Inflammatory Bowel Disease (IBD), comprised of Crohn’s disease and ulcerative colitis, is manifested by chronic inflammation of the gastrointestinal tract with significant morbidity and, at times, life-threatening complications. Accumulating evidence indicates that an altered immune response to microbial factors plays a major role in IBD. This evidence includes genetic studies that identified several IBD-associated genes, including autophagy genes (ATG16L1 and IRGM) and tumor necrosis factor superfamily member 15 (TNFSF15). Autophagy describes a biological process that involves trafficking and handling of macromolecules, including bacteria. Recent studies show that autophagy plays an important role in the clearance of microbial pathogens. Alterations in the autophagic machinery, such as in ATG16L1 and IRGM, may result in the ineffective elimination of pathogens and the persistent activation of the inflammatory cascade in the intestine, which may contribute to the pathogenesis of IBD. My research group uses combinatorial approaches to study the mechanism of gut mucosal immunobiology, using inflammatory bowel disease (IBD) as a disease model. The approaches are described below.
In Vivo Target Development and its Characterization
Genome-wide association study (GWAS) has identified over 160 genetic loci that may be important in the pathophysiology of IBD. One of the main challenges resulting from GWAS studies is to determine whether these putative disease loci are causative (and not just associative) and to determine their role in gut mucosal immunology. Our laboratory aims to develop genetically modified mice to characterize IBD-associated gene function in vivo and in vitro. We have generated several murine models in the areas of mucosal immunology, fibrostenosis, and bacterial-host interaction. Table 1 lists the murine models that were generated by our group.
|Table 1. List of Murine Models|
Host and gut flora have a mutualistic relationship, and disturbance of this coexistence can lead to human diseases such as IBD. GWAS studies identified several IBD-associated genes involved in microbial-host interaction, including intracellular pattern recognition sensor NOD2, autophagy genes (ATG16L1 and IRGM) and TL1A. We have shown that bacteria, both pathogenic and commensal, can induce TL1A expression in antigen presenting cells. We also show that TL1A expression can be further increased by autophagy in the presence of bacteria. This indicates that autophagic processing of bacteria is important for the optimal expression of TL1A, a critical factor in the modulation of gut mucosal inflammation. Our overall hypothesis is that autophagy handling of microbial organisms is important for optimal TL1A expression that determines the severity of chronic intestinal inflammation and fibrosis. Below are brief descriptions of the ongoing research in this area.
a. Determine the role of autophagy in epithelial cells, dendritic cells, and macrophages on the development and severity of murine chronic colitis: We have generated tissue-specific Atg16l1 deficiency in epithelial cells, dendritic cells, and macrophages (Table 1). We will assess the inflammatory phenotype using DSS colitis and Salmonella typhimurium infectious models.
b. Characterize germ free transgenic mice that constitutively express TL1A in T cells (L-TL1A Tg): We have shown that L-TL1A Tg mice develop intestinal inflammation and fibrostenosis in 2 murine models of colitis. We are collaborating with Dr. Balfor Sartor (UNC) and Dr. Jonathan Braun (UCLA) to re-derive the L-TL1A Tg mice germ free and characterize the effect of gut microbiota on intestinal inflammation and fibrosis.
c. Determine the roles of a naturally occurring antibacterial peptide, cathelicidin, in intestinal inflammation: In collaboration with Dr. Koon and Dr. Pothoulakis (UCLA), we have shown that cathelicidin protects against murine colitis through activation of TLR9-ERK signaling by bacterial DNA. Additionally, we have preliminary evidence that cathelicidin ameliorates C. difficile infection in mice. C. difficile infection is common in IBD patients. We are currently evaluating further the anti-inflammatory mechanisms of cathelicidin.
Mechanisms of Intestinal Fibrostenosis
Crohn’s disease (CD) is a chronic inflammatory condition with pathological features such as patchy transmural inflammation and fibrostenosis. TL1A, a member of the TNF superfamily, binds to death domain receptor 3 (DR3) and modulates the adaptive immune response. A particular TNFSF15 haplotype is associated with higher TL1A expression, increased risk of CD, intestinal fibrostenosis, and greater need for surgery. In addition to human reports, studies in mice also implicate the TL1A/DR3 signaling pathway in mucosal inflammation and fibrosis. Constitutive TL1A expression in mice leads to mild spontaneous ileitis and increased collagen deposition. Our overall hypothesis is that TL1A is a pro-fibrogenic gene which may be a therapeutic target to treat both gut mucosal inflammation and fibrosis, thereby altering the natural history of Crohn’s disease (Figure 1). Below are brief descriptions of the ongoing research in this area.
a. In vivo analysis of the mechanisms of TL1A/DR3 pathway in fibrogenesis and pathological healing: We have generated conditional knockouts of TL1A and DR3 in fibroblasts, epithelial cells, T cells, and antigen presenting cells. Investigation is underway to characterize these genetically modified mice in fibrogenic mechanisms.
b. In vitro analysis of the mechanisms of TL1A/DR3 pathway in fibrogenesis and pathological healing: We have established methods to isolate IMF from mice and humans. We will be analyzing the mechanisms of fibrogenesis using isolated fibroblasts from genetically modified mice. In addition, we will isolate IMF from genetically defined IBD patients with and without intestinal fibrosis to confirm our findings in mice.
|Figure 1. Gut Fibrosis Model - Under chronic injury, intestinal fibrogenesis occurs, leading to intestinal fibrostenosis. This is due to an increase in the number of activated intestinal myofibroblasts (IMF). During the healing process, the number of activated IMF decreases. This is due, at least in part, to resetting the fibrogenic program by reducing TGF-β and IGF-I signaling.|
Investigators at the F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute have performed GWAS studies on IBD patients who are followed at Cedars-Sinai. In addition to genotypic information, the patients are also clinically prospectively serotyped and phenotyped, with tissues being deposited in a bio-repository. All the clinical, serological, genotypic, and banked tissue information is being or has been populated in the IBD database. I have been using this valuable information and these reagents in hopes of developing“personalized medicine” programs to treat our IBD patients. The translational research studies that are currently underway in my research group include:
a. Clinical outcomes of concomitant infection (e.g., CMV) in IBD flare using genetic, serologic, and clinical parameters.
b. Metabolism of thiopurine drugs (6MP and azathiopurine) to minimize adverse effects and optimize drug dosing.
c. Genetic, serological, and clinical factors predicting extraintestinal manifestations of IBD and its management.
d. Genotype-phenotype association of H. pylori infection in IBD.
e. Assessment of synergy with concomitant anti-TNF/thiopurine therapy in IBD.