Final Progress Report

Proposal No. IBD-0102
Principal Investigator:   Andrew T. Gewirtz, Ph.D.
Applicant Organization:  Emory University (Atlanta, Georgia, U.S.A.)
Project Title:  Adaptive immunity to flagellin in inflammatory bowel disease
Period of Award:  May 1, 2004 – April 30, 2006

A. Project Aims:

The goal our BMRP project were to define whether bacterial flagellin was a target of the adaptive immune response in Inflammatory bowel and mouse models of colitis. Further, we sought to define the mechanisms that regulate such responses. Our specific aims were as follows:

Specific Aim I: Examine serum antibody titers to flagellin in humans in health and disease. We will build upon our preliminary data to i) Define titers to flagellin in healthy controls and various states of IBD. Control samples will include unaffected relatives and spouses. These will be further compared to titers of persons having recent acute resolved Salmonella infections. ii) Determine the emphasis of flagellin titers toward mucosal vs. systemic immunity as well as acute vs. chronic immunity by measuring the relative levels of IgG, IgA, and IgM antibodies to flagellin. iii) Determine whether flagellin antibody titers may originate from epithelial barrier dysfunction by examining whether antibody titers to flagellin correlate with barrier function in IBD and control patients. iv) Investigate whether high antibody titers to flagellin derive from repeated infectious episodes by examining serum from critically ill patients with such recent history of infection.

Specific Aim II: Measure murine antibody titer to flagellin in well-defined experimental models of gut inflammation. We will take advantage of the wealth of models of acute and chronic gastroenteritis mediated by epithelial dysfunction, immunological dysregulation, or pathogenic bacteria to mechanistically understand when and how the adaptive immune system sees flagellin in health, normal immunity, and IBD. We will i) Define normal murine antibody titers (serum and fecal) in healthy mice. ii) Determine antibody titers to flagellin following acute sub-lethal Salmonella infection. Antibody titer to homo and heterotypic flagellin serotypes will be measured. iii) Determine flagellin antibody titers in IBD models (DSS and TNBS induced colitis) whose pathogenesis is thought to involve prolonged (DSS) or acute (TNBS) epithelial breach as an initiating event in chronic colitis. iv) Determine flagellin titers in the spontaneous IL-10 knockout colitis model in which immune dysregulation initiates colitis (including measuring the effect of altering epithelial barrier function with NSAIDS in this context. v) Determine the role of TLR5 activation in developing anti-flagellin antibodies.

B. Accomplishments:

These aims were, largely successfully completed. Briefly, we found that bacterial flagellin is indeed one specific target of the Crohn’s disease associated adaptive immune response (1). Further, we observed that immune responses to flagellin are indeed induced in mice by inducing epithelial barrier dysfunction with chemicals such as DSS (2). Moreover, we went substantially beyond our proposed aims in that we mechanistically defined how generation of adaptive immune responses to flagellin are regulated and demonstrated the relevance of these findings in humans in that we observed that carriage of a TLR5-stop polymorphism lowered both immune responses to flagellin and was negatively associated with Crohn’s disease (3). This latter finding suggests that pharmacologic inhibition of TLR5 might be able to treat or prevent CD.

C. A list of Significant results:

   1.    Persons with CD, but not UC, have elevated serum antibodies (IgG, IgA, and IgM) to common bacterial flagellin.
   2.    Flagellin is the major target of the Crohn’s disease associated humoral immune response that is reactive with E. coli.
   3.    Among persons of Jewish ethnicity, carriage of a common TLR5 polymorphism may provide protection against Crohn’s disease.
   4.     In mice, epithelial barrier dysfunction is sufficient to result in antibodies to endogenous flagellins.
   5.    Generation of anti-flagellin antibodies requires activation of T-cells and innate immunity.

CCFA Fellowship Lay Summary:

The symptoms of active flares of inflammatory bowel disease (IBD) are generally similar to those of persons who’ve been infected with food-borne bacteria such as Salmonella even though extensive laboratory testing on samples from IBD patients consistently fail to detect the presence of any known food-borne infectious agents. Whether occurring in Salmonellosis or IBD these symptoms result, in large part from the recruitment/actions of cells of the immune system. When occurring in response to Salmonella, this immune response is a health-promoting event as it clears the infection. However, similar immune responses seem to occur in IBD even though appropriate triggers appear not to be present. It has been observed that, in mouse models of IBD, disease does not develop if mice are kept in germ-free housing leading to the theory that IBD results from an aberrant immune response to the bacteria that normally live in the human intestine. While a substantial amount of research supports this hypothesis, the reasons underlying how and/or why the immune system might so respond to normal intestinal bacteria remains largely unknown. My mentor’s approach has been to try to understand the mechanisms that control the healthy immune response to Salmonella in the hope that it might allow for insight into how things go awry in IBD. In vitro studies in this vein, have indicated that an important means by which the immune system normally recognizes Salmonella is by detecting a protein called flagellin that is released by these bacteria. Although both normal intestinal bacteria and Salmonella make flagellin, only Salmonella can get flagellin across the lining of the intestine where it must be to trigger an immune response. However, if intestinal barrier function is lacking as is known to occur in IBD, flagellin from normal bacteria may be able to trigger immune response and thus may be a trigger for active flares of IBD. In this proposal, I plan to test this hypothesis in vivo by examining immune responses to flagellin in humans with IBD as well as in mouse models of this disorder.

CCFA Senior Award Lay Summary:

Crohn’s disease and ulcerative colitis, collectively referred to as inflammatory bowel disease (IBD) is thought to result from the intestinal immune system “attacking” the commensal bacteria that normally “peacefully” exist in the intestine. Understanding the mechanisms by which this inappropriate immune response occurs will likely allow for development of new drugs treat and/or prevent IBD. Research on cultured cells, mice, and humans suggest that one bacterial protein in particular, flagellin, plays an important role in driving the inappropriate immune response associated with IBD. Thus, the goal of this research proposal is to understand how and why flagellin is targeted by the immune system. Specifically, the role of flagellin and its receptor in driving the acute flares of IBD and driving the long-lasting immune responses that maintain IBD will be investigated as will the cell types that mediate this response. A major focus of the proposal will be to study the molecule toll-like receptor 5, which serves as the host’s sensor for flagellin and thus likely normally plays a role in host defense. Our primary experimental approach will utilize mouse models of IBD in which we have the advantage of using genetically-engineered mice to allow a rigorous reductionist approach to be applied to a complex disease process.