IL-10 plays a crucial role in the modulation of immune responses and recently has been implicated as one of the cytokines mediating mucosal T-cell tolerance (17
) to bacterial antigens. Our results show that normal enteric bacteria are essential for the development of spontaneous colitis in IL-10-deficient mice since germfree IL-10-deficient mice had no histologically detectable colitis. In contrast, IL-10 knockout mice maintained in SPF environments had moderate colitis, comparable in character and severity to that of SPF IL-10-deficient mice from previous reports (3
). Unlike some previous descriptions in which inflammation was limited to the proximal colon of IL-10-deficient mice (27
), inflammation in this study was found in all regions of the colon. Interestingly, severely inflamed areas could lie adjacent to nearly normal areas of mucosa, similar to the focal nature of the “skip lesions” of Crohn’s disease. Also, we present novel data regarding the absence of mucosal immune responses, including IL-12 p40 secretion, in the absence of normal luminal bacteria. These data also illustrate the importance of age at the time of bacterial colonization, the ability of Helicobacter
-free bacteria to stimulate colitis in this model, and the different capacities of defined bacterial species to induce colitis in different genetically engineered rodents.
Germfree IL-10-deficient mice displayed none of the immune system activation observed in the SPF IL-10-deficient mice, indicating that subclinical colitis was not present in the germfree state. In the absence of viable bacteria, markers of T-cell activation and in vitro immunoglobulin secretion were not different from those of germfree heterozygote or SPF wild-type mice. Although IL-10-deficient SPF mice had increased concentrations of immunoglobulins in their colonic culture supernatants, B lymphocytes are not essential to the pathogenesis of colitis in IL-10-deficient mice (15
). Therefore, elevated levels of circulating antibody and enhanced mucosal immunoglobulin production should be considered markers of immune system activation rather than effector molecules causing tissue damage. Recent observations that IL-10-deficient mice have anticolonic epithelial cell antibodies (15
) thus may mean that these autoantibodies are a secondary result of epithelial damage in the setting of a dysregulated immune response. Moreover, the pattern of in vitro immunoglobulin secretion with abundant IgG2a in SPF IL-10-deficient mice is consistent with a Th1-gamma interferon (IFN-γ)-driven immune response (40
In keeping with a Th1 profile of cytokines, we found that IL-10-deficient mice with colitis had readily detectable amounts of IL-12 p40 in culture supernatants of inflamed colon fragments, presumably reflecting increases in biologically active IL-12 p70 heterodimer. It should be emphasized that these cultures had no in vitro stimuli to promote IL-12 production, suggesting that cells residing in the colon were activated in vivo to produce and secrete IL-12 p40. The production of IL-12 p40 mRNA and biologically active p70 IL-12 from macrophages or dendritic cells is upregulated in vitro in response to many stimuli. Live bacteria and bacterial products such as LPS and lipotechoic acid, as well as prokaryotic DNA, have all been demonstrated to increase production of IL-12 in vitro (9
). Our results suggest that in vivo exposure to resident luminal bacterial components may likewise stimulate IL-12 production. IL-12 has recently been incriminated as a mediator of autoimmune disease induced by bacterial products (46
) and loss of tolerance to resident luminal bacteria in experimental colitis (17
). Furthermore, treatment with neutralizing antibodies to IL-12 prevents the development of colitis in IL-10-deficient mice if treatment is initiated at an early age before development of severe colitis (40
), prevents the development of hapten-induced colitis in IL-2-deficient mice (18
), and reverses established inflammation in trinitrobenzene sulfonic acid-treated mice (37
). Because IL-10 promotes the development of Th2 responses by inhibiting macrophage production of IL-12 (14
), it is not surprising that in the absence of IL-10, unregulated colonic production of IL-12 could occur in response to microbial stimulation.
An unexpected finding in this study was the increased severity of cecal inflammation observed in gnotobiotic IL-10-deficient mice that had been colonized with SPF bacteria as adults compared with those colonized at weaning with the same organisms. The difference in the intensity of inflammation may have been related to the age of the animals and the maturity of their mucosal immune system at the time of colonization. Not only is cecal inflammation more aggressive with submucosal inflammation and mucosal ulceration more common following colonization of older IL-10-deficient mice, but the onset of inflammation is more rapid (5 weeks) than in germfree IL-10-deficient mice colonized at weaning (8 to 12 weeks). The submucosal pattern of colitis is more aggressive than that seen in 3-month-old HLA-B27 transgenic rats colonized with SPF bacteria, which developed cecal inflammation confined to the mucosa which was not significantly worse than colitis in B27 transgenic rats born in an SPF environment (39
has been recently shown to be associated with cecal inflammation in several immunodeficient mouse strains and has been advanced as a putative cause of experimental colitis in genetically engineered mice (54
). However, the development of aggressive typhlitis, and to a lesser degree colitis, in a Helicobacter
-free environment does not support an essential role of Helicobacter
in the development of colitis in IL-10-deficient mice. However, a potential contribution of Helicobacter
infection in the pathogenesis of colitis in IL-10-deficient mice is not excluded. Our observations that resident bacteria in the absence of Helicobacter
can cause colitis are particularly important given the widespread colonization of IL-10-deficient mice with this opportunistic pathogen (39a
The combination of the six colitis-related bacteria used in the present study readily provoked colitis in HLA-B27 transgenic rats which approximated that seen with SPF bacteria (39
). The observation that adult germfree IL-10-deficient mice colonized with these same colitis-related flora had delayed onset of very mild colitis compared to that of the germfree mice moved to SPF conditions suggests that all bacterial strains do not have equal capacity to induce intestinal inflammation. Importantly, the mild nature of colitis induced in these mice colonized with the six identical organisms which induce aggressive colitis in the HLA-B27 transgenic rat further supports the notion that other factors, in particular the host genetic background, play a critical role in the development of experimental colitis in response to any given bacterial stimulus. Indeed, inbred C57BL/6 mice with the IL-10 deletion develop mild, delayed-onset colitis in striking contrast to the early and aggressive colitis of IL-10-deficient mice of the 129 Sv background (3
). Similarly, we did not observe colonic adenocarcinomas in either the SPF or germfree IL-10-deficient outbred (C57BL/6 × 129) mice followed for up to 38 weeks of age, as were observed in 6-month-old 129 Sv mice (3
). Alternatively to differences in genetic background, quantitative differences in the numbers of each bacterium (not measured in this study) may have influenced the development of inflammation, particularly if pro-inflammatory bacteria were limited in their proliferation by noninflammatory bacteria. Further studies will be necessary to determine the dominant enteric bacterial strains that induce or perpetuate colitis in IL-10-deficient mice.
These results point out the inflammatory consequences of unbalanced immune responses to resident nonpathogenic bacteria but do not completely explain the mechanisms by which inflammation is mediated. Possible unregulated mucosal production of IL-12 as suggested by our results could result in excess production of IFN-γ, a proinflammatory Th1 cytokine known to alter epithelial barrier integrity in vitro. Preliminary observations with IL-10-deficient mice suggest that even in the germfree state these mice have lower colonic mucosal electrical resistance than do wild-type mice (46a
). Recombinant IL-10 has been shown to prevent IFN-γ-mediated damage to epithelial barrier integrity in cultured T84 cells (31
). It thus is possible that IL-10-deficient mice have inherent defects in mucosal barrier function that allow endogenous luminal bacteria and/or bacterial products to induce colonic inflammation. Alternatively, IL-10-deficient mucosal macrophages exposed to bacterial antigens could have overly aggressive cytokine production and antigen-presenting activities in the absence of immunosuppressive IL-10, perhaps resulting in activation of T lymphocytes to endogenous bacterial antigens. Exacerbations of pathologic immune responses have been described in IL-10-deficient mice infected with Trypanosoma cruzi
) or Toxoplasma gondii
). These results have been attributed to an intrinsic lack of down regulation of Th1 immune responses, since administration of exogenous recombinant IL-10 or neutralizing anti-IL-12 antibodies abrogates pathologic responses in these models and disease in the infected mice is not attributable to uncontrolled proliferation of the infecting organism. Lastly, an unbalanced cytokine environment could alter antigen-presenting cell function (13
) to lead to immune responses to normally nonimmunogenic antigens. Applying these observations to resident luminal bacteria, we speculate that in the genetically susceptible host, a dysregulated immune response could establish the cytokine milieu which promotes pathologic responses to normally nonpathogenic luminal bacterial antigens.