Data from both humans and experimental animals underscore the critical role of intestinal bacteria in the establishment and maintenance of IBD (2
). The molecular mechanisms underlying this clear bacterial dependence on mucosal inflammation are likely to relate to the intrinsic ability of these organisms to trigger both acquired and innate immune responses in the gut. Using an unbiased molecular screen to search for bacterial antigens relevant to IBD, the dominant antigens identified were a family of related, novel flagellins. A strong, IgG2a-biased serological response to these specific flagellins was seen in multiple models of experimental colitis across several distinct MHC haplotypes. In addition, marked reactivity against these flagellins was seen at the T cell level, and flagellin-specific T cells were able to induce colitis when adoptively transferred into immunodeficient animals. Interestingly, while these flagellins were identified from mouse cecal bacteria, there was clear, specific reactivity against these molecules in patients with CD (but not in patients with UC or in NCs). Given the activity of flagellin as a specific ligand for TLR5, these data provide a potentially important link between adaptive and innate immune responses in mucosa in the pathogenesis of IBD.
Among bacterial antigens, flagellin is an interesting candidate to play a role in mucosal immune responses. Specifically, flagellin is a common bacterial antigen present on most motile bacteria in the gut (22
). Moreover, flagellin is highly antigenic; indeed, responses against flagellin are protective in Salmonella
infections in mice (23
). Furthermore, flagellin has been identified as a pathogen-associated molecular pattern and has been identified as the specific ligand for TLR5. Monomeric, translocated flagellin is capable of stimulating cytokine and chemokine production by TLR5-positive intestinal epithelial cells (8
), and flagellin has been demonstrated to induce the maturation of dendritic cells via TLR5 (25
). Indeed, we have observed that full-length flagellin Fla-X (endotoxin free) is capable of stimulating TNF-α production by human macrophages in vitro (M.J. Lodes and R.M. Hershberg, data not shown). It is tempting to speculate that the intrinsic “adjuvanticity” of flagellin is likely to contribute to its antigenicity. While flagellin molecules clearly have the capacity to stimulate the production of proinflammatory cytokines via TLR5, we favor the hypothesis that the B and T cell responses to flagellin contribute more directly to the chronic intestinal inflammation seen in IBD.
Our clinical data (Figure ) are consistent with the fact that the aberrant response in patients with CD is specific to the subgroup of flagellins identified in our molecular screen. Specifically, there was no correlation between IBD and a response to flagellin from Salmonella muenchen
, which is very similar (84–91%) in the NH2
conserved region to the flagellin from the commensal organism Escherichia coli
. It must be emphasized that the flagellins identified were from a source of material devoid of known bacterial pathogens. The bacteria with genes that “encode” the flagellins CBir1 and Fla-X (the two dominant flagellins tested) are unknown; however, preliminary phylogenetic data suggest that these flagellins are most closely related to the flagellins of bacteria in the genera Butyrivibrio, Rosburia, Thermotoga
, and Clostridium
and fall within the Clostridium
subphylum XIVa cluster of Gram-positive bacteria (Figure B). This group of bacteria contains many of the butyrate-producing bacteria that are found in the human gut (27
). We speculate that the aberrant response to the flagellin molecule(s) from these organisms is related to a combination of the intrinsic property of the molecules themselves (as immunogens and adjuvants) and an underlying genetic susceptibility. The question of the basis for selective responses to an antigen present in the normal commensal flora of most (if not all) individuals is a challenging one. Using monoclonal antibodies directed against CBir1, we have demonstrated that this antigen is present in the stool of wild-type strains (FVB, C57BL/6, BALB/c, and C3H/HeJ) and colitic strains (mdr1a–/–
, and C3H/HeJBir) (data not shown). These data indicate that the presence of the antigen itself does not strictly correlate with colitis. Still, the widespread presence of these antigens does not preclude the possibility of enhanced colonization of organisms expressing these flagellins in CD lesions. This idea is certainly consistent with the recent observations by Swidsinski and colleagues (29
). Alternatively, alterations in barrier function (30
) or intrinsic innate immune responses (32
) may enhance the likelihood of the response to these flagellin molecules in patients prone to develop IBD.
In general, the data are consistent with the hypothesis that IBD is associated with a defect in tolerance to commensal organisms (34
). The IgG2a-biased antibody against Fla-X and CBir1 highlights the Th1 bias of the T cell responses seen. The broad recognition of these flagellins in several different mouse models and in humans with CD indicates that these flagellins are among the immunodominant antigens of the microbiota. However, the exact role of these flagellins in the pathogenesis of IBD (e.g., whether they are predominant or obligatory for disease) compared with that of other microbial antigens remains to be defined. We favor a model in which a T regulatory response to specific flagellins (and/or other bacterial antigens) may be selectively impaired in IBD. In this context, specific flagellin molecules may represent novel targets for antigen-directed therapy in IBD. Experiments are currently underway to address this possibility.
Clearly, flagellin is not the only bacterial antigen involved in the pathogenesis of IBD. It is likely that there will be specific subsets of patients with IBD (both CD and UC) that display patterns of seroreactivity against the molecular determinants that we have identified, similar to the patterns currently seen with responses to oligomannans from Saccharomyces cerevisiae
, I2, and ompC for E. coli
). Indeed, the identification of flagellin complements the efforts Braun and co-workers in discovering bacterial antigens relevant to the pathogenesis of IBD. One interesting example is I2, an antigen derived from Pseudomonas fluorescens
that appears to have “superantigen-like” activity (16
). As we observed with the specific flagellins identified here, only a subset of patients with CD show specific seroreactivity against I2. It should be noted that an unbiased molecular approach, RDA, was also used to identify I2. In contrast to the technique of SEC, which depends upon the selective antibody responses to a given antigen, RDA depends upon the selective presence of bacteria in anatomic sections of IBD. Given the heterogenous nature of IBD and the complexity of the intestinal microflora, we are continuing our efforts to identify additional antigens in patients that do not respond to flagellin. It will be of considerable interest to determine how the reactivity against these antigens correlates with genetic determinants, clinical phenotypes, and responses to treatment.