatory cells, coupled with shortened survival of enterocytes, indicates that both hypothesized mechanisms of actions are valid (372, 373). 60 The histopathological and immunopathological responses described in the TNBS model make it a suitable model for studying CD. However, methodology and strain variations are widespread in the literature, which likely accounts for some of the discrepancies in findings related to the role of TNF in the model. In addition, the mechanism of action of TNF antibodies appears to illicit strikingly different results as compared to utilizing TNF deficient mice. Finally, because the TNBS compound binds to autologous intestinal components (likely bacterial), flora differences amongst mice related not only to housing, but also to strain, could contribute significantly to findings generated in this model. The Oxazolone Mouse Model of Colitis In contrast to TNBS colitis, which elicits at Th1/Th17 effector response, oxazolone is used as a model of ulcerative colitis and induces at Th2 polarized response (Table 1.2) (374). This model has been shown to be flora dependent in zebrafish. Zebrafish maintained in filtered and ultra-violet radiated water were resistant to the development of oxazolone induced colitis. However when maintained in a standalone filtered tank, overall changes in the microbial composition resulted in an enhanced susceptibility to oxazolone colitis (375). In addition, treatment with vancomycin, while not altering bacterial load, resulted in a flora dominated by Fusobacteria and a concomitant decrease in enterocolitis (375). Oxazolone is a haptenating agent, similar to TNBS, and is also delivered intra-rectally dissolved in an ethanol vehicle. As with TNBS, strain susceptibility differences exist and resistant strains (e.g. C56Bl/6) require presensitization prior to IR treatment and optimization of dose for effective induction and perpetuation of disease (368, 376). Intrarectal administration of oxazolone results in weight loss and diarrhea which peaks by two days after the IR treatment. The chemical produces severe hemorrhage and inflammation, limited to the distal colon. 61 Significant histological features include: patchy ulceration and a reduction in the number of goblet cells and glands. Mucosal inflammation was mostly neutrophilic and lymphocytic with edema and similar inflammation extending to the submucosa (377). As stated above, the development of oxazolone colitis is associated with a Th2 effector response in the colon. Lamina propria T cells were shown to produce elevated levels of IL-4 and IL-5 (377). Increased production of IL-13 was also noted in the colons and determined to be produced by NKT cells (374, 378). This model elicits a strict Th2 response, in fact there is little production of IFN-γ or IL-12 and treatment with anti-IL-12 antibodies resulted in no effect or exacerbation of disease (377). This extent and duration of disease is effectively limited by TGFβ which is increased production 20-30 fold by T cells in the lesion. In addition, treatment with antiTGFβ antibodies not only results in more colitis, but the limiting effect is lost and lesions extend throughout the colon (377). Reports defining the role of TNF in the oxazolone model are limited and have also produced varied results. Studies which used anti-TNF antibodies that resulted in remission of TNBS induced colitis, showed no effect on oxazolone induced colitis (370). However, Noti et al showed (347) that while oxazolone treatment did not result in an increase in TNF (or IFN-γ), when mouse TNF was administered daily to colitogenic mice there was a significant decrease of colitis, as compared to WT controls, accompanied by an increased in colonic steroid production. The latter findings suggest that TNF is not necessary for stimulation of intestinal glucocorticoids production which are capable of ameliorating disease. Similar to the ability of TNBS to replicate CD, oxazolone appears to be an effective inducer of disease which mimics UC. As indicated with the TNBS model, strain susceptibility and protocol optimization are key points to consider when using this model. Similarly, the 62 intestinal flora likely plays a key role in this model, primarily because of the haptenating nature of the chemical used. CONCLUSIONS Many factors contribute to the development of IBD. Affected individuals essentially have a “perfect storm” comprised of genetic abnormalities and aberrant responses to their host flora, which may or may not contain a specific pathogenic component. The cellular contributors to the disease are many and are members of both the innate and adaptive immune system. In addition cellular alterations affect not only infiltrating cells but resident cells as well. Studies utilizing mouse models of colitis have allowed us to greatly advance our knowledge of this multifaceted disease. However, many issues with these models still exist, including, but not limited to: discrepancies in strain and protocol usage; contributions of variation of the flora; and mixed immuno- and histopathological responses resulting in phenotypes of more than one disease process (i.e. features of both UC and CD). Taken together these factors significantly confound our ability to develop maximally effective therapies which also have minimal adverse effects. It is clear that much work is still needed to be done in the field in order to better understand the pathogenesis of development and cessation 63 of both components of IBD. Table 1.2: The Role of TNF in Selected Mouse Models of IBD Mouse Model Model of Human Disease ΔARE TNF Mouse Cytokine Response Effect on disease using antiTNF antibody Effect on Possible disease using Reasons for TNF-/- mice vs discrepancies WT CD (and RA) Th1/Th17 ----- ---- ---- CD Th1 3wk and 3mo: no effect ---- • Age of initiation of treatment • length of time of treatment (311, 313) IL-10 -/- Mouse (319, ↑ TNF, IL17, IFNγ, IL-10 ↑ TNF, IL1, IFNγ 324, 325) 4wk-20wks: ↓ colitis T cell Transfer (332, Model 334) CD/UC Th1 ↓ colitis ↑ TNF, IFNγ ↓IL-10, IL-4 64 Absence of ---disease (both hematopoietic and nonhematopoietic TNF-/sources) Table 1.2 (continued) DSS Colitis (342-347) UC Acute: Th1/Th17 (Th2) Acute: ↑ colitis ↑ colitis ---- Acute: ↓ colitis; no difference in inflammation • Variation in protocols • Chronic: ↓ colitis Variation in mouse strain • Variation in mouse source ---- ---- Chronic: ↓ ↑ TNF, IFNγ, IL-12, colitis IL-1, IL-4, IL-17 Chronic: Th2 (Th1) ↑ IFNγ, IL6, IL-10, IL4 TNBS (344, Colitis CD Th1/Th17 367, 369, ↑ TNF, IL18, IL-2 IFNγ, IL-12, IL-6 370) ↓ colitis ↓IL-4 347, 349, 356, 357, Oxazolone UC (370, Colitis 374, 377, Th2 No effect ↑ IL-4, IL-5, IL-13 378) 65 CHAPTER TWO Materials and Methods ANIMALS Animal experiments were performed using 6- to 8-week-old, specific pathogen free mice of mixed sexes, which had been maintained in a temperature controlled room with an alternating 12-hr dark/light cycle. The animal experiments were conducted in accordance with the animal care and use committee guidelines of the National Institutes of Health. Mice with a C57BL/6 background with genetic ablation of TNF (Tnf -/- ) were generated as previously described (41). Briefly, two synthetic loxP sites were inserted flanking the critical coding region of the TNF gene and the adjacent, inserted neomycin cassette. LoxP mediated deletion of the gene and neomycin cassette was performed without affecting the function of the nearby lymphotoxin gene to derive mice with complete deficiency of the TNF gene (Tnf -/- ). Littermates (Tnf +/+ ) were used as wild type (WT) controls. Mice with cell-specific deletion of TNF were generated as previously described (41). Briefly, mice with the TNF gene floxed by two loxP sites (TNF flox/flox; Tnf fl/fl ) were crossed to cell-type-specific Cre deleter mice [CD4-Cre for specific deletion in T cells (T Tnf -/- ), CD19-Cre mice for B cells (B Tnf macrophages/neutrophils (MN Tnf -/- ), Lyzozyme-Cre for -/- ) and villin-Cre for enterocyte (E Tnf experiments, both wild type C57BL/6 littermates of Tnf -/- and littermate Tnf fl/fl -/- )]. For all mice were used as WT controls. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of National Cancer Institute (Frederick, Maryland). Animal care was 66 provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985). The expression of TNF was analyzed in different leukocyte populations in the spleen, peritoneal macrophages and bone marrow and enterocytes of untreated mice with deletion of TNF in specific cell types (enterocytes, T cells, or macrophages/neutrophils). In E Tnf 86% of the TNF expression in enterocytes was deleted; in T Tnf -/- expression was blocked in CD4 and CD8 T cells; and in MN Tnf -/- mice, mice, over 96% of the TNF -/- mice, the percentage of inhibition of TNF in macrophages and neutrophils was extremely variable depending upon the organ evaluated (Table 2.1). 67 Table 2.1: TNF Expression Levels in selected subsets for each strain of mouse Mouse T-TNF -/- TNF KO T cell-specific Cell type % of TNF deletion 5 CD4 T cell 96 splenic CD8 T cell 98 splenic Macrophage 4 splenic pDC 28 splenic CD4 DC 7 splenic CD8 DC 5 peritoneal Neutrophil 7 splenic B cell 9 splenic CD4 T cell 7 splenic CD8 T cell 6 splenic Macrophage 49.5 peritoneal Macrophage 82 Bone marrow Macrophage 70 splenic pDC 9 splenic CD4 DC 28 splenic CD8 DC 30 peritoneal Enterocyte-specific TNF KO B cell-specific TNF KO E-TNF -/- B cell splenic MN-TNF -/- TNF KO M/N-specific splenic Neutrophil 86 colonic splenic 86 97 splenic splenic splenic splenic peritoneal splenic Enterocytes B cell (enriched) CD4 T cell Splenocytes CD8 T cell B cell Macrophage CD4 T cell Neutrophil CD8 T cell splenic Macrophage 3 splenic 68 0 14 0 4 0 4 0 3 INDUCTION OF TRINITROBENZE SULFONIC ACID (TNBS) ACUTE AND CHRONIC COLITIS Induction of TNBS colitis was performed according to the protocol of Wirtz et al with minor modifications (368). On day 1, animals were anesthetized with isopropanol and a 1.5 x 1.5 cm field of the skin on the back between the shoulders was shaved. 100 μl of the TNBS (SigmaAldrich) presensitization solution (acetone and olive oil in a 4:1 volume was mixed rigorously by vortexing; four volumes of acetone/olive oil solution was mixed with 1 volume of 5% TNBS solution to obtain 1% (w/v) of TNBS) was applied to the shaved area. Control mice (SHAM) were treated with presensitization solution without TNBS. On day 7, mice were anesthetized as previously stated and, using a plastic feeding gavage needle affixed to a 1-ml syringe, 100 μl of the TNBS intra-rectal (IR) solution (1 volume of 5% (w/v) TNBS solution mixed with 1 volume of absolute ethanol) was slowly instilled into the colon. The mice were kept with the head down in a vertical position for 60 seconds to ensure that the solution stayed in the colon. As a control (SHAM), the same volume of 50% ethanol in PBS solution was administered to additional mice. For induction of acute colitis, mice were given one IR injection and tissues were harvested 2 or 3 days post IR injection. For induction of chronic colitis, mice were given one IR injection per week for 5 or 10 weeks and tissue were harvested 2 or 3 days post the last IR injection. Body weights were recorded every other day, and all mice were sacrificed 2 or 3 days after IR TNBS administration. At necropsy, the colons were removed, opened and washed with PBS. The colons were cut in half longitudinally and 4 centimeters of one-half of the colon was fixed in 4% paraformaldehyde (or 10% buffered formalin) for histological examination. The remaining onehalf of the colon was divided into two, 2-cm segments, which were either snap frozen in liquid ° nitrogen and stored at -20 C until use for protein, RNA or DNA analysis, or were cultured for 69 ° analysis of supernatant. Cultured colons were incubated at 37 C for 6 hours in 2ml of 10% RPMI ° with charcoal stripped serum or normal goat serum and the supernatants were stored at -20 C for future use. Blood samples were obtained by periorbital venous draw just prior to necropsy. ° Serum was separated from whole blood by centrifugation and stored at -20 C for future use. AZOXYMETHANE/DEXTRAN SODIUM SULFATE CHRONIC COLITIS ASSOCIATED COLON CANCER (AOM/DSS) MODEL OF Mice were given an intraperitoneal (IP) injection of AOM (Midwest Research Institute) (10 mg/kg) diluted in 0.2 ml saline. One week after AOM administration, DSS at 2% concentration was administered in the drinking water for 5 consecutive days, thereafter mice received reverse osmosis (RO) water. Four DSS cycles were administered, with intervals of 16 days on RO water between cycles. In some experiments AOM (10 mg/kg) was administered IP once a week for 6 consecutive weeks. During the course of the experiment mice were monitored for body weight loss/gain, rectal prolapse, diarrhea, macroscopic bleeding as well as occult bleeding via hemoccult assays (Hemoccult Sensa, Beckman Coulter). At the time of harvest, colons were resected, flushed with PBS, opened longitudinally and measured. Polyps were counted using a stereomicroscope. Colon sections were fixed in formalin or snap frozen. In some experiments polyps were harvested individually and snap frozen in liquid nitrogen. TNBS MODEL OF CHRONIC COLITIS ASSOCIATED COLON CANCER Mice (6-8 weeks old and of mixed gender) with a heterozygous mutation in the adenomatous polyposus coli (APC) allele (APC min/+ ) were obtained from Jackson Labs (Bar Harbor, MA) or from the National Cancer Institute (NCI). Induction of colitis in this resistant strain (C57Bl/6) of mouse was achieved by presensitization of the skin with a TNBS solution as 70 previously described (368) and intrarectal (IR) injections of 100 μl of TNBS dissolved in ethanol (1:1 ratio). Mice were given 5 or 10 IR injections (1 per week) and euthanized 2 days subsequent to the last injection. Alternatively, WT C57Bl/6 mice obtained from Jackson Labs or NCI were given 1 IP injection of AOM at a concentration of 10 mg/kg, dissolved in saline. One week post IP injection, mice were presensitized. One week post presensitization, mice were given 8 or 10 weekly IR injections and euthanized 2 days subsequent to the last IR injection. Colons were harvested as described above. Colons were also assessed for the presence or absence of tumors (gross evaluation) or histological evidence of atypia or dysplasia/metaplasia. Tumors noted grossly were measured and counted. Histological features of atypia or dysplasia/metaplasia were assessed and the percentage of mice having either lesion was presented graphically. Diagnosis of dysplasia or aplasia was made based on description of these features in the literature (379, 380). Consultation/advice regarding diagnosis of atypical glandular hyperplasia or dysplasia was provided by Drs. Philip Martin and Jerrold Ward. RNA ISOLATION AND NANOSTRING® ANALYSIS Previously snap frozen sections of colon (~30 mg of tissue) were placed in 2 ml Eppendorf tubes containing 1.0 ml of glass beads (Biospec) and 600 μL of RLT buffer (Qiagen RNeasy Kit). Samples were homogenized using a mini-bead beater (Biospec) for 50 seconds. Supernatants were removed from the bead mixture and centrifuged for 10 minutes at 14000 rpm to remove any remaining beads or tissue fragments. RNA was extracted from this supernatant according to the “animal tissue” protocol described in the Qiagen RNeasy Kit. RNA concentration was quantified using a spectrophotometer (Grace Scientific). 100 ng of RNA was submitted to the Laboratory of Molecular Technology SAIC-Frederick, Inc. for NanoString® 71 analysis. Briefly, biotinylated capture probes and color coded reporter probes were mixed in excess with RNA samples and allowed to hybridize overnight. Subsequent to washing steps to remove unbound probe sets, samples were analyzed for gene expression. Gene expression levels were tabulated using the nCounter™ Digital Analyzer. Raw data was provided in units of “counts” and normalized to the HPRT housekeeping gene subsequent to subtraction of the background signal. Gene expression was considered detectable with a count ≥20. Genes below counts of 20 were excluded from evaluation (with the exception of TNF). A total of 150 preselected genes, including 5 housekeeping genes (Table 2.2) selected for their importance in various inflammatory conditions and colon cancer were evaluated. Data is presented for all genes (except TNF) with a ±2 fold change of either TNBS treated animals to SHAM treated animals. Data from analysis groups (acute and chronic Tnf represent combined results from 2 experiments each. 72 -/- : WT and E Tnf -/- : Tnf fl/fl ) Table 2.2: Preselected gene set for Nanostring® Ang4 Aim2 Akt1 Apc Areg Arg1 Atm Atr Bad Bak1 Bax Bcl2 Bmp7 Bmp8b Brca1 Brca2 Casp8 ccl2 Ccl28 Ccnb1 Ccnb2 Ccnd1 Ccne1 CCR6 ccr7 Cd19 Gene List Frzb GAPDH Gata3 Gsk3b Gsta3 Hgf hif1 Hpgd HPRT icos Ifi16 Ifi202b Ifi30 ifna4 ifnb Ifng Il10 il11 Il12a Il12b Il13 il15 Il17a il17e Il18 Il18bp 73 Mnda Msh2 Msh6 Muc1 Mx1 Myc nos2 Ogg1 Parp1 Ppara Pparg Psen1 Pten Ptgs2 Rb Reg3g Relmb rorgc Rpl27 Rpl30 Rpl9 S100a11 S100a16 Smad1 smad2 Smad3 Table 2.2 (continued) Cd3e Cd40 Cd40lg Cdk4 Cdkn1a Cdkn2a Cdkn2b Chek2 Cldn2 Crp csf2 (GMCSF) csf3 (G-CSF) Ctnnb1 cxcl10 cxcl9 Ddah1 Dsh E2f2 eomes Esr1 Fancb FancF Fas Foxp3 Il1a Il1b Il1rl1 il1rn il21 IL22 Il23a Il27 ebi3 il27p28 il33 Il4 Smad4 Smad5 Smad6 Smad7 Socs2 socs3 Socs4 Socs6 Sod1 SOD2 stat1 Il6 indo kc Lta Ltb Maf Met Mlh1 mmp10 Mmp15 mmp2 Mmp3 mmp9 Stat3 Tbx21 Tcf Tgfb1 timp1 Timp4 Tnf Trp53 vegfa Wisp Wnt1 Wnt3 Wnt5a DNA ISOLATION AND REAL-TIME PCR (RT-PCR) DNA was isolated from the feces on day 1 of each experiment and one or two days post IR injection of TNBS using the DNeasy kit (Qiagen). For each sample, RT-PCR was performed using 12.5 ng of cDNA, the SYBR Green PCR Master Mix (Applied Biosystems) and the forward and reverse primers at a final concentration of 0.3 μM, in a sample volume of 25 μl. The primers (see Table 2.3 for sequences) were located from specified sources in the literature. Identified primer sequences were submitted to GeneBank and checked in BLAST to confirm the 74 total gene specificity. PCR was performed using a StepOnePlus machine (Applied Biosystems) under the cycling conditions required in the protocol for SYBR Green PCR Master Mix. The expression of candidate genes in each group of mice was normalized to 16s RNA to obtain a ΔCt value and to calculate a 2^-(Mean ΔCt). Data are normalized to the levels of expression of 16sRNA for each sample. Data is presented as fold change of treated to untreated for normalized samples. 75 Table 2.3: Oligonucleotide sequences for RT-PCR analysis of the expression of selected bacterium Bacterium Forward Sequence Reverse Sequence ACT CCT ACG GGA GGC ATT ACC GCG GCT AGC AGT GCT GGC Eubacteria (381) Eubacterium rectale/ Clostridium coccoides ACT CCT ACG GGA GGC (381) AGC GGT TCT GAG AGG Bacteroides (381) AGG TCC C Bacteroides/Prevotella GGT GTC GGC TTA AGT /Porphyromonas (382) GCC AT Mouse intestinal CCA GCA GCC GCG Bacteroides (381) GTA ATA Segmented filamentous bacteria GAC GCT GAG GCA TGA (381) GAG CAT Salmonella enterica TGT TGT GGT TAA TAA Typhimurium (381) CCG CA Enterobacteriaceae GTG CCA GCM GCC (381) GCG GTA A Enterobacteriaceae CAT GAC GTT ACC CGC (382) AGA AGA AG CAT GCC GCG TGT ATG Escherichia coli (383) AAG AA Lactobacillus/Leucono stoc/Pediococcus AGC AGT AGG GAA TCT (383) TCC A Lactobacillus/Lactococ AGC AGT AGG GAA TCT cus (381, 384) TCC A Streptococcus TTA TTT GAA AGG GGC salivarius (383) AAT TGC T CCC TTA TTG TTA GTT Enterococcus (383) GCC ATC ATT TCG CGT C(C/T) GGT Bifidobacterium (382) GTG AAA G ACC GCT TTC AGC AGG Atopobium (384) GA GGA TGA CAC TTT TCG Campylobacter (384) GAG Clostridium butyricum GTG CCG CCG CTA (385) ACG CAT TAA GTA T 76 GCT TCT TAG GGT ACC GTC AT GCT GCC TCC AGG AGT CGG A(C/T) GTA GCC GTG C CGC ATT CCG ACT TCT C GAC GGC ACG TGT TAT TCA GAC TAC CAG ATC TAA TCC GCC TCA AGG CAA CCT CCA AG CTC TAC GAG CAA GCT TGC CGG GTA ACG ATG AGC AAA TCA CGT AGG CAT GAT GGT GCA ACT TCA CGC CAC TGG TGT TCY TCC ATA TA CAC CGC TAC ACA TGG AG GTG AAC TTT CCA CTC TCA CAC ACT CGT TGT ACT TCC CAT TGT CCA CAT CCA GC(A/G) TCC AC ACG CCC AAT GAA TCC GGA T AAT TCC ATC TGC CTC TCC ACC ATG CAC CAC CTG TCT TCC TGC C Table 2.3 (continued) Clostridium coccoides/Eubacteriu m rectale group (384) Clostridium cellulosi (386) Clostridium clostridiiforme (385) Clostridium difficile (384) Clostridium perfringens (384) Clostridium perfringens (382) Desulfovibrio desulfuricans (384) CGG TAC CTG ACT AAG AAG C GCA CAA GCA GTG GAG T AAT CTT GAT TGA CTG AGT GGC GGA C TTG AGC GAT TTA CTT CGG TAA AGA ATG CAA GTC GAG CGA (G/T)G CGC ATA ACG TTG AAA GAT GG GGT ACC TTC AAA GGA AGC AC C(A/T)A ACG CGA TAA Fusobacterium (384) GTA ATC Fusobacterium CCC TTC AGT GCC GCA prausnitzii (384) GT Helicobacter/Flexispira TGG GAG AGG TAG /Wollinella (384) GTG GAA TTC T Helicobacter pylori GAA GAT AAT GAC GGT (384) ATC TAA C CTT AAC CAT AGA ACT Helicobacter (381) GCA TTT GAA ACT AC A(C/T)C AAC CTG CCC Veillonella (384) TTC AGA Faecalibacterium GGA CTG AGA GGT TGA prausnitzii (386) CA Ruminococcus albus CAG GTG TGA AAT TTA (385) GGG GC AGT TT(C/T) ATT CTT GCG AAC G TTG ATA AAA CGG AGG AAG CCA TCT CAC ACT ACC GGA GTT TTT C CCA TCC TGT ACT GGC TCA CCT TAT GCG GTA TTA ATC T(C/T)C CTT T CCT TGG TAG GCC GTT ACC C GGG ATT TCA CCC CTG ACT TA TGG TAA CAT ACG A(A/T)A GGG GTC GCA GGA TGT CAA GAC GTC GCC TTC GCA ATG AGT ATT C ATT TCA CAC CTG ACT GAC TAT GGT CGC CTT CGC AAT GAG TA CGT CCC GAT TAA CAG AGC TT CTC AAC AAG GAA GTG ACG GTC AGT CCC CCC ACA CCT AG PYROSEQUENCING OF BAR-CODED 16S RRNA GENE AMPLICONS. Bacterial rRNA sequences (16S) were amplified from stool genomic DNA using primers 27F (AGAGTTTGATCCTGGCTCAG) and 534R (ATTACCGCGGCTGCTGG) were used for PCR amplification of the V1-V3 hypervariable regions of the 16S rRNA gene . Each primer included also 454 sequencing primers A or B, start key sequence and a 7-9 mer barcode. 16S rRNA genes were amplified using AccuPrime™ 77 Taq DNA Polymerase High Fidelity (Invitrogen) and 50 ng of template DNA in a total reaction volume of 25 µl. Reactions were run in a PTC-100 thermal controller (MJ Research) using the following cycling parameters: ° ° ° 5 min of denaturation at 95 C; followed by 25 cycles of 30 s at 95 C (denaturing), 30 s at 56 C ° ° (annealing), and 90 s at 72 C (elongation); with a final extension at 72 C for 7 min. Negative controls without a template were included for each reactions. The presence of amplicons was confirmed by gel electrophoresis on a 2% agarose gel and staining with Ethydium bromide, quantified by using Picogreen quantification system (Bio-Rad), and equimolar amounts ( 100 ng) of the PCR amplicons were mixed in a single tube. Amplification primers and reaction buffer were removed by processing the amplicons mixture with the AMPure kit (Agencourt). The purified amplicon mixtures were sequenced by 454 XLR pyrosequencing using 454 Life Sciences primer A by the Molecular Diagnostics Laboratory, SAIC, NCI-Frederick, Frederick, MD using protocols recommended by the manufacturer. 454 SEQUENCING AND DATA ANALYSIS Initial 454 data was processed using XLR v2.3. Sequences were further processed using Mothur software (387). Briefly, reads longer than 300bp and with quality score >20 were selected and primers and barcodes were trimmed and assigned to samples. ChimeraSlayer algorithm was used to remove possible chimeras. Sequences were aligned using ARB software and each sequence was assigned to corresponding toxomomies using RDP CLASSIFIER at 0.7 threshold. Data was further analyzed using Partek and GraphPad Prism software. HISTOLOPATHOLOGICAL, IMMUNOHISTOCHEMICAL, CYTOCHEMICAL AND MORPHOMETRICAL ASSESSMENT FOR TNBS COLITIS Fixed sections of colonic tissues were embedded in paraffin, cut into 6-μm sections, stained with hematoxylin–eosin (H&E) for histological analysis via light microscopy. The 78 degree of inflammation in cross sections of the colon was assessed by Yava Jones semiquantitatively from 0 to 12 as described (Table 2.4). The scoring method was established by Yava Jones. Literature search resulted in a variety of scoring methods. The most applicable method was selected (346) and minor modifications were made based on the predominant features seen in this model. Colons were evaluated beginning at the colorectal junction and proceeding adorally 4cm to the middle colon. The proximal colon was not evaluated due to the lack of the ability of the IR TNBS instillation to significantly reach the area and elicit pathology. Briefly, the severity of the leukocytic infiltrate in the mucosa was subjectively assessed as mild, moderate or severe and the distribution was evaluated and denoted as focal/locally extensive, multifocal, or diffuse and scored accordingly. The distribution of ulceration was also assessed as focal, multifocal or diffuse. If necrosis was present it was subjectively assessed as mild, moderate or severe and scored accordingly. Total disease score per mouse was calculated by summation of each parameter for each mouse. Consultation/advice for scoring (and Aperio analysis) was provided by Drs. Matti Kiupel, Joshua Webster, and Mark Simpson. 79 Table 2.4: Histological assessment of colonic inflammation (Colitis Score) Criteria Distribution of leukocytes a Degree of Inflammation 1 2 3 Focal/locally Multifocal Diffuse extensive Severity of infiltrate Mild Moderate Severe Distribution of epithelial erosion Focal/locally extensive Multifocal Diffuse Severity of necrosis Mild Moderate Severe a. A score of 0 was assigned for each criterion not represented in the section Immunohistochemical staining was performed on formalin- fixed paraffin-embedded tissues by Yava Jones, at Histoserv Inc. (Germantown, MD) or by The Pathology and Histotechnology Laboratory (Frederick, MD) following the general immunohistochemical staining protocol described below: Paraffin was removed and sections were rehydrated; endogenous peroxidase was blocked; eptitopes were retrieved with steam; non-specific binding was blocked; avidin and biotin were blocked (Vector Labs); sections were incubated with the primary antibody (Table 2.5) for 30-60 minutes; sections were incubated with the appropriate secondary antibody (Vector Labs) at 1:100 dilution for 15 minutes; ABC (Vector Labs); color reaction with DAB (Invitrogen Inc); dehydration; cleared with xylene. Hematoxylin was used as a counterstain. Positively stained cells were identified via manual count in the distal colon. 80 Table 2.5: Primary antibodies with dilutions and manufacturers Antibody Dilution Company CD 45 F4/80 Gr1 CD3 CD45R/B220 Ki-67 1:100 1:100 1:50 1:50 1:200 1:500 BD Biosciences AbD Serotec BD Pharmingen Abcam Abcam Abcam Cytochemistry was performed on unstained slides of paraffin embedded tissues. Sections were stained for collagen deposition according to manufacturer’s instruction using the Masson’s Trichrome Staining kit (Sigma-Aldrich). Briefly, deparaffinized slides were incubated in Bouin’s solution for 15 minutes then washed; slides were incubated in Working Weigert’s Iron Hematoxylin Solution for 5 minutes then rinsed in deionized (DI) water; sections were stained with Biebrich Scarlet-Acid Fucshin, for 5 minutes then rinsed in DI water; slides were placed in Working Phosphotungstic/Phosphomolybdic Acid Solution followed by Aniline Blue Solution for 5 minutes each; slides were placed in 1% acetic acid solution for 2 minutes, rinsed, dehydrated and then cleared with xylene. The percentage of collagen was quantified within a defined area utilizing algorithms with Aperio analysis system (Aperio Technologies, Inc, Vista, CA). The trichrome value represents the percent positivity (blue stain) divided by the area analyzed in the distal colon. HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL (IHC) ASSESSMENT FOR AOM/DSS COLITIS ASSOCIATED COLON CANCER IHC staining was performed as described above on paraffin embedded sections. In situ hybridization for TUNEL was performed at Histoserv, Inc, Germantown, MD using the antidigozigenin-AP kit (Roche) at 1:1000 dilution on paraffin embedded sections. 81 The distal and middle sections of colon (4cm adoral from the anal-rectal junction) were analyzed as described above with one modification (additional category). The depth of inflammation was scored as follows: 1=mucosal only; 2=extends to submucosa; 3=extends to muscle. Total disease score per mouse was calculated by summation of each parameter for each mouse. Colons were also assessed for the presence or absence of tumors (gross evaluation) or histological evidence of atypia or dysplasia/metaplasia. Tumors noted grossly were measured and counted. STATISTICAL ANALYSIS Comparisons of clinical disease and histological scores were statistically analyzed using a two-tailed, nonparametric Mann-Whitney U test. All data in bar graph or dot plot format are expressed as mean ± S.E.M. Weights are presented with standard error bars. Differences were considered statistically significant at P <0.05. For statistical analysis or RT-PCR, a two-tailed Mann Whitney test was performed comparing the ΔCt values. The comparison of the mean incidents of tumors was performed using a Poisson regression model was performed by Data Management Services, Frederick, MD. The experiments were blocks in a randomized block experimental design, and “experiment” was included in the model as a random factor. Treatment group was entered into the model as a fixed 2 factor. An overdispersion parameter σ was included in the model in case the variance increased at a faster rate than the means increased. Penalized quasilikelihood estimation of the parameters (388) was performed using the GLIMMIX macro in the SAS software system (389). A Bonferroni adjusted p-value, p=0.0125, was used to control for multiple comparisons since 4 group means were compared with the floxed group’s mean incidence of tumors. The Poisson model is: 82 Let Yijk represent the number of tumors of the k’th mouse in the i’th treatment group in the j’th experiment. Log (E(Yijk )) = μ + αI + δj Var ((Yijk )) = σ2 μ The blocking factor experiment, δj is assumed to be normally distributed: 2 δj ~ N (0, σδ ) 83 CHAPTER THREE The effect of tumor necrosis factor in the pathogenesis of colitis is dependent upon on the source of production and the chronicity of disease Yava Jones, Amirnan Dzutzev, Rosalba Salcedo, Caroline Salter, Sergei Nedospasov, and Giorgio Trinchieri ABSTRACT Tumor necrosis factor (TNF), a pleiotropic cytokine, is upregulated in the colon of and systemically in patients with inflammatory bowel disease. Additionally, anti-TNF therapy has proven effective for the treatment of Crohn’s Disease (CD). However, TNF has been shown to have a differential role in the promotion or inhibition of acute versus chronic disease. Different biological functions of TNF have also been demonstrated depending on the cellular source of production. Here, we used mice with complete abrogation of TNF or deletion of TNF from specific cell types to elucidate the protective or deleterious contribution of TNF in the trinitrobenzene sulfonic acid (TNBS) model of CD. In acute colitis, TNF is protective against disease and the enterocytes are the main source of the TNF required for this protection. Mice lacking TNF production completely (Tnf -/- ) or from enterocytes (E Tnf -/- ) developed more inflammation, with increased leukocytic infiltrates in the mucosa and upregulation of many proinflammatory genes. As disease progresses to chronicity, however, the protective function of TNF was lost and by 10 weeks Tnf -/- mice had less inflammation and less atypical glandular hyperplasia. This study reveals the complexity of TNF production is dependent not only on the length of time it is produced, but also on the cellular source of production. 84 INTRODUCTION Inflammatory bowel disease (IBD) is a lifelong condition consisting of repeated bouts of inflammation and healing in the gastrointestinal (GI) tract. The two components of IBD are Crohn’s disease (CD) and Ulcerative Colitis (UC) (1). The pathogenesis of these diseases has not been fully elucidated, however, it is generally accepted that disease develops in genetically susceptible individuals with a hyper-responsiveness to their intestinal flora (1, 5, 7, 390). Patients with IBD have disrupted cytokine paradigms in their intestinal tract, resulting in an overproduction primarily of cytokines in the T-helper (Th)-1/Th-17 pathway in CD patients or, the Th-2 cytokine pathway in UC patients (1, 390). Tumor necrosis factor (TNF) is a pleiotropic cytokine considered to be a master regulator of cytokine production and this cytokine has been shown to be elevated in both the serum and mucosa of IBD patients (281-284). Inhibition of TNF has proven to be an effective therapy for certain CD patients (287). However, this cytokine can have diverse functions, including, but not limited to, stimulation of apoptosis and induction of proliferation. Additionally, several studies have shown that the cellular source of production can have varied effects on disease progression or inhibition (41, 391, 392). Thus, long term, pan-blockade of TNF production may prove detrimental to patients. The trinitrobenzene sulfonic acid (TNBS) mouse model induces an immunopathology akin to what occurs in patients with CD and is thus a suitable model for studying this disease (349, 376). Mice deficient in TNF production (Tnf -/- mice) or receiving neutralizing TNF antibodies have been used in this model and results have varied. Studies utilizing Tnf -/- mice or neutralizing antibodies have reported that TNF deficiency results in decreased inflammation 85 (347, 366, 369, 370) or no change in inflammation (367) in acute TNBS colitis. In contrast, in chronic (TNBS induced) disease, TNF deficient mice were resistant to inflammation (356). Here we use the TNBS model of colitis (368) to study the role of TNF in the pathogenesis of CD. We sought to evaluate the role of TNF both in acute and chronic disease, as well as elucidate the contribution of different cellular sources of TNF to the progression or inhibition of disease in this model. We found that TNF is protective in acute colitis, but detrimental in chronic disease. Additionally, enterocytes are the primary source of TNF production and is this source that is required for protection. TNF from T cells and macrophages/neutrophils has either no effect or a redundant effect on disease progression. RESULTS TNF is required for protection against acute TNBS colitis Acute TNBS colitis was induced in presensitized mice via intrarectal (IR) instillation of TNBS. Mice were euthanized and tissues were harvested 2 or 3 days post the last IR injection. Tnf -/- mice lost more weight than the WT control mice subsequent to IR injection (Figure 3.1 A). Histological evaluation (Table 2.4) of the distal and middle colon of TNBS and control treated animals revealed significantly higher colitis scores (Figure 3.1 B) and increased leukocytes in the colonic mucosa (Figure 3.1 C) in TNBS treated Tnf -/- mice as compared to TNBS treated WT mice. Colitis scores were higher in TNBS treated animals than in SHAM treated animals (Figure 3.1 A). Representative photomicrographs of inflammation in the distal colon of TNBS treated WT (Figure 3.1 D) and Tnf -/- mice (Figure 3.1 E); SHAM treated WT (Figure 3.1 F) and Tnf 86 -/- mice (Figure 3.1 G) ; and CD45+ cells in the mucosa of TNBS treated WT (Figure 3.1 H) and Tnf -/- mice (Figure 3.1 I) are depicted. Enterocyte-derived TNF is determinant in conferring protection against TNBS-induced acute colitis Acute TNBS colitis was induced and evaluated in enterocyte-specific Tnf and Tnf Tnf -/- fl/fl mice as described above. The disease E Tnf -/- mice, but was more severe. E Tnf -/- -/- (E Tnf -/- ) mice was similar to what was seen in mice lost significantly more weight than Tnf fl/fl mice (Figure 3.2 A). The increased weight loss was accompanied by increased colitis scores (Figure 3.2 B) and increased leukocytes in the colonic mucosa (Figure 3.2 C). photomicrographs of inflammation in TNBS treated Tnf (Figure 3.2 E); SHAM treated Tnf fl/fl Tnf -/- -/- (Figure 3.2 D) and E Tnf (Figure 3.2 F) and E Tnf CD45+ cells in the mucosa of TNBS treated Tnf I) are depicted. When compared Tnf fl/fl fl/fl Representative -/- -/- mice mice (Figure 3.2 G); and (Figure 3.2 H) and E Tnf -/- mice (Figure 3.2 mice, the inflammation induced by TNBS treatment in E mice resulted in significantly higher colitis scores (Figure 3.2 J) and significantly more leukocytes in the mucosa (Figure 3.2 K). 87 Figure 3.1 88 Figure 3.1 (continued) -/- Figure 3.1. TNBS treated Tnf mice have more acute colitis than TNBS treated WT or SHAM treated mice (A) Time course of the average percent weight loss/gain in TNBS and -/SHAM treated WT and Tnf mice. IR injection was given on day 7. (B) Colitis scores (data are combined from two independent experiments) and (C) leukocytes (CD45+), neutrophils (Gr1), B cells (B220), macrophages (F480) and T cells (CD3) infiltrating the mucosa (graph shows the -/number of positive cells/5 hpfs;) in TNBS treated WT and Tnf mice. Representative -/- photomicrographs (100x) of inflammation in the distal colon are shown in WT (D) and Tnf (E) mice. Data in A-C are shown as means ± SE from 2 independent experiments combined. For 89 -/- -/- colitis scores, n=13 (WT TNBS), n=14 (Tnf TNBS), n=4 (WT SHAM), and n=3 (Tnf SHAM). For IHC with n=3 mice per group. *p<.05; **p<.01. For IHC graphs, * represent -/comparisons between WT and Tnf mice. Mann-Whitney test was performed for colitis scores. Un-paired t-test was performed for individual leukocyte counts. Photomicrographs at 200x magnification. Bar= 50μm Colitis in Tnf -/- and enterocyte derived Tnf deficient mice is accompanied by an up or dowregulation of several key mediators and markers of inflammation Sections of the distal colon were homogenized and RNA expression levels of several genes (Table 2.2) were analyzed by the nCounter Gene Expression Assay (Nanostring®) subsequent to induction of TNBS colitis. Several genes were up- or downregulated (genes with a fold change of <2 were excluded from analysis) due to TNBS treatment compared to SHAM treatment in both Tnf E Tnf -/- fl/fl : mice. -/- and WT mice. These genes were then compared in Tnf -/- : WT mice and Many key markers or mediators of inflammation, mucosal homeostasis, tissue repair and cell death were found to be differentially expressed dependent upon TNF expression. Chemokines play an important role in the recruitment and activation of leukocytes (393) and there was significant upregulation of several chemokines in Tnf -/- and E Tnf -/- mice compared to WT mice. The “inflammatory chemokine” monocyte chemoattractant protein-1 (MCP-1/CCL2), which is primarily responsible for recruitment of monocytes/macrophages to sites of tissue injury (394) was upregulated 6 fold in Tnf (Figure 3.3) and 4 fold in E Tnf -/- -/- mice as compared to WT mice mice as compared to floxed mice (Figure 3.3). 90 CCL2 has been shown to be induced in IBD patients and mice lacking the receptor for CCL2 have amelioration of colitis in some IBD models (395). Proinflammatory stimulation has been shown to increase the expression of CXCL10 in intestinal epithelial cells in vitro (396) and is upregulated in affected colonic areas of UC patients (397). CXCL10 was upregulated 4 fold in Tnf -/- mice as compared to WT mice (Figure 3.3) and 2 fold in E Tnf floxed mice (Figure 3.3). -/- mice as compared to CXCL1 (keratinocyte-derived chemokine; KC) is constitutively expressed in many mouse tissues (398) and is among the cytokines considered critical for neutrophil recruitment (399). This cytokine was found to be 7 fold higher in Tnf compared to WT mice (Figure 3.3) and significantly higher (4 fold) in E Tnf -/- -/- mice mice as compared to floxed mice (Figure 3.3). Greater upregulation of these inducers of leukocyte infiltration in Tnf -/- mice is congruent the increased leukocytic infiltration noted histologically. 91 Figure 3.2 92 Figure 3.2 (continued) 93 Figure 3.2 (continued) -/- Figure 3.2. TNBS treated E Tnf mice have more severe acute colitis than TNBS treated Floxed or SHAM treated mice (A) Time course of the average percent weight loss/gain in -/TNBS and SHAM treated WT and E Tnf mice. IR injection was given on day 7. (B) Colitis scores (data are combined from two independent experiments) and (C) leukocytes (CD45+), 94 neutrophils (Gr1), B cells (B220), macrophages (F480) and T cells (CD3) infiltrating the mucosa -/(graph shows the number of positive cells/5 hpfs;) in TNBS and SHAM treated WT and E Tnf mice. Representative H&E photomicrographs of inflammation in the distal colon are shown in -/-/TNBS treated WT (D) and E Tnf (E) mice as well as sham treated WT (F) and E Tnf (G) mice. Representative photomicrographs of CD45+ staining cells of TNBS treated WT (H) and E -/-/-/Tnf (I) mice are depicted. Colitis scores comparing Tnf to E Tnf mice (J) show more colitis in E Tnf -/- mice. The higher colitis score is supported by more infiltrating CD45+ cells in -/- -/- the mucosa of E Tnf mice compared to Tnf mice (K). Data in A-C are shown as means ± SE from 2 independent experiments combined. For colitis scores, n=7 per group (TNBS) and n=5 per groups (SHAM). For IHC with n=3 mice per group. *p<.05; **p<.01. For IHC graphs, -/* represent comparisons between WT and Tnf mice. Mann-Whitney test was performed for colitis scores. Un-paired t-test was performed for individual leukocyte counts. Photomicrographs at 200x magnification. Bar= 50μm As stated above, cytokine dysregulation is the underlying pathogenic component of the progression of IBD. Several cytokines and factors associated with cytokine signaling were more highly upregulated in Tnf -/- mice compared to WT mice. Interleukin (IL)-33, a cytokine in the IL-1 family can enhance the production of Th2 type cytokines, such as IL-4, by natural killer (NK) cells and NKT cells (400, 401). We suspect that in this Th1 mediated disease, elevation of this Th2 cytokine serves as a compensatory mechanism to drive the immune response towards equilibrium and disease resolution. Therefore, it is not surprising that levels of IL-33 were higher (2 fold in Tnf -/- and E Tnf -/- ) in the mice with greater Th1 inflammation (Figure 3.3). Additionally, an increase in suppressors of cytokine signaling (SOCS) proteins can dampen cytokine responses. In our case, SOCS3, a negative regulator of inflammation, was increased 3 fold in Tnf -/- mice (Figure 3.3) and significantly higher (2 fold) in E Tnf -/- mice (Figure 3.3), possibly in an attempt to control inflammation. SOCS3 can be induced by and cause inhibition of interferon (IFN)-γ and IL-6 (402), cytokines which are important in TNBS colitis (349). Mcuin 95 (MUC) 1 is overexpressed and hypglycosylated in IBD, adenomatous dysplasia and colorectal cancer (404-407). The correlation with increased inflammation and increased Muc1 expression was also seen in this study. Tnf -/- and E Tnf -/- mice had significantly higher inflammatory scores and significantly higher levels of Muc1 (2 fold each) in their colons (Figure 3.3). Serum levels of IL-6 are frequently elevated in both acute and chronic disease processes (408). Furthermore, elevated IL-6 has been seen in both CD and UC patients (8, 409, 410). In the progression of IBD, IL-6 functions primarily to enhance T cell survival and resistance to apoptosis at sites of inflammation (411, 412). In our study we found significantly elevated (>100 fold) colonic IL-6 levels in Tnf -/- mice compared to WT mice (Fig 3.3), consistent with the elevated inflammation seen in these mice. However, IL-6 levels were essentially the same in E Tnf -/- and floxed mice (Fig 3). Although IL-6 can signal through its specific membrane bound receptor IL-6Rα, a broad range of its effects are mediated through the formation of a soluble receptor complex with a soluble form of IL-6Rα that bind to the second shared chain of its receptor - gp130 - that can be found on the surface of virtually all cells (408). Interestingly, another IL-6 family member that shares with IL-6 the gp130 chain, IL-11 (413), was also elevated in both Tnf -/- mice and E Tnf -/- mice compared to WT and floxed mice. IL-11 is a pleiotropic cytokine of produced by bone marrow stromal cells and other cells of mesenchymal origin (414). IL-11 has been shown to downregulate proinflammatory Th1 cytokines such as TNF, IL-12, and IFN-γ and reduce the effector functions of activated macrophages, having a protective role in IBD (413, 415). In our studies, Tnf -/- -/- mice and E Tnf mice each had a 6 fold higher induction of the expression of IL-11 in the colon as compared to 96 WT and Tnf fl/fl mice. This finding suggests that IL-11 increased subsequent to induction of inflammation in order to mitigate the severity of inflammation and restore mucosal homeostasis. Other cytokines of importance that were increased in E Tnf 1β (significant in E Tnf -/- -/- mice include: IL- mice), IL-1α and IL-22 (2 fold each) (Figure 3.3). IL-1β and IL-1α are potent pro-inflammatory cytokines that are elevated in the mucosa of IBD patients (416-418). In addition to initiating and propagating intestinal inflammation, elevated IL-1β results in increased permeability of tight junctions in the intestine (419), which is suspected to allow increased permeation of luminal antigens to the submucosa (420). IL-22 was shown to be elevated in the mucosa of IBD patients, but not normal colons and is thought to elicit proinflammatory and remodeling roles in IBD (421). Resistin-like molecule beta (RELMβ) is a protein which expressed by intestinal goblet cells and is secreted into the intestinal lumen upon induction by microbes (422, 423) and by Th2 cytokines (424, 425). RELMβ is overexpressed in IBD patients (390) and loss of RELMβ has been shown to reduce the severity of DSS colitis (426). This gene was upregulated 2 fold in E Tnf -/- mice compared to Fl mice (Figure 3.3), but was downregulated 2 fold in Tnf (Figure 3.3). The relevance of upregulation of Relmb in E Tnf dowregulation of the gene in Tnf -/- -/- -/- mice mice—compared to mice is not completely clear. However, possible explanations include: enterocyte derived TNF is a mediator of production of this protein, increased inflammation seen in mice that lack of enterocyte derived TNF is due to decreased Relmb, and/or the inflammation in E Tnf -/- results in downregulation of Relmb. 97 Transforming growth factor (TGF) β is a pleiotropic cytokine that regulates a variety of cellular processes such as cell growth, differentiation, and apoptosis, and also arbitrates the immune response (427). Mothers Against Decapentaplegic Homolog (SMADs) are a group of proteins involved in TGFβ signaling (428). SMADs 6 and 7 are induced by the TGβ family of cytokines, but serve to inhibit its signaling through establishment of a negative feedback loop (427). We found that E Tnf -/- mice had significant downregulation of both SMAD 6 and 7 (Figure 3.3), possibly as a result of the increased inflammation and the greater need for TGF-β production (which was significantly upregulated) to dampen the inflammatory response. However, these SMADs were slightly increased in Tnf increased Tnf -/- -/- mice, but SMAD4 was significantly mice (Figure 3.3). This data indicates that the colitis mediated specifically by enterocyte loss of TNF differs from that mediated by a total loss of TNF. TNBS colitis results in an increase in Enteriobacteriacea in the feces of both Tnf -/- -/- and E Tnf mice, but not WT mice To evaluate the role of the microbiota in the induction of colitis or secondary to colitis induction, we sampled feces from 3 WT and 3 Tnf -/- mice (littermates) 2 days post IR treatment. We performed 454 sequencing to compare the bacterial compositions in these mice. Compositional analysis at the phylum (Figure 3.4 A), and class (Figure 3.4 B) levels showed very little variability. However, more heterogeneity was noted at the genus level (Figure 3.4 C). Although not significant, the highest difference between the groups was noted in Tnf -/- mice for the presence of Escherichia/Shigella. Subsequent species analysis using the Basic Local Alignment Search Tool (BLAST) showed 97% homology with the O26:H11 variant of E. coli. 98 This enterohemorrhagic E. coli (EHEC) is a common cause of both hemolytic uremic syndrome and diarrhea (429, 430). Figure 3.3 99 Figure 3.3 (continued) Figure 3.3. RNA expression levels of selected genes in acute TNBS colitis: Data shows the -/-/fl/fl (dark blue relative fold induction of genes in Tnf vs WT (dark red bar) and E Tnf vs Tnf bar) TNBS-treated mice. Genes with p .05 are shown in the lighter color corresponding to the ≤ -/genotype. Only genes that had at least a ± 2 fold change in response to TNBS treatment in Tnf and/or in WT mice compared to sham-treated mice were depicted. TNBS treatment induced upregulation and downregulated relative to the expression of TNF. Un-paired t-test used for statistical analysis. Data combined from 2 independent experiments for each genotype. N≥5 per treatment group. 100 In addition to 454 sequencing, we evaluated the levels of E. coli and 32 other bacterial categories (Table 2.3) in WT, Tnf -/- , and E Tnf -/- mice post-treatment versus pre-treatment by qRT-PCR. All strains had decreased expression levels of Segmented filamentous bacteria (SFBs) post-treatment (Figure 3.4 D-F). SFB are apathogenic bacteria indigenous to the small intestine of many species, including mice (431) and are potent inducers of T helper (Th)17 cells (432). In the ileum, SFBs are firmly attached to the intestinal epithelium and have predilection for epithelium covering lymphoid foci, such as Payer’s patches. The bacteria are separated from the gut lumen by a thin film of mucous (431). Colonization of the intestine with both SFB and a defined cocktail of specific pathogen free (SPF) bacteria induces colonic inflammation that is not observed in germ free mice or mice monoassociated with either SFB or SPF (433). We speculated that this loss was primarily due to the physical location of the bacteria. Subsequent to IR instillation of the chemical and inflammation, the bacteria were possibly mechanically disrupted from their habit and shed prior to sampling. E. coli and Enterbacteriaciae were most significantly altered in Tnf E Tnf -/- mice (Figure 3.4 F), but not WT mice (Figure 3.4 D). -/- (Figure 3.4 E) and Enterobacteriaceae is a large family of bacteria which include many pathogens among them, many (e.g. Salmonella, E. coli, Klebsiella, and Proteus) are important in various types of colitis, enteritis, and enterocolitis (434437). These findings suggest that the microbial flora participate in colonic inflammation and/or changes secondary to TNBS induced inflammation. The increase in TNF deficient mice, but not WT mice indicates, also that this change in the flora is TNF dependent. What is not clear, however, is if the increase in bacterial load in the feces is proportional to an increase of colonization of the colon or if it results in fewer bacteria in the colon (due to fecal loss). Further 101 studies to evaluate bacterial levels in the colonic tissue and distant sites (i.e. local and peripheral lymph nodes, the liver, and spleen) are needed to address these issues. The lack of B cell derived, T cell derived, or macrophage/neutrophil derived TNF has no effect on the severity of colitis Mice with deletion of TNF in B cells (B Tnf neutrophils (MN Tnf -/- ), T cells (T Tnf -/- ) or macrophages and -/- ) or mice that completely lack B cells (μMT mice) were subjected to acute TNBS colitis as described above. The lack of these sources of Tnf or of B cells did not result in pathology significantly different from the fl/fl or WT controls (Figure 3.5 A-D). These data indicate that each cellular source of TNF is redundant in function in the pathogenesis of this disease and underscore the importance of the enterocyte source of TNF in IBD. 102 Figure 3.4 103 Figure 3.4 (continued) 104 Figure 3.4 (continued) 105 Figure 3.4 (continued) 106 Figure 3.4. TNBS acute colitis induces changes in fecal microbiota composition which are both dependent and independent of TNF: 454 sequence analysis showed that TNBS treatment did not induce significant variability in (A) phylum or classes of bacteria. (B) Species analysis -/showed increased Escherichia/Shigella in Tnf mice compared to WT mice. We used feces from 3 WT and 3 for evaluation. qRT-PCR analysis of 33 bacteria showed a significant (p<0.001) decrease in SFB post-treatment in all genotypes (C) 2 or 3 days post IR TNBS compared to pre-treatment. The lack of TNF and enterocyte derived TNF resulted in a -/significant (p<0.05) increase in E. coli and Enterobacteriaceae (ENTERO) in Tnf (D) and E -/- (E) mice 2 or 3 days post IR TNBS compared to pre-treatment. Each point represents a Tnf bacterium. Feces was taken from the same mouse pre and post IR treatment. Fecal bacteria -/-/composition of mice in each group (WT: n=5; Tnf :n=6; and E Tnf : n=4) was analyzed, normalized to 16s RNA expression, and results were averaged. Mice were littermates in each genotype. Paired t-test was used for statistical analysis. A p< .05 was considered significant. 107 Figure 3.5 108 Figure 3.5. TNF from B cells, T cells, and macrophages/neutrophils and B cells had either insignificant or redundant roles in acute TNBS colitis: Mice with a deficiency of B cell derived Tnf (A) or B cells (B; μMT mice); deficiency of T cell derived Tnf (C); and macrophage/neutrophils derived Tnf (D) were subjected to TNBS and SHAM treatment. Colitis scores did not differ between TNBS treated mice or between. Means ± SEM are shown. Each experiment was performed once. TNF deficiency results in increased colonic pathology at 5 weeks, similar to what is seen in acute disease To induce chronic colitis, presensitized WT and Tnf -/- mice were given an IR injection of TNBS once per week for 5 weeks or 10 weeks. Mice were euthanized and tissues were harvested 2 or 3 days post the last IR injection. At 5 weeks of disease, Tnf -/- mice lost more weight than the WT control mice subsequent to each IR injection (Figure 3.6 A). Histological evaluation of the distal and middle colon of TNBS and control treated animals revealed significantly higher colitis scores (Figure 3.6 B) and significantly more macrophages in the colonic mucosa (Figure 3.6 C) in TNBS treated Tnf -/- mice as compared to TNBS treated WT mice. Colitis scores were higher in TNBS treated animals than in SHAM treated animals (Figure 3.6 A). Representative photomicrographs of inflammation in the distal colon of TNBS treated WT (Figure 3.6 D) and Tnf -/- mice (Figure 3.6 E); SHAM treated WT (Figure 3.6 F) and Tnf -/- mice (Figure 3.6 G) ; and CD45+ cells in the mucosa of TNBS treated WT (Figure 3.6 H) and Tnf -/- mice (Figure 3.6 I) are depicted. Fibrosis was significantly upregulated at 5 weeks as compared to acute colitis (Figure 3.7 A), providing evidence of chronicity, although the degree of fibrosis did not differ between WT and Tnf -/- mice. Inflammation was also higher in Tnf 109 -/- and WT mice at 5 weeks compared to acute disease, as noted by the increased numbers of CD45+ leukocytes in the mucosa (Figure 3.7 B). The gene expression profile at 5 weeks is similar to what occurs in acute colitis Colons were homogenized post 5 weeks of TNBS colitis and the expression level of RNA was analyzed as described above. Most genes were similarly up- or downregulated at 5 weeks as they were in acute disease (Figure 3.8). However, several key inducers of TNBS colitis were increased at 5 weeks which were not seen in acute disease or were present at lower levels. This is consistent with our finding of increased inflammation at 5 weeks of colitis as compared to acute colitis. As previously stated, CD (1, 390) and TNBS colitis (349, 376) are both Th1/Th17 mediated diseases. Additionally, Alex et al (344) showed that the intensity of Th1/Th17 disease in TNBS colitis increases as disease progresses from acute to chronic. In our study we saw changes in genes at 5 weeks that were not seen in acute disease. The Th17 cytokines, IL-17a (2 fold) and IL-22 (significant; 3 fold), were increased in Tnf -/- mice as compared to WT mice (Figure 3.8). Also, the mRNA for the two chains, IL-12α and IL-12β of the Th1-inducing cytokine IL-12, was increased in Tnf -/- mice compared to WT mice (Figure A). IL-6 (438) and IL-1β (439) are also important proinflammatory cytokines in the pathogenesis of IBD. Both cytokines were significantly upregulated (6 fold and 2 fold, respectively) in Tnf compared to WT mice at 5 weeks (Figure 3.8). 110 -/- mice Figure 3.6 111 Figure 3.6 (continued) -/- Figure 3.6. TNBS treated Tnf mice have more severe chronic (5 week) colitis than TNBS treated WT or SHAM treated mice at 5 weeks: (A) Time course of the average percent weight -/loss/gain in TNBS and SHAM treated WT and Tnf mice. IR injections were given on days 8, 13, 21, 29, 35, and 38. (B) Colitis scores (data are combined from two independent experiments) and (C) leukocytes (CD45+), B cells (B220), macrophages (F480) and T cells (CD3) infiltrating -/the mucosa (graph shows the number of positive cells/5 hpfs;) in TNBS treated WT and Tnf mice. Representative photomicrographs of inflammation in the distal colon are shown in TNBS -/-/treated WT (D) and Tnf (E) mice as well as SHAM treated WT (F) and Tnf (G) mice. -/- Photomicrographs of CD45+ celsl in the mucosa of WT (H) and Tnf (I) mice are also depicted. Data in A-C are shown as means ± SE from 2 independent experiments combined. For -/-/colitis scores, n=22 (WT TNBS), n=21 (Tnf TNBS), n=4 (WT SHAM), and n=5 (Tnf 112 SHAM). For IHC with n=4 mice per group. *p<.05; **p<.01. For IHC graphs, * represent -/comparisons between WT and Tnf mice. Mann-Whitney test was performed for colitis scores and individual leukocyte counts. Photomicrographs at 200x magnification. Bar= 50μm Figure 3.7 113 Figure 3.7. Chronic (5 week) TNBS colitis induces significantly more fibrosis and mucosal -/inflammation in WT and Tnf mice than observed in acute colitis: Sections of colon of -/-/TNBS treated Tnf and WT mice were stained for collagen deposition. Both Tnf and WT TNBS treated mice have more collagen deposition at 5 weeks than Tnf -/- and WT mice do in -/- acute colitis (A). However, there was no difference between the levels of fibrosis between Tnf and WT mice at 5 weeks. CD45+ cells were counted in the distal colonic mucosa. There were -/leukocytes in the mucosa at 5 weeks in both Tnf and WT mice (B) as compared to acute colitis. Means ± SEM are shown. N=at least 6 mice/group. Mann Whitney test with *p<.05 comparing each genotype in acute versus chronic disease. Both IL-11 and IL-6 signaling can induce phosphorylation of Signal Transducers and Activators of Transcription (STAT)3 (413). Similar to the divergent roles of IL-6 and IL-11 in IBD, STAT3 can play a differential role in its pathogenesis. Activated STAT3 can contribute to the progression of disease by promoting survival of pathogenic T cells or induce suppression of colitis by activation of the innate immune system (440). In our study, STAT3 mRNA was upregulated 2 fold in Tnf -/- mice as compared to WT mice (Figure 3.8). Given the promoting versus inhibiting role of STAT3 in IBD, it’s not clear if this upregulation was a protective measure in response to the increased inflammation or if the upregulation was a factor causally associated with the increase in inflammation. Some genes were similarly affected in acute colitis and at 5 weeks in Tnf Upregulated genes in Tnf -/- -/- mice. mice compared to WT mice included: Muc1 (significant; 2 fold), IL- 33 (significant; 3 fold), SOCS3 (3 fold), CXCL 9 and 10 (both 2 fold) and CD3e (2 fold) (Figure 3.8). Relmb was slightly downregulated (1 fold) at 5 weeks in Tnf seen in Tnf -/- mice with acute disease (Figure 3.8). 114 -/- mice, similar to what was The protective role of TNF is lost as disease progresses and chronic production of TNF promotes inflammation and glandular atypia At 10 weeks of disease, Tnf deficient mice have less pathology than WT mice, indicating TNF promotes disease at this stage of disease. TNBS treated Tnf -/- mice lost less weight (Figure 3.9 A) and had lower colitis scores (Figure 3.9 B) than TNBS treated WT and had significantly fewer CD45+ leukocytes and CD3+ T cells in the mucosa (Figure 3.8 C). Representative photomicrographs of inflammation in the distal colon of TNBS treated WT (Figure 3.9 D) and Tnf -/- mice (Figure 3.9 E); SHAM treated WT (Figure 3.9 F) and Tnf -/- mice (Figure 3.9 G) ; and CD45+ cells in the mucosa of TNBS treated WT (Figure 3.9 H) and Tnf -/- mice (Figure 3.9 I) are depicted. Atypical glandular hyperplasia was more prevalent in WT mice than in Tnf deficient mice (Figure 3.9 J). A representative photomicrograph of squamous metaplasia (Figure 3.9 K) atypical glandular hyperplasia in WT mice (Figure 3.9 L) compared to relatively normal glandular architecture in Tnf -/- mice (Figure 3.9 M) is depicted. DISCUSSION Many cell types are capable of TNF production in vivo (392), thus, different cell subsets are likely to contribute differentially for systemic versus mucosal TNF production, as well as for TNF-mediated responses in IBD and under physiological conditions. In addition, TNF is known to have opposing effector roles, contributing to cellular proliferation, activation and apoptosis (441). Grivennikov et al (41) showed that systemic TNF is produced primarily by macropages and neutrophils in response to stimulation by bacterial components, such as lipopolysaccharides. These cellular sources are critical in resisting infection by Listeria. Additionally, 115 macrophage/neutrophil and T cell derived TNF promote autoimmune hepatitis with non-redunant functionality. These examples highlight the distinct role of TNF from different cellular sources, as well as the importance of appropriate amounts of the cytokine being produced at the appropriate times. In this study, we employed the same panel of mice as used by Grivennikov et al (41) with highly specific and efficient deletion of the TNF gene from the genome or in several types of leukocytes or enterocytes in a model of acute colitis. We concluded that TNF is protective in acute TNBS colitis and is responsible for limiting the severity of inflammation. We also found that neither T cell, nor macrophage/neutrophil, nor B cell derived TNF (nor B cells) were independently sufficient or necessary to affect TNBS colitis. Results in the literature comparing Tnf -/- mice to WT mice in the TNBS colitis model have produced varied results. Some groups, contrary to our findings, report that Tnf deficient mice either had decreased colitis (347, 356) or no significant difference (367) in colitis as compared to WT controls. The difference in our findings could be attributed to different microbiota, different concentrations of TNBS used (347), or strain variation responses to treatment (356). As well, treatment with an anti-TNF monoclonal antibody resulted in decreased mucosal cellular infiltration and down regulated proinflammatory cytokine levels (369, 370). In those studies, however, antibody administration resulted in significant apoptosis of macrophages (370) in the lamina propria or of enterocytes (369), which was considered to be the most likely mechanism of disease remission. Decreased inflammation in Tnf -/- mice in our study, however, was not attributable to either increased apoptosis or decreased proliferation. Kinoshita et al (366) reported that Tnf -/- mice had lower levels of colonic myeloperoxidase and lower colonic levels of proinflammatory cytokines such as IL-1β and IL-6. As stated above, the differences in 116 our results may relate to differences in the intestinal flora and/or differences in the protocol. In our study, the lack of TNF production in acute TNBS colitis resulted in an increase in all parameters of clinical disease that were assessed, with induction of mRNA levels of several proinflammatory cytokines in the colon. We believe that these findings support the theory that TNF is a master regulator and is critical to control an acute inflammatory response. The lack of TNF results in the inflammation proceeding uninhibitedly, with the continued production of proinflammatory cytokines, results in propagation of inflammation. Enterocytes are the first line of defense against mucosal bacteria and are capable of producing many proinflammatory cytokines, including TNF, in response to lipopolysaccharide (LPS) stimulation (442). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) a transcription factor responsible for the regulation of transcription of many pro-inflammatory cytokines, has been shown to be upregulated in enterocytes of IBD patients (443). Previous studies have shown that TNF activation of NF-κB in vitro in a non-transformed small intestinal crypt cell line is more rapid than that induced by LPS (444) and blockade of the p65 subunit of NF-κB has been shown to reduce the severity of colitis in mice (445). A major role of NF-κB in enterocytes in regulating induced inflammation was further confirmed by showing that transgenic mice with an enterocyte specific ablation of IKKβ, which blocks activation of the canonical NF-κB pathway, are protected against systemic inflammation following intestinal ischemia-reperfusion (446). Congruent with published results of the predominant role of enterocytes in mucosal immunopathology, we now show that the enterocyte source of TNF is a key mediator of inflammation in the TNBS acute colitis model. The lack of enterocyte derived TNF resulted in a significant increase in colitis scores. Notably, the lesions in E Tnf more severe than those seen in Tnf -/- -/- mice were mice. This increased intestinal response was also evident in 117 the expression pattern of pro-inflammatory genes in the colon. Many more genes which promote inflammation were significantly upregulated and several that inhibit inflammation were significantly downregulated in E Tnf -/- mice as compared to Tnf Figure 3.8 118 -/- mice. Figure 3.8 (continued) Figure 3.8. RNA expression levels of selected genes: Data shows the relative fold induction of -/-/genes in Tnf vs WT (red bar) with acute colitis and Tnf vs WT (red bar) with chronic colitis (blue bar) TNBS-treated mice. Genes with p≤ .05 are shown in the lighter color corresponding to the genotype. Only genes that had at least a ± 2 fold change in response to TNBS treatment in -/and/or in WT mice compared to sham-treated mice were depicted. TNBS treatment Tnf induced upregulation and downregulated relative to the chronicity of treatment. Un-paired t-test used for statistical analysis. Data combined from 2 independent experiments for each condition. 119 Figure 3.9 120 Figure 3.9 (continued) 121 Figure 3.9 (continued) Figure 3.9. TNF promotes inflammation at 10 weeks of chronic TNBS colitis: (A) Time -/course of the average percent weight loss/gain in TNBS and SHAM treated WT and Tnf mice. IR injections were given on days 7, 15, 22, 29, 36, 42, 50, 57, 64, and 71. (B) Colitis scores (data are combined from three independent experiments) and (C) leukocytes (CD45+), macrophages (F480) and T cells (CD3) infiltrating the mucosa (graph shows the number of -/positive cells/5 hpfs;) in TNBS treated WT and Tnf mice. Representative photomicrographs of -/- inflammation in the distal colon are shown in WT (D) and Tnf (E) mice. Data in A-C are shown as means ± SE from 2 independent experiments combined. For colitis scores, n=8 (WT -/-/TNBS), n=7 (Tnf TNBS), n=3 (WT SHAM), and n=4 (Tnf SHAM). For IHC, n=4 mice per -/- group. *p<.05; **p<.01. For IHC graphs, * represent comparisons between WT and Tnf mice. Mann-Whitney test was performed for colitis scores and individual leukocyte counts. The distal and middle sections of colon were evaluated for the presence or absence of atypical glandular hyperplasia. Atypical glandular hyperplasia was present in significantly more WT mice treated 122 -/- with TNBS than in Tnf mice treated with TNBS or SHAM treated mice (J). Representative photomicrographs of the squamous metaplasia (K) and atypical glandular hyperplasia (L) in the -/colons of WT mice, but not and Tnf (M) mice are shown Data represents 3 independent experiments. Mann-Whitney test with **p<.01. Photomicrographs at 200x magnification. Bar= 50μm The results showing no difference in colitis induction in B Tnf -/- mice or μMT mice were not surprising considering the accepted, T cell mediated pathology of the TNBS colitis model. These data are consistent with the previous studies (447) using mice with overexpression of TNF (Tnf ΔARE mice), which developed a Crohn’s like ileitis. In these mice, removal of the B cell compartment had no effect on disease, suggesting that B cells did not contribute to IBD induction or progression. We were surprised, however, to note that the T cell derived TNF was not an important mediator of inflammation in this model. We hypothesized that independent deletion of this source of TNF could possibly be compensated for by production from macrophages/neutrophils, enterocytes or stromal cells. Of particular interest, however, it has been shown that TNBS colitis can be induced in mice lacking B cell and T cells [Severe -/- Combined Immunodeficiency (SCID), Rag 1 ] to the same severity as WT (Balb/c) mice (448). These findings in lymphocyte deficient mice and our findings in mice which lack T or B cell derived TNF are striking and highlight the fact that while the requirement for T cells in the pathogenesis of IBD is compelling, it has not been directly proven. The ability to induce TNBScolitis in lymphocyte- or lymphocyte Tnf deficient mice indicates that T and B cells are unimportant in the initiation and development of IBD and suggests that disease can also develop through interaction with other cell types. 123 The pathogenic role of TNF in chronic colitis, as seen in patients affected by IBD (1) as well as in mouse models of chronic colitis (356, 449, 450) and colitis associated colon cancer (254), is well established. Indeed, unlike in acute colitis, we observed that with increased chronicity of disease, TNF transitions from a protective mediator to a promoter of inflammation and glandular atypia. At 5 weeks of disease, Tnf deficient mice still have significantly more inflammation than WT mice. However, as disease is allowed to progress to 10 weeks, TNF deficiency protects against inflammation. In addition, Tnf -/- mice have significantly less glandular atypia than WT mice. This finding is noteworthy considering the high rate of mixed hyperplastic and adenomatous lesions in patients which eventually develop colon cancer (451). The presence of these lesions in colorectal cancerous patients supports the possible adenomatous transformation of hyperplastic lesions. Taken in context, these data support findings by us (see chapter 4) and others (254) in which the lack of TNF results in a lower rate of colonic polyp formation in the axozymethane/dextran sodium sulfate model of colitis associated colon cancer. We observed that in acute colitis and/or chronic (5 week) colitis many genes known to be important in the pathogenesis of IBD were more upregulated in TNF deficient mice than in control mice. We will highlight 3 of particular interest in IBD and this study, MUC1, IL-11, and STAT3. The human mucin gene family contains 16 known members which can be divided into secreted gel-forming mucins, cell-surface mucins, and secreted non-gel-forming mucins (452). Mucins form a physical protective layer in the intestine between the surface of the mucosa and the luminal components (453). In the healthy colon, membrane bound MUC1 is expressed at low levels and is heavily glycoslyated (452, 454). In IBD, adenomatous dysplasia and colorectal cancer, MUC1 is overexpressed and hypoglycosylated (404-407), possibly because -/- proinflammatory cytokines induce MUC1 expression by epithelial cells (455, 456). Il-10 124 mice develop IBD like colitis, but lack MUC1 expression (454). To evaluate the role of MUC1 in this model, human MUC1 was introduced into the Il-10 -/- model (454). The results showed more similarity to the human disease in that MUC1 was increased in inflamed areas and MUC1 expression by Il-10 -/- mice resulted in more severe inflammation (454). Although we did not evaluate in this study possible changes in the structure of the overexpressed mucins; studies have shown that structure of mucins in IBD patients may reduce the effectiveness of the mucus barrier, possibly making it more susceptible to bacterial degradation (457, 458). In mice, reduction in the sulfonation of mucin resulted in impaired ability to prevent colonization of the small and large intestines and hepatic translocation by the pathogen C. jejuni. However, despite a mild increase in intestinal permeability, hyposulfonation did not result in an inability to contain normal flora to the lumen (459). Applying these studies to our findings suggests that the perturbations in the intestinal flora which we noted in both Tnf -/- and E Tnf -/- mice with acute colitis may be associated not only with the lack of TNF production, but also with changes in Muc1 production. Activation of the transcription factor NFκB appears to be a major component in the pathogenesis of IBD. Activation primarily results from an imbalanced immune response to luminal antigens (460-462). IL-11 inhibits NFκB expression, resulting in downregulation of cytokines produced by macrophages secondary to LPS stimulation (463). A polymorphism in the IL-11 gene has also been shown to be significantly associated with genetic predisposition to the development of UC, but not CD (464). Taken together these results suggest that IL-11 effector functions may represent a major regulatory pathway in mucosal responses to enteric bacteria. In the present study, Il-11 was upregulated in the animals with more acute inflammation 125 (possibly in an attempt to limit inflammation). As well, these mice had increased, potentially pathogeneic, fecal bacteria. Thus it is also plausible that the elevated levels of IL-11 seen in Tnf -/- and E Tnf -/- mice with acute colitis were an attempt to limit NFκB responses to luminal antigens. Of note, IL-11 is capable of inducing STAT3 production by epithelial cells, which may result in suppression of experimental colitis by enhancement of mucosal repair or by inhibiting apoptosis of epithelial cells (465, 466). STAT3 has been shown to be elevated in the mucosa of patients with IBD (467, 468). Mudter et al (469) showed both STAT3 and pospho-STAT3 were elevated in patients with CD and that the level of pospho-STAT3 expression, observed in both T cells and epithelial cells, correlated with the severity of severity of inflammation. STAT3 can have disparate roles in IBD, however, which can be partially explained by the cell type producing the protein. In cases of adaptive immune cells, such as T cells, activation of STAT3 leads to activation of IL-6 and prolonged survival of pathogenic T cells via inhibition of apoptosis (411, 470). In contrast, activation of STAT3 can repress the activation of macrophage and enhance the ability of the epithelial cells to maintain the barrier that limits translocation of luminal antigens (471, 472). In fact, inactivation of STAT3 in colonic epithelial cells or macrophages results in induction of intestinal inflammation and mucosal damage (471-474). These data indicate a regulatory role for STAT3 in the development of colitis that is dependent upon its activation in innate immune cells. In the present study, Stat3 was not significantly induced in acute disease, but was upregulated in Tnf -/- mice with 5 weeks of colitis. It is not clear which cells expressed STAT3 in this case, but it is possible that the elevation could be a promoting factor resulting in the elevated colitis if it was predominantly produced by T cells. 126 On the whole, the present study has explored some of the effects of TNF production during acute colitis by individual cell types as well as during acute versus chronic colitis. Our data demonstrate the protective role of TNF in acute colitis is, at least in part, dependent upon TNF production by enterocytes. We also found, that alterations in TNF production that resulted in more severe colitis were associated with significant changes in the microbial flora. Comparatively, although TNF is protective in acute disease, this cytokine promotes inflammation in chronic disease and induces glandular atypia. These findings indicate that a more targeted cellular and temporal approach of TNF blockade will likely prove to be a more beneficial therapeutic approach for IBD patients. MATERIALS AND METHODS Materials and methods are outlined in Chapter 2. STATEMENT OF CONTRIBUTION: Colitis experiments and accompanying data analyses were conducted by Yava Jones, with comments and advice by Giorgio Trinchieri; Matti Kiupel, Mark Simpson, and Joshua Webster (for colitis scoring and aperio analysis); and Philip Martin and Jerrold Ward (for dysplasia/atypia/neoplasia). Mice were originally provided by Sergei Nedospasov, however current breeding and colony management was done by Yava Jones. RT-PCR for bacterial sequences was performed by Caroline Salter with supervision and assistance by Yava Jones and Amiran Dzutsev. 454 sequencing and analysis was performed by Amiran Dzutsev. 127 CHAPTER FOUR Promoting Versus Protective Role of Tumor Necrosis Factor in the Pathogenesis of Colitis Associated Colon Cancer *Zsofia Gyulai, *Yava Jones, Rosalba Salcedo, Sergei Nedospasov, Giorgio Trinchieri *Equally contributing co-first authors ABSTRACT Tumor necrosis factor (TNF) is a key inflammatory mediator and has been shown to have both promoting, as well as inhibiting roles in tumor development. The observation of opposing roles for TNF based on the cellular source of production during disease is of key importance to understanding the seemingly disparate contributions of TNF in various disease processes. We investigated the role of TNF in a murine model of chronic colitis associated cancer (CAC), which utilizes the mutagenizing agent azoxymethane (AOM) in conjunction with colitis induced by dextran sodium sulfate (DSS). The inflammation instituted by DSS, however, develops at least in part independently of adaptive immunity. Thus, we also attempted to develop a model of colitis using AOM in combination with the chemical Trinitrobenzene sulfonic acid (TNBS) or using APC min/+ mice and TNBS. TNBS induces a T helper (Th)1/Th17 mediated colitis, reminiscent of Crohn’s disease. Although, the AOM/TNBS (and APC min/+ mice with TNBS) induced CAC model was unsuccessful, we did find striking results in the AOM/DSS model. Our results suggest that a contributing factor in the duality of TNF contributions to disease may be determined by the cellular source of TNF. We found that under basal conditions and during 128 AOM/DSS induced colitis, enterocytes are the major producers of TNF. Additionally, the complete absence of TNF and deficiency of enterocyte derived TNF resulted in decreased numbers of colonic polyps, induced by AOM/DSS treatment. In contrast, deficiency of T cell derived TNF resulted in enhanced polyp formation. Therefore, in colitis associated colon cancer, specific blockage of enterocyte derived TNF might have greater advantages than systemic blockage and as an added beneficial effect, the protective effects of T cell derived TNF could be preserved. INTRODUCTION Inflammatory bowel disease (IBD) is comprised of two disease entities, Ulcerative Colitis (UC) and Crohn’s Disease (CD), which have both distinct and overlapping characteristics. Both diseases result in lifelong bouts of relapse and remission of gastrointestinal (GI) inflammation (8). While the pathology of UC is restricted to the distal colon and rectum, CD can affect any portion of the GI tract from the mouth to the anus, with a predominance of disease in the distal ileum and colon (80). The incidence of IBD has increased in recent years and affects approximately 1 million Americans with an average onset in individuals of 15-30 years old and about 30,000 new cases per year (475, 476). The chronic, relapsing inflammation and healing of both conditions can often lead to dysplasia of epithelia, which can result in the development of colorectal cancer (CRC) (477). In fact, the probability of CRC increases from 2 to 18% in individuals affected from 10 to 30 years, respectively (229). It is thus important to understand the link between chronic inflammation and cancer in order to develop therapies to prevent cancer development in IBD patients. Mouse models of IBD have been widely used because they often express many features which recapitulate human disease. Administering dextran sodium sulfate (DSS) in the water of 129 mice and rats induces a colitis with diarrhea and ulceration, with features similar to those seen in patients with UC (337). However, in contrast to UC which is a T helper (Th) 2 mediated disease process, DSS colitis has features of both Th1 and Th17 during acute disease and switches to Th17 during chronic disease (344). As well, the pathogenesis of this model has been shown to be primarily mediated by the innate immune system and occur independently of adaptive immunity (338). More appropriate models for UC and CD use the haptenating agents Oxazolone and Trinitrobenze Sulfonic Acid (TNBS), respectively. Both agents are administered intra-rectally and bind to autologous proteins in the colon, rendering them self reactive. Oxazolone induces pathology and a Th2 cytokine response reminiscent of UC, whereas TNBS induces pathology and a Th1/Th17 response which mimics CD (349, 374). Despite the obvious inconsistencies with the DSS model, the administration of repeated rounds of DSS alone or subsequent to an intra-peritoneal injection of the mutagenizing agent azoxymethane (AOM) has been shown to induce CRC and the latter model is often used to study the pathogenesis of colitis associated cancer (CAC) seen in humans (478, 479). Tumor necrosis factor (TNF) is a key cytokine in the both the induction and propagation of CAC seen in patients with IBD. TNF has been found to be upregulated in the serum, stool and mucosa of IBD patients (283, 480, 481). Additionally, neutralization of circulating TNF has been shown to be highly effective as therapy for many patients with CD (482). Moreover, a recent article by Popivanova et al (254) showed that when mice were subjected to AOM/DSS treatment and were treated with soluble human tumor necrosis factor receptor (TNFR)2 (Etanercept) or mice were deficient in deficient for TNFR1, there was a significant reduction in tumorigenesis. In this study this reduction in polyp number and size was accompanied by a decrease in intestinal inflammation, suggesting a link between the two processes (254). Onizawa et al (255) showed 130 that anti-TNF antibodies decreased the formation of polyps but not inflammation in the AOM/DSS model of colitis, suggesting that the role of TNF and other ligands of the TNFRs might be dissociated in the inflammatory mechanisms leading to colitis or carcinogenesis. Systemic blockade of TNF, however, may be detrimental to patients. In light of the fact that several studies have shown that TNF can be produced by a variety of sources, studies have sought to elucidate the specific role of TNF from various cellular sources (41, 391, 392). Grivennekov et al (41) have shown that macrophages and neutrophils are the primary source of TNF in response to lipopolysaccaride (LPS) and this source is required for protection against Listeria infection. In contrast, T cell derived TNF is required to protect against high bacterial loads. In a model of autoimmune hepatitis, it was shown that both T cell and macrophage derived TNF are required for promoting the disease and these sources are nonredundant (41). Additionally, Togbe et al (483) have shown that macrophage/neutrophil and T cell derived TNF have differential functions in the model of LPS induced airway disease. Deletion of the macrophage/neutrophil sources of TNF resulted in reduction of vascular leakage, bronchochonstriction, and neutrophil recruitment to the lungs. Whereas, deletion of T cell derived TNF resulted in augmentation of these pathologies (483). These studies suggest that targeted deletion of TNF can have beneficial or detrimental effects depending on the nature of the disease. Here we investigate the role of TNF in the AOM/DSS model of CAC with a focus on the cellular source of TNF. We hypothesized that the source of TNF may play a differential role in the promotion or inhibition of the pathogenesis of CAC using mice which have TNF deleted from certain cells. Here we show that, indeed, the cellular source of TNF is an important factor in mediating the pathogenesis of CAC. Mice with total abrogation of TNF (Tnf 131 -/- ) and mice with deletion of TNF from enterocytes (E Tnf (WT) mice (Tnf +/+ or Tnf -/- ) have decreased polyps as compared to wild type fl/fl ). However, mice with deletion of TNF from T cells (T Tnf macrophages/neutrophils (MN Tnf -/- -/- ) had increased tumorigenesis. In addition, we attempted to use the chemical TNBS rather than DSS, with or without AOM, administered to WT, Tnf APC min/+ ) and -/- , or mice to establish a novel model of CAC. However, we were unable to establish carcinogenesis in mice using TNBS. RESULTS AOM/TNBS colitis associated colon cancer We attempted to develop a model of CAC by using IR injections of TNBS to elicit chronic inflammation, rather than DSS. Following presensitization of APC min/+ mice were given 5 weekly IR injections of TNBS and euthanized either 3 days post the last IR injection or 10 weeks post the last IR injection. Mice treated for 5 weeks lost weight subsequent to each IR injection, however, after cessation of IR injections, the mice gained weight steadily until they were euthanized at 15 weeks. Histopathological analysis of colon revealed modest inflammation in mice harvested at 5 weeks, however this inflammation had resolved by 15 weeks (Figure 4.1 A). Additionally, foci of dysplasia (Figure 4.1 B and C) were present randomly in the distal colon of 100% of mice harvested at 5 weeks, but these lesions were not seen in mice harvested at 15 weeks (Figure 4.1 B). Neoplasia was not seen in any animals. In other experiments, the following protocols were performed: a) presensitized APC min/+ mice were given 10 weekly IR injections and euthanized 3 days post the last IR injection; 132 b) presensitized WT, Tnf fl/fl or Tnf -/- mice were given one IP injection of AOM followed by10 weekly IR injection of TNBS; c) presensitized WT mice were given 10 weekly IR injection of TNBS without receiving an AOM injection; and d) presensitized APC Tnf -/- min/+ mice or Tnf fl/fl and mice were given SHAM solution IR for 10 weeks. Mice lost variable amounts of weight post IR injection, but gained weight throughout the course of treatment. Changes in body weight was variable amongst the groups and conclusions attributable to treatment (TNBS vs SHAM) or genotype (WT, Floxed, Tnf -/- , or APC min/+ ) were not noted. Grossly (and histologically) tumors were rare and were only noted in one Tnf fl/fl mouse treated with TNBS and AOM and 2 WT mice treated with AOM and SHAM solution (not shown). Glandular atypia was noted at a low to moderate rate in most mice independent of treatment (TNBS versus SHAM) or genotype (not shown). Overall, neoplastic or pre-neoplastic lesions were not induced to a degree which would make this a useful model for the studying CAC. Enterocytes are the major source of TNF constitutively and post AOM/DSS treatment. We utilized the AOM/DSS model to investigate the role of TNF and specific cellular sources of TNF in the pathogenesis of CAC. The expression of TNF was first analyzed in different leukocyte populations in the spleen, peritoneal macrophages and bone marrow and enterocytes of untreated mice with deletion of TNF in specific cell types (enterocytes, T cells, or macrophages/neutrophils). In E Tnf deleted; in T Tnf -/- -/- mice, 86% of the TNF expression in enterocytes was mice, over 96% of the TNF expression was blocked in CD4 and CD8 T 133 cells; and in MN Tnf -/- mice, the percentage of inhibition of TNF in macrophages and neutrophils was extremely variable depending upon the organ evaluated (Table 2.1). To determine the effect of deletion of TNF production by specific cell types in a model of CAC, conditional Tnf knockout and complete Tnf knockout mice were injected with the carcinogen AOM, on day 0, and seven days later, DSS was administered in drinking water at cycles of 5 days on and 16 days off, for a total of 2 cycles. The induction of TNF by AOM/DSS was also remarkably lower E Tnf mice and T Tnf -/- and MN Tnf -/- -/- in comparison to the levels observed in the treated Tnf fl/fl mice; indicating that enterocytes constitute the predominant source of TNF induced by colitis (Figure 4.2). Not surprisingly, however, AOM/DSS treatment resulted in increases of TNF production in Tnf fl/fl , T Tnf to basal levels. These increases were significant in Tnf -/- and MN Tnf fl/fl -/- mice when compared mice (p=.0274) and T Tnf (p=.0009) (Figure 4.4). Levels pre and post treatment in Tnf treatment) and E Tnf -/- -/- -/- mice (absent basally and post mice, however were not significantly different (Figure 4.2). The lack of TNF promotes chronic inflammation but depletion of TNF from specific cell types does not affect colitis induction Histological analysis of the colons post two DSS cycles revealed no significant difference in the severity of colonic inflammationin the Tnf -/- mice in comparison with the Tnf fl/fl mice (p value = 0.052) and similar levels of inflammation in the cell specific deleter mice compared with Tnf fl/fl mice and compared with each other (Fig 4.3 A.). However, histological analysis of the level of colonic inflammation post four DSS cycles showed a significant increase in colitis in Tnf 134 -/- mice compared to Tnf fl/fl mice (Figure 4.3 B) (although there was considerable overalp in colitis scores) with significantly more T cells in Tnf C). -/- fl/fl mice compared to Tnf mice (Figure 4.3 Consistent with results post 2 DSS cycles, Tnf deficiency in enterocytes, T cells or macrophages/neutrophils did not result in colonic inflammation significantly different in severity from Tnf fl/fl mice (Figure 4.3 B). Thus, in these studies, TNF production may be required to protect against the chronic inflammation induced by AOM/DSS (4 cycles) treatment. It also appears that the lack of TNF from specific cellular sources is insufficient to protect against (or promote) colonic inflammation. The lack of TNF promotes chronic inflammation but depletion of TNF from specific cell types does not affect colitis induction Histological analysis of the colons post two DSS cycles revealed a tendency toward less severe inflammation, although not statistically significant, in the Tnf the Tnf fl/fl -/- mice in comparison with mice (p value = 0.052), but similar levels of inflammation in the cell specific deleter mice compared with Tnf fl/fl mice and compared with each other (Fig 4.3 A.). However, histological analysis of the level of colonic inflammation post four DSS cycles showed a significant increase in colitis in Tnf with significantly more T cells in Tnf -/- -/- mice compared to Tnf mice compared to Tnf fl/fl fl/fl mice (Figure 4.3 B) mice (Figure 4.3 C) . Consistent with results post 2 DSS cycles, Tnf deficiency in enterocytes, T cells or macrophages/neutrophils did not result in colonic inflammation significantly different in severity from Tnf fl/fl mice (Figure 4.3 B). Thus, in these studies, TNF production is required to protect 135 against the chronic inflammation induced by AOM/DSS (4 cycles) treatment. It also appears that the lack of TNF from specific cellular sources is insufficient to protect against (or promote) colonic inflammation. 136 Figure 4.1 min/+ Figure 4.1. APC mice treated for 5 week and harvested at 5 weeks had moderate inflammation and foci of dysplasia, however mice harvested at 15 weeks had neither min/+ inflammation nor dysplasia: APC mice were presensitized and given 5 weekly IR injections of TNBS. Animals euthanized 3 days post the last injection exhibited moderate inflammation which had resolved completely in animals harvested at 15 weeks (A). 100% of mice harvested at 5 weeks exhibited multifocal areas of dysplasia which was not seen in mice harvested at 15 weeks (B). Dysplasia is shown in a representative photomicrograph (C) taken at 400 x magnification. 137 Figure 4.2 Figure 4.2. Enterocytes are the major source of TNF constitutively and post AOM/DSS treatment: TNF mRNA accumulation in the colon tissue was analyzed by Nanostring® in untreated mice and mice treated with AOM /DSS (2 cycles). The gene expression of TNF in treated and untreated mice is depicted. The data shown corresponds to a representative experiment out of two experiments performed. Data shows the means of each sample. MannWhitney test was performed analysis with *p < 0.05 and ***p < 0.001. 138 Enterocyte derived TNF promotes intestinal polyp formation, whereas T cell and Macrophage/Neutrophil derived TNF inhibits tumorigenesis Examination of the colons at day 90-100 post-AOM administration indicated a marked decrease in the number of colonic polyps in the TNF deficient mice in contrast to Tnf (mean number of polyps: 4.9 ± 3.2 in TNF -/- controls (mean number of polyps: 3.7 ± 2.7 in E Tnf Tnf fl/fl mice vs. 9.2 ± 3.7 in control; p=0.0188) (Figure 4.4 A and B). Significantly fewer polyps developed in the E Tnf (Figure 4.4 A and B). In contrast, T cell Tnf fl/fl -/- -/- -/- mice compared to the Tnf fl/fl mice vs. 9.2 ± 3.7 in TNF fl/fl; p=0.0015) mice develop significantly more polyps than the littermates (mean number of polyps: 19.3 ± 7.7 in T cell specific; p = 0.0027) (Figure 4.4 A and B). Using a Poisson regression model to compare multiple experiments (for description see Chapter 2), we found that the decreased polyp number seen in Tnf -/- compared to Tnf fl/fl was significant (p <0.0001) (Table 4.1). In addition the lower polyp count seen in E Tnf compared to Tnf fl/fl -/- mice mice mice seen in observed was also significant (p <0.0001) (Table 4.1). Using the same model to compare T Tnf -/- and MN Tnf -/- to Tnf fl/fl mice the increased polyp count was significant in both (p <0.0001 and p=0.0188, respectively) (Table 4.1). These data indicate that TNF produced by different cell types exhibited different biological activities regarding polyp formation. While enterocyte-specific TNF appears to have a promoting effect on tumorigenesis, T cell and MN-specific TNF inhibit tumor formation. 139 Figure 4.3 140 Figure 4.3 (continued) Figure 4.3. TNF protects against colon chronic inflammation but depletion of TNF from specific cells neither ameliorates nor augments colitis: Mice were injected with AOM on day 0, followed by DSS as indicated in the methods section. At the end of the second or fourth DSS cycle, colons were evaluated. Post two DSS cycles there was no significant differences in -/inflammation (A); however post 4 cycles of DSS Tnf mice had significantly more colitis (B). The elevated colitis featured significantly more CD3+ T cells (and slightly more F4/80+ and -/GR1+ cells) infiltrating the mucosa and submucosa of the distal cm of the colon in Tnf mice (C). Means ± SEM are shown. Mann-Whitney test was performed for colitis scores and IHC. 141 Figure 4.4. Enterocyte derived TNF promotes CAC, while T cell and MN derived TNF protects against CAC: Mice received AOM/DSS treatment as indicated in the methods. One representative experiment of 12 is shown. Poisson Regression Analysis is shown in Table 4.1. At the end of the forth DSS cycle, colons were dissected and visible polyps were counted (A) -/-/fl/fl and Tnf mice had significantly fewer polyps than Tnf and photographed (B). E Tnf -/- mice. T Tnf and MN Tnf represent means ± SE. -/- mice had significantly more polyps than Tnf 142 fl/fl mice. Data Table 4.1: Poisson Regression Model comparing group tumor means with the control group tumor mean Label Mean ratio estimate Number of Experiments DF* tvalue Pr** > |t| (95% Confidence Interval) Tnf -/- 0.493 0.581) (0.419 - 11 332 -8.48 <.0001 E Tnf -/- 0.466 0.597) (0.364- 5 332 -6.06 <.0001 T Tnf -/- 1.568 1.854) (1.327- 5 332 5.29 <.0001 M/N Tnf -/- 1.254 1.513) (1.038- 4 332 2.36 0.0188 *Degree of Freedom; **Poisson Regression Enterocyte, but not T cell or macrophage/neutrophil derived, TNF mediates the promotion of tumor growth and progression of malignancy In general, the size distribution of polyps was similar among the strains, although the E Tnf -/- mice had fewer polyps which were greater than 4 mm in size than the other strains (Fig 5 A). Despite the relative similarity in polyp size, the Tnf -/- mice had a higher rate of malignant tumors (28%) when compared to the other strains (Figure 4.5 B). Surprisingly, although E Tnf mice mimicked Tnf -/- in most features evaluated, only 2% of the polyps in E Tnf 143 -/- -/- mice were adenocarcinomas, strikingly fewer than what was seen in Tnf -/- MN Tnf -/- and Tnf fl/fl mice developed adenocarcinomas at a frequency similar to Tnf mice. T Tnf fl/fl -/- and mice (7.6% and 12.9%, respectively) (Figure 4.5 B). Thus, although TNF favors increased tumor development, tumors are more often benign. Whereas, TNF produced by enterocytes not only positively influences tumor growth and development, but also promotes malignancy. In contrast, T cell and MN derived TNF, while protecting against the formation of polyps, have very little influence on tumor size or malignancy. AOM/DSS treatment induces upregulation of several key mediators and markers of inflammation and carcinogenesis Sections of the distal colon were homogenized and RNA expression levels of several genes (Table 2.2) were analyzed by the nCounter Gene Expression Assay (Nanostring®) preand post DSS/AOM treatment. Several genes were upregulated due to treatment in all strains of mice, most of which were markers/mediators of inflammation (Figure 4.6). Overall, there were no statitistically significant changes in inflammatory gene expression. Notably, however, there were 3 genes important in mucosal defense against bacterial antigens which were upregulated in Tnf -/- and E Tnf -/- mice. Angiogen 4 (Ang4) and Resistin-like molecule beta (Relmb) were both upregulated only in Tnf E Tnf -/- and E Tnf -/- mice (but also in MN Tnf and Reg3g in Tnf -/- and E Tnf -/- -/- mice (Figure 4.6). Reg3g was also upregulated Tnf and T Tnf -/- and -/- ). The upregulated expression of Relmb, Ang4 -/- mice may indicate increased exposure to luminal bacterial antigens in these mice. 144 Figure 4.5 Figure 4.5. Enterocyte, but not T cell or macrophage/neutrophil derived, TNF mediates the promotion of tumor growth and progression of malignancy: Mice received AOM/DSS 145 treatment as indicated in the methods. One representative experiment of 12 is shown. At the end of the forth DSS cycle, colons were dissected and visible polyps’ sizes were measured (A). Polyps were analyzed histologically and the percentage of mice with adenocarcinomas is shown (B). The data shows one representative experiment of two. Figure 4.6 Figure 4.6. RNA expression levels of selected genes: Data shows the fold induction of genes comparing each treated strain to treated untreated mice. Gene depicted are only those with a ± 1.5 or more fold change induced by AOM/DSS treatment in any strain. Several genes are upregulated or downregulated in response to treatment independent of TNF expression in all 5 strains. DISCUSSION 146 As previously stated, administration of DSS in the drinking water has been shown to induce colitis with ulceration as one of the primary pathological features (337). Colitis occurs in this model independent of the adaptive immune system and is thought to cause direct toxicity to colonic crypt epithelia, as well as disrupts mucosal integrity (484). However, both CD and UC develop as a result of dysregulation of cytokine production by CD4+ T cells (10-12). In light of the deficiencies of the AOM/DSS model, we attempted to develop a model of CAC, using the chemical TNBS as the mediator of chronic inflammation due to the inflammatory qualities induced which are more similar to CD than those initiated by DSS. Despite utilizing various protocols and genotypes of mice, we were unable to produce significant neoplasia with models using TNBS. We speculate that this is because TNBS does not mimic DSS in the induction of marked inflammation and mucosal ulceration to a degree which results in significant mutations occurring during healing and repair. Alternatively, C57Bl/6 mice may be resistant to development of CAC using TNBS, as they are, in general, resistant to TNBS colitis. Unfortunately, attempts to use a more susceptible strain (SJL) were unsuccessful due to the high rate or morbidity/mortality following 2 IR injections of TNBS. In the present study, we evaluated the role of TNF in general, as well as TNF production from specific cell types, in the pathogenesis of promotion of chronic inflammation and cancer using the AOM/DSS model of CAC. TNF can have cancer promoting and, as the name implies, cancer necrotizing functions (45). It has also been shown that the cellular source of TNF production plays a differential role in the propagation or inhibition of various disease processes (41, 391, 392, 483). Here we used mice with complete abrogation of TNF or mice with deletion of TNF from specific cell types to address the question of how TNF plays a role in the AOM/DSS model of CAC. 147 We found that after AOM injection and 4 cycles of DSS, mice lacking TNF developed fewer colonic polyps and most were of moderate size, but the polyps that developed were more often malignant. There was also more severe inflammation in the Tnf -/- mice. Thus, our study indicates a disparate role for TNF in this model. While chronic production of TNF can promote tumorigenesis, TNF is also needed to protect against perpetuation of chronic inflammation (evident by the increase in severity of inflammation from 2 cycles to 4 cycles of DSS) and malignancy. The increased colitis scores noted in Tnf -/- mice were mostly attributable to increased numbers of T cells. Overall, however there weren’t significant changes in inflammation as evidenced by similar infiltrating numbers of macrophages and neutrophils and similar mRNA expression levels of pro-inflammatory genes. These results are in agreement with previous studies in which blockade of TNF by monoclonal antibodies failed to prevent or reduce the severity of AOM/DSS chronic colitis, but did result in decreases in size and number of polyps and suppression of NF- B activation, suggesting that the role of TNF and other ligands of the TNFRs might be dissociated in the inflammatory mechanisms leading to colitis or carcinogenesis (255, 485). Nevertheless, there are conflicting reports regarding the promoting vs. inhibitory effects of TNF in colonic inflammation induced by DSS. Neutralization of TNF by using antibodies or antisense oligonucleotides has been shown to reduce inflammation in chronic DSS-induced colitis (345, 450). In a study by Popivanova et al (254), the decrease in polyp formation observed in mice deficient for TNR1 or treated with Etenercept was accompanied by a decrease in inflammation, specifically in infiltrating macrophages and neutrophils. Conversely, Murthy et al showed (486) that blockade of TNF with a single treatment of an inhibitor of TNF release or a TNF monoclonal antibody resulted in decreased 148 disease activity in an acute model of DSS colitis, however blockade of TNF with multiple injections of the TNF antibody, after 3 cycles of DSS had pro-inflammatory effects. Using cell specific deleter mice, we attempted to delineate the contribution of TNF from different cellular sources. Mice lacking TNF production from enterocytes mimicked Tnf -/- in that they had significantly fewer polyps and most were moderately sized. However, the lack of enterocyte derived TNF had no effect on inflammation. Mice deficient in enterocyte derived TNF had the lowest rate of adenocarcinoma formation, suggesting that this source promotes maligancy. Evaluation of T Tnf -/- and MN Tnf -/- mice revealed that these sources had similar effects. Both cellular sources appeared to protect against tumorigenesis. However, neither source affected the progression or severity of inflammation, the size of tumors which developed or the rate of malignancy. Here we show a protective role for T cell and MN derived TNF in the AOM/DSS model, but previous observations in an animal model of inflammatory bowel disease showed both T cell- and macrophage-derived TNF drove the pathology of the disease by induction of IL-12- and IFN-γ- mediated Th1 responses (311). The latter study (311), however evaluated TNF contributions in acute diseases, whereas the DSS/AOM model is a model of chronic disease, possibly explaining the differences in the contribution of these sources. Using Nanostring® technology we evaluated the expression levels of several preselected genes. We found that DSS induced expression of many pro-inflammatory genes as well as suppressed expression of some inflammatory genes and genes which are instrumental in tissue remodeling and repair, independent of TNF expression. 149 The correlation between the development and IBD and microbial antigens is well established (487-489). Ang4 is a protein with potent microbicidal properties that is produced abundantly by Paneth cells in the intestine, primarily in response to gram positive bacteria (490). Ang4 and Reg3g are both induced by inflammation and bacterial challenge, suggesting that these proteins are key players in host defense (491). Relmb is a protein which is expressed by intestinal goblet cells and is also secreted into the intestinal lumen upon induction by microbes (422, 423) and by Th2 cytokines (424, 425). RELMB is overexpressed in IBD patients (390) and loss of RELMB has been shown to reduce the severity of DSS colitis (426). In addition, mice that lack IL-10 develop chronic inflammation and a higher rate of colonic cancer than WT mice which is dependent upon the flora (492). In the latter study treatment with probiotics significantly decreased tumor formation in colitic mice (492). In our study, there was upregulation of 3 genes which play important roles in host defense and protection against microbes seen in the mice with suppression of tumor formation. When considering the established role for the microbiota in tumor promotion, we speculated that upregulation of these genes may have afforded the Tnf -/- and E Tnf -/- mice greater capacity to combat microbial invasion, resulting in a decreased tumor incidence. In conclusion, our results indicate that a tight balance in cell-type-restricted TNF production is an important regulatory mechanism to control the beneficial vs. the deleterious effects of TNF. During colitis, enterocytes, a major source of TNF, exert pro-tumorigenic activities (Figure 4.7). Whereas, TNF from T cells and macrophages/neutrophils is partially protective and can inhibit tumorigenesis (Figure 4.7). Our studies indicated that selective blocking of TNF expression could be beneficial in IBD patients, but systemic blockade of TNF 150 would negate the potential beneficial effects achieved by T cell and macrophage/neutrophil production of the protein. MATERIALS AND METHODS Materials and methods are outlined in Chapter 2. STATEMENT OF CONTRIBUTION Experimental design and gross evaluation was conducted by Yava Jones for the TNBS experiments and by Zsofia Gyulai for the AOM/DSS experiments. Histopathological analysis of tumors and colonic inflammation was conducted by Yava Jones. Immunohistochemical analysis was performed by Zsofia Gyulai. Mice were originally provided by Dr. Nedospasov; however breeding and colony management for these experiments was done by Zsofia Gyulai and Yava Jones. Nanostring evaluation was conducted by Yava Jones with assistance from Rosalba Salcedo and Marco Cardone. Statistical assistance was provided by Octavio Quiñones. 151 Figure 4.7 152 Figure 4.7 (continued) Figure 4.7. Divergent Roles of TNF in Tumorigenesis: In response to injury, TNF production by enterocytes can have a pro-tumorigenic effect. In contrast, TNF production by CD3+ T cells and possibly macrophages, can inhibit tumor formation. 153 CHAPTER FIVE Conclusions and Future Directions Many cell types are capable of tumor necrosis factor (TNF) production in vivo (392) and TNF is known to contribute to cellular proliferation and activation or induce apoptosis (441). Grivennikov et al (41) showed that in response to stimulation by bacterial components, TNF is produced primarily by macropages and neutrophils. Additionally, macrophage/neutrophil and T cell derived TNF promote autoimmune hepatitis. These examples highlight the distinct role of TNF from different cellular sources. In addition to the source of TNF, the chronicity of production as disparate effects on colitis. Studies using a variety of IBD models have shown that blocking TNF function can promote (345, 347, 493), inhibit (325, 334, 366, 369, 370), or have no effect on (324, 367) acute colitis. In the context chronic colitis and colon cancer, blocking TNF has been shown to inhibit (114, 254, 255, 345, 356) or have no affect on (253) disease progression. In this study we utilized mice with complete abrogation of TNF (Tnf -/- ) mice or mice with specific cellular deletion of TNF in models of acute and chronic colitis and colitis associated colon cancer (CAC). Our data shows that TNF is protective in acute disease and the enterocyte derived Tnf is the determinant source of this protection. In our study Tnf -/- -/- and E Tnf mice had increased colonic inflammation, increased expression levels of pro-inflammatory genes in the colon and significant changes in their microbiota. B cell, T cell and macrophage/neutrophil derived Tnf, however, did not augment or ameliorate acute colitis. 154 In contrast, in models of chronic colitis, TNF has a promoting role in disease. Tnf -/- mice had less inflammation and less evidence of epithelial changes which could potentially promote neoplasia, such as glandular atypia and squamous metaplasia. We further expanded our studies to evaluate the role of TNF in CAC. We found that enterocyte derived Tnf promoted the development of colonic polyps. In contrast, T cell derived Tnf protects against the development of CAC. These disparate roles of TNF in colon cancer development overall occurred independently of the level of colitis. From these studies we conclude that regulation of TNF production is a tightly balanced event. Variation in sources of TNF production or length of time or production determines the possible beneficial versus the deleterious effects of TNF which are seen in different diseases. Thus, we suggest that selective cellular or temporal blocking of TNF expression would be more advantageous therapy for IBD patients than systemic blockade of TNF, which could counteract the potential beneficial effects achieved by T cell and macrophage/neutrophil production of the protein. We’ve established a predominantly protective role for TNF production, particularly by enteryocytes, during acute TNBS colitis. This effect appears to be dependent upon or mediated by, at least in part, the microbial flora of the colon. Future projects will expand our microbiome analysis of the feces and colon to better understand the flora composition in our genetically altered mice both pre and post TNBS treatment. In addition will would like to evaluate the flora of mesenteric lymph nodes, the serum and distant organs such as the spleen and liver the establish the level of bacterial translocation pre and post acute colitis. We will pursue the microbiome evaluation with 454 sequencing analysis, using PCR to confirm results as necessary. 155 Although, we did not detect a contributory component of TNF when it was deleted from either B cells or CD4+ T cells alone, we will pursue evaluation of TNF production from the hematopoietic compartment as a whole using mice which express the Vav-Cre transgene and are crossed with Tnf fl/fl mice. These mice will delete TNF from hematopoietic cells and their progenitors. Using our data from the E Tnf -/- mice and these mice, we should be able to better assess the role of hematopoietic cells as well as estimate the role of the stromal compartment in the pathogenesis of acute TNBS colitis. If a striking phenotype is observed in these mice, we may pursue microbiome evaluation as stated above. We have established a model of chronicity (10 weeks) which results in a phenotype in our Tnf -/- mice, which is opposing to what we have observed in acute colitis and is in line with the therapeutic benefits observed with anti-TNF treatment and in Tnf -/- mice subjected to models of colitis associated colon cancer. We would like to first repeat the 10 week experiments using our Tnf -/- mice to increase our sample size and possible achieve differences in colitis which reach statistical significance. In addition, we hope to evaluate the role of enterocyte derived TNF, and possibly hematopoietic derived TNF (vav-cre mice) at 10 weeks. Our findings emphasize the complexity of TNF in promotion and protection during various stages of inflammation, as well as the potential integrated role of the microbiota in promoting or preventing inflammation. In addition, these results underscore the need for a more targeted approach to therapy, rather than broad, long-term blockade of this multifunctional cytokine. As well, these studies indicate a potentially beneficial role for patient specific therapy driven by the patient’s commensal flora. 156 APPENDIX 157 APPENDIX PUBLISHED CO-AUTHORED PAPERS 1. Salcedo, R., Worschech, A., Cardone, M., Jones, Y., Gyulai, Z., Dai, R., Wang, E., Ma, W., Haines, D., Ohuigin, C., Marincola, F., and Trinchieri, G. (2010) MyD88-Mediated Signaling Prevents Development of Adenocarcinomas of the Colon via Interleukin-18. Journal of Experimental Medicine. 207: 1625-1636. ABSTRACT Signaling through the adaptor protein MyD88 promotes carcinogenesisin several chemically induced cancer models. In contrast, inthe AOM/DSS model of chronic colitis induced cancer, we observeda protective role for MyD88 in the development of colitis-associatedcancer (CAC). The inability of Myd88-/- mice to heal ulcersgenerated upon DSS treatment creates an altered inflammatoryenvironment that induces profound early alterations in expressionof genes encoding pro-inflammatory factors as well as pathwaysregulating cell proliferation and apoptosis. Additionally, AOM/DSStreatment induced alterations in DNA repair in MyD88 deficientmice, resulting in increased frequency of mutations and a dramaticincrease in adenoma formation and progression to invasive phenotype.Others have reported that Tlr4 deficient mice share similarsusceptibility to colitis as Myd88 deficient mice but, unlikethe latter, are resistant to CAC. We observed that mice deficientfor Tlr2 or IL-1R do not show a differential susceptibilityto colitis or CAC. In contrast, AOM/DSS treatment resulted inincreased susceptibility to colitis development and enhancedpolyp formation in Il-18-/- mice. This indicates that the phenotypeof Myd88 -/- mice is in part due to their inability to signalthrough the IL-18 receptor. This study revealed a previouslyunknown level of complexity surrounding MyD88 activities downstreamof different receptors that differentially impact tissue homeostasisand carcinogenesis. 158 STATEMENT OF CONTRIBUTION: The experimental design and the majority of data analysis for this study were conducted by Rosalba Salcedo. Yava Jones’ contribution to this paper was confined to histopathological evaluataion of colon tumors (also analyzed by a staff pathologist at the Pathology and Histotechnology Laboratory at NCI, Frederick) and colon inflammation. Immunohistochemical analysis was performed by Rosalba Salcedo with assistance from Yava Jones. 2. Young, M., Ileva, L., Bernardo, M., Riffle, L., Jones, Y., Kim, Y., Colburn, N., and Choyke, P. (2008). Monitoring of tumor promotion and progression in a mouse model of inflammationinduced colon cancer with Magnetic Resonance Colonography. Neoplasia. 11(3): 237-246 ABSTRACT Early detection of precancerous tissue has significantly improved survival of most cancers including colorectal cancer (CRC). Animal models designed to study the early stages of cancer are valuable for identifying molecular events and response indicators that correlate with the onset of disease. The goal of this work was to investigate magnetic resonance (MR) colonography in a mouse model of CRC on a clinical MR imager. Mice treated with azoxymethane and dextran sulfate sodium were imaged by serial MR colonography (MRC) from initiation to euthanasia. Magnetic resonance colonography was obtained with both T1- and T2-weighted images after administration of a Fluorinert enema to remove residual luminal signal and intravenous contrast to enhance the colon wall. Individual tumor volumes were calculated and validated ex vivo. The Fluorinert enema provided a clear differentiation of the lumen of the colon from the mucosal lining. Inflammation was detected 3 days after dextran sulfate sodium exposure and subsided during the next week. Tumors as small as 1.2 mm(3) were detected and as early as 29 days after initiation. Individual tumor growths were followed over time, and tumor volumes were measured by MR imaging correlated with volumes measured ex vivo. The use of a Fluorinert enema during 159 MRC in mice is critical for differentiating mural processes from intraluminal debris. Magnetic resonance colonography with Fluorinert enema and intravenous contrast enhancement will be useful in the study of the initial stages of colon cancer and will reduce the number of animals needed for preclinical trials of prevention or intervention. STATEMENT OF CONTRIBUTION: Experimental design and the majority of data analysis for this project were conducted by Matthew Young. Yava Jones’ contribution to the study was confined to histopathological evaluation of colonic tumors and colonic inflammation. 3. Jones, Y. Fitzgerald, S. D. (2009). Articular Gout, Metastatic Calcification, and Suspected Pseudogout in a Basilisk Lizard (Basilicus plumifrons). Journal of Zoo and Wildllife Medicine. 40(3): 576–578. ABSTRACT A 9-yr-old male Basilisk lizard (Basilicus plumifrons) with a history of painful and limited mobility for approximately 4 mo, which had seemed to be more pronounced in the front limbs, was presented for necropsy. The animal had exhibited moderate weight loss and anorexia before euthanasia. Postmortem examination revealed yellow-to-white, soft-to-semifirm nodules within the periarticular fascia and musculature of the left and right shoulder joints, hip joints, and stifle joints. Several other joints, including the left and right tarsi, left and right elbow joints, and the left carpus had calcified, white material present on the articular surfaces. Histopathologic evaluation of representative sections of all organs and the joints confirmed tophaceous articular gout and articular pseudogout. The differentiation between articular gout and pseudogout was based on histologic appearance, histochemical staining for calcium, and birefringence under polarized light. 160 This article was a case report of a Basilisk lizard that presented for necropsy at the Diagnostic Center for Population and Animal Health at Michigan State University. The animal was determined to have lesions of gout and suspected lesions of pseudogout in its joints and several soft tissue sites. The lesions were thought to be dietary induced. STATEMENT OF CONTRIBUTION: The case was evaluated by Yava Jones and Scott Fitzgerald. The report was written by Yava Jones under the purview of Scott Fitzgerald. SUBMITTED OR IN PROGRESS CO-AUTHORED PAPERS 4. Butts, CL, Jones, YJ, Lim, JK, Salter, CE, Belyavskaya, E, and Sternberg, EM. (Nov 2010) Tissue expression of steroid hormone receptors is associated with differential immune responses. Brain, Behavior and Immunity (In Press). ABSTRACT Glucocorticoids have been used as treatments against a number of diseases, especially autoimmune/inflammatory conditions in which the immune system is overactive. These treatments have varying degrees of responsiveness among individuals and in different tissues (including brain); therefore, it is important to determine what could account for these differences. In this study, we evaluated expression of stress hormone receptors in immune cells from lymphoid and non-lymphoid tissues (including brain) as a possible explanation. We analyzed leukocytes (CD45(+)) in kidney, liver, spleen, and thymus tissues from healthy mice for expression of the receptor for stress hormone (glucocorticoid-GR) as well as other steroid hormones (androgen-AR, progesterone-PR) and found that all tissues expressed these steroid hormone receptors but with varying patterns. To determine whether tissue-specific differences were related to immune cell composition, we examined steroid hormone receptor expression in T lymphocytes from each of these tissues and found similar patterns of expression in these cells 161 regardless of tissue source. Because glucocorticoids can also impact brain function, we further examined expression of the stress hormone receptor in brain tissue and found GR expressed in immune cells at this site. In order to investigate the potential impact in an area of neuropathology, we utilized a mouse model of West Nile Virus (WNV). We observed pathological changes in brains of WNV-infected animals and T lymphocytes in the areas of inflammation; however, these cells did not express GR. These data indicate that tissue-specific differences in steroid hormone receptor expression by immune cells could determine responsiveness to steroid hormone treatment. STATEMENT OF CONTRIBUTION: Experimental design and the majority of data analyses for this study were conducted by Cherié Butts. Yava Jones performed the immunohistochemical staining for and histopathological evaluation of the brain, thymuses and spleens. Thymuses and spleens had been previously analyzed by a staff pathologist at the Pathology and Histotechnology Laboratory at NCI, Frederick. Their findings of apoptosis and lymphoid depletion were confirmed and Yava Jones assisted with interpretation of these lesions in the context of this study. 5. Tami, C., Butts, C., Pedras-Vasconcelos, J., Jones, Y., Puig, M., Wang, V., and Verthelyi, D., (2010). MyD88 expressing T cells are required for neuropathogenesis of Tacaribe virus. (In Progress). This study evaluated the role of MyD88 expressing T cells in the pathogenesis of Tacarabe virus (TCRV) infection. The study concluded that T cells mediate disease and death in TCRV-infected mice without affecting viral replication and brain tropism and suggest a critical role of MYD88 expression on T cell in the pathology. STATEMENT OF CONTRIBUTION: Experimental design and the majority of data analyses for this study were conducted by Cecelia Tami and Cherié Butts. Yava Jones performed the 162 immunohistochemical staining for and histopathological evaluation of the brains of infected animals. 163 REFERENCES 164 REFERENCES 1. 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