Overview
The CSIBD brings together investigators with expertise in the key areas integral to understanding IBD. The leadership and organization of the Biomedical Cores have been developed to capitalize on the progress made during the current funding period in addressing the Center's underlying IBD hypothesis. The Molecular Biology, Immunology and Morphology Cores ensure the ability of Center investigators to apply the most sophisticated techniques to define the molecular and cellular basis of immune/inflammatory responses and epithelial cell function. In particular, the Genetic Animal Model, Morphology and Immunology Cores form a closely coordinated consortium in which the morphologic and biochemical assessment of murine IBD models enables CSIBD investigators to understand the association of immune, inflammatory and epithelial cell function with the establishment of intestinal inflammation. The Clinical/Tissue Core is central in enabling the Center to also pursue parallel and complementary studies in man. With ongoing basic advances, the support of translational research aimed at improving approaches to the care of patients with IBD is of paramount importance.
Epithelial Function and Mucosal Barrier
Accumulated evidence underscores the critical role that the alterations in epithelial cell function and barrier integrity can play in the pathogenesis of intestinal inflammation. Thus, studies of patients with inflammatory bowel disease using inert markers suggest that these individuals may have enhanced intestinal permeability and that this alteration can preexist the actual development of inflammatory bowel disease. The continuity of the epithelium itself in which the constituent cells are joined by tight junctions provides the most essential defense as a physical barrier to the luminal contents. Over the past several years considerable further molecular detail of tight junction structure and regulation have been acquired. It is clear that these structures are formed by a complex assortment of proteins, which can regulate paracellular passage of small molecular weight substances from the lumen, presumably including bacterial and diet derived products. In addition to the epithelial cells and their tight junctions, barrier function is enhanced by the presence of the pre-epithelial layer formed primarily by mucus glycoproteins and trefoil peptides.
It is noteworthy that each of the three genes (see below) so far identified for current linkage to IBD (NOD2, OCTN1 and DLG5) is expressed within the epithelial compartment, and at least one of these (OCTN1) which serves as a transporter of small cationic molecules is apparently almost exclusively expressed within the epithelial compartment.
Work in this center and elsewhere has led to the recognition that the epithelial cell compartment participates in a broader variety of functions than was historically appreciated including an active contribution to immune responses, especially those of the innate immune system mediated by both Toll-like and Nod receptor families. Their presence and characterization of their function has underscored the pivotal role that the epithelium plays in the interaction with the luminal flora, an interface which is increasingly recognized to be of central importance in the maintenance of normal homeostasis or the eventual development of IBD as highlighted below. Other epithelial functions contribute to host defense, including the production of antimicrobial peptide most especially by the Paneth cells (a subpopulation of epithelial cells which also express various innate immune receptors such as NOD2). Therefore, achieving a comprehensive understanding of the molecular basis for epithelial cell function and its alteration in association with inflammatory bowel diseases is needed.
Host-Microbial Interaction
Compelling evidence from clinical experience and direct experimental data supports the conclusion that alterations in the normal homeostatic interaction between the mucosal surface and the luminal flora play key roles in the pathogenesis of inflammatory bowel disease. Increasingly, suspicion is focused on the possibility that microbial agents that are normally non-pathogenic, perhaps even normal intestinal commensal flora, might be pathogenic in a susceptible host. Furthermore, bacterial flora appears to be a prerequisite for the development of chronic inflammation in many genetically susceptible murine strains. Thus genetically susceptible murine lines do not develop colitis when maintained in a germ-free environment but do manifest the colitis when a "normal" luminal flora is established, providing direct evidence that bacteria which are not pathogens in a conventional sense (i.e. are not aggressive invaders or toxin producers) play a necessary role in pathogenesis of inflammatory bowel disease. Of great interest, a number of groups have recently demonstrated increased numbers of mucosal adherent and invasive E. coli in patients with IBD. It is also well known that bacterial products including those recognized by innate immune receptors (so called PAMPS, or pathogen associated molecular pattern molecules) can stimulate many mucosal cell populations including surface epithelium (as noted above) and dendritic cells which may sample these products directly. Therefore it is of critical importance to define the mechanisms of mucosal-microbial interactions, including both those involving surface epithelium and those involving the underlying lamina propria.
Innate and Adaptive Immunity and IBD
Recently the central importance of activation of innate immune responses in the pathogenesis of IBD and the close interplay with subsequent activation of adaptive immune responses has become clear.
In the past few years rapid progress has been made in defining the mechanisms of the innate immune response. These are diverse and range from the production of antimicrobial peptides (e.g. defensins by Paneth and myeloid cells) as well as the secretion of other peptide products, including Mannose Binding Protein (MBP) and Decay Accelerating Factor (DAF). However, PRRs are increasingly recognized as being central to innate immune responses. These evolutionarily ancient receptors provide a "hard wired" response to microbial species and other foreign constituents by recognizing common molecular patterns. Many of the PRRs recognize so-called pathogen associated molecular patterns (PAMPs). However, it should be noted that many of the molecular patterns recognized by the PRRs are, in fact, shared by commensal microbial species. A critical question as yet unresolved is how these receptors distinguish between commensal and pathogenic bacteria. An elucidation of the basis of the homeostatic co-existence of the intestinal mucosa with the luminal flora will be essential to achieve an understanding of the mechanisms leading to chronic inflammation in the context of IBD which appears, on the basis of the studies of murine models, to be driven by these very same commensal bacteria.
The Toll like receptor (TLR) and Nod receptor families play key roles in innate immune responses. Although initially presumed to be present almost exclusively within the myeloid cell compartments (especially the dendritic cell populations), it is now clear that these PRRs are also expressed more broadly within the intestinal mucosa. Their importance is certainly underscored by the finding that the IBD 1 locus encodes Nod2 (also designated CARD15) a cytoplasmic receptor for muramyl dipeptide (MDP) (a fragment of proteoglycan derived from bacterial cell walls). The PRR NOD2 activates innate immune signaling pathways and, recent evidence suggests, may indirectly modulate responses induced by the TLRs a separate family of transmembrane PRRs as well as cross activate adaptive immune responses. Germ line mutations within the leucine rich repeat (LRR) ligand binding domain are present in a minority of Crohn's disease patients (particularly those with ileal involvement). These mutations are associated with defective activation of NF- k B and, as demonstrated in this center, concomitant defects in the ability to control intracellular bacterial survival. A detailed understanding of the pathways activated by these innate immune receptors and their functional effects will be essential to understand their role in inflammatory bowel disease, and their integration with adaptive immune responses.
Inflammatory Pathways Leading to IBD
It is clear that collectively cytokines play a major role in the development of IBD and recruitment of lymphocytes, macrophages and neutrophils . They facilitate amplification of immune responses by enhancing recruitment and activation. Many of these same cytokines have been demonstrated to be produced by and/or to activate fibroblasts, endothelial cells, smooth muscle cells and epithelial cells. Indeed the recognition of the close integration of the epithelium with cell populations within the lamina propria as a result of both their production and response to cytokines has been an important development in which CSIBD investigators have made significant contributions. The pro-inflammatory cytokines also mediate systemic manifestations of inflammation, e.g. fever and hepatic production of acute phase reactants. Other important effects include enhanced production of collagen and other extracellular matrix components by smooth muscle and other cells that may result in fibrosis and stricture formation in CD.
In addition to cytokines and chemokines, it is increasingly apparent that neuropeptides may play an important role in regulating many of these same inflammatory responses and modulating the functional effects of cytokines and other mediators. Increased concentrations of some neuropeptides are present in IBD tissues while their receptors may be found on many cell populations, e.g. substance P, somatostatin and VIP. These peptides may substantially modulate lymphocyte activation and other mucosal inflammatory responses.
Recruitment of acute inflammatory cells leads to the production of additional key mediators of inflammation with resulting amplification of these processes . These products include additional cytokines and chemokines as well as "non-specific" mediators of inflammation. Among the latter, arachidonic acid metabolites appear to be especially important. Thus leukotrienes are important chemotactic compounds while prostaglandins along with other metabolites and some cytokines promote electrolyte and fluid secretion contributing to diarrhea.
Although an understanding of the full spectrum of immune and inflammatory mediators which contribute to the pathogenesis of IBD, requires further research, a broad schema of these pathways is already possible. Elucidation of these pathways and the relative functional importance of these mediators are important goals of the CSIBD.
Genetic Determinants and IBD
The CSIBD emphasizes further characterization of the processes that lead to IBD to facilitate the search for specific genetically regulated pathophysiologic processes in IBD patients. Identification of specific genes and their products have enabled development of new more refined hypotheses of IBD pathophysiologic mechanisms, in particular innate immune mechanics. Importantly implicating innate immune receptor activated pathways by genetic studies coincides with the independent recognition of their importance resulting from efforts focused on studies of functional properties of intestinal mucosa and their alterations in conjunction with IBD and other disorders. Understanding these mechanistic pathways is a major thrust of many CSIBD investigators, and the center's program facilitates these efforts by fostering collaboration and providing key support services.
Many observations support the inference that genetically determined factors contribute to the development of IBD . In man, these include (1) variations in prevalence of disease among different populations, (2) the co-segregation of IBD with disorders known to be genetically determined in some kindreds and (3) the increased risk of disease in first degree relatives of a patient with either UC or CD. The high degree of concordance of disease in monozygotic twin pairs reported from Sweden (as high as 67% in those pairs in which the proband had CD and 20% concordance for UC in identical twins compared to 8% for non-identical twins) provides especially compelling support for the conclusion that IBD can result from, or depends upon, genetically defined factors.
These and other observations also indicate that genetically defined factors may result in a predisposition for disease but development of disease requires either cooperative interaction among multiple genetic loci and/or initiating "environmental" (most likely luminal) factors. The interaction between two or more genetic loci is suggested by the still relatively low frequency of disease in first degree relatives of patients with IBD (generally 5-10%, too low to reflect recessive inheritance of a simple Mendelian "disease gene" even with variable penetrance). Furthermore, the lack of uniform disease concordance even within identical twin pairs (especially UC) clearly implies that disease development is not solely a reflection of genetically defined host susceptibility.
The specific genes responsible for three susceptibility loci have been identified and high risk alleles (mutations) identified. Interestingly, it appears that the products encoded by at least two of these genes belong to a pathway with genetic epistasis demonstrated among the disease associated alleles. The latter are also both selectively associated with ileal Crohn's disease rather than UC or Crohn's involving other segments of the GI tract. As noted above, the first of these, IBD1 encodes NOD2, (also designated CARD15), which serves as a cytoplasmic receptor for muramyl dipeptide (MDP) a fragment of bacterial cell wall proteoglycan. As a pattern recognition receptor, NOD2 activates innate immune signaling pathways [and recent evidence suggests may indirectly modulate responses induced by Toll-like receptors (TLRs)], as well as cross-activate adaptive immune responses. Germline mutations in the MDP binding leucine rich repeat (LRR) domain are present in a significant minority of Crohn's disease patients with ileal involvement and may be somewhat more common in those with the fibrostenotic phenotype. NOD2 is expressed in both epithelial and monocytic lineages in the intestinal mucosa. Work in this center has suggested that Nod2 activated pathways play an important role in decreasing intracellular bacterial survival.
Although it remains unclear whether all of these same genes will be relevant to the majority of patients with IBD, who lack a family history of IBD, identification of these susceptibility genes represent significant advances in providing a foundation for better understanding of IBD mechanisms. However, identification of genetically implicated products is not sufficient to understand how they fit into the etiopathogenetic events that result in IBD.
Murine Models of IBD
Murine models offer a powerful tool for the analysis of genetic processes in IBD. Over the past several years, a number of murine models of IBD have been developed which support the concepts underlying the theme of the CSIBD; i.e. the interaction of genetic, immune and environmental factors most notably luminal bacterial flora can result in chronic intestinal inflammation . The opportunities for progress in the next few years are especially promising due to the availability of murine models offering the potential to study both factors necessary for the initiation of chronic intestinal inflammation and the interrelationships between the mediators, which contribute to inflammatory amplification and tissue injury.
Development of these models has already been essential to many of the recent advances in IBD research. These include the finding of an IBD-like colitis in T-cell receptor a (TCR a ) deficient mice first documented by this center. Early characterization of these models and others now available in this center (e.g. the CD3 e 26 transgenic line developed in the CSIBD and CD45RB hi + adoptive transfer model) demonstrates that the manifestation of "IBD" resulting from gene deletion is significantly influenced by other genetic loci. Developmental work by the CSIBD Genetic Animal Model Core has demonstrated that the prevalence and severity of colitis in TCR a / b , MHC Class II and IL-2 deficient mice varies significantly among different inbred mouse strains when the original lines are cross-bred to other genetic backgrounds. In addition, the ability of the microbial environment to modulate the phenotype of the gene deficient mice was demonstrated by the finding that many (if not all) of these mutant murine lines do not develop colitis when maintained in a germ-free environment.
The further study of these and other animal models to advance understanding of the mucosal immune response, inflammation and other dimensions of mucosal biology is central to the continuing goals of the CSIBD in the next five years. Progress in the study of the pathogenesis of disease in the murine models will not only provide further insight into the interrelationships among different components of mucosal immune and inflammatory responses, but will also provide a guide to which of these components may be critical to examine in the study of genetic alterations in man.
