- Top of page
- MATERIALS AND METHODS
Background: Monoassociating gnotobiotic IL-10-deficient (−/−) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T-cell-mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently.
Methods: Germ-free (GF) inbred 129S6/SvEv IL-10−/− and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3–7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL-12/IL-23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4+ cells were stimulated with antigen-presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN-γ and IL-17 levels were measured in the supernatants.
Results: Dual-associated IL-10−/− (but not WT) mice developed mild-to-moderate pancolitis by 3 weeks that progressed to severe distal colonic-predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF-κB was activated in the duodenum and colon in dual-associated IL-10−/− × NF-κBEGFP mice. The aggressiveness of intestinal inflammation and the degree of antigen-specific CD4+ cell activation were greater in dual- versus monoassociated IL-10−/− mice.
Conclusion: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL-10−/− mice act additively to induce aggressive pancolitis and duodenal inflammation.
The IL-10-deficient (−/−) mouse is a well-characterized colitis model that develops intestinal inflammation when exposed to normal commensal bacteria, unlike its normal wildtype (WT) counterpart,1 suggesting that endogenously produced IL-10 prevents colitis. IL-10−/− mice maintained in sterile germ-free (GF) conditions remain free of intestinal inflammation and have no evidence of immune activation.2 However, these mice develop chronic progressive colitis (predominantly cecal) after colonization with specific pathogen-free (SPF) bacteria. This colitis is evident within a week after colonization and is severe by 3–5 weeks after initial colonization.2, 3 Of considerable importance, gnotobiotic IL-10−/− mice do not develop intestinal inflammation when monoassociated with Bacteroides vulgatus, which previously has been shown to selectively induce colitis in HLA-B27/β2 microglobulin TG rats4, 5 or Pseudomonas fluorescens, a bacterial species that expresses the I2 superantigen that has been implicated in Crohn's disease (CD).6, 7 We have previously shown that monoassociating IL-10−/− mice with either nonpathogenic strains of E. faecalis or E. coli induce variable disease phenotypes in IL-10−/− mice, as manifested by different kinetics and localization of colitis in E. faecalis- versus E. coli-monoassociated IL-10−/− mice.7E. faecalis induced a slower-onset (10–12 weeks post-inoculation), distal colon-predominant inflammation, which progressed to severe distal colitis and duodenal inflammation by 24 weeks. In contrast, E. coli-monoassociation induced earlier onset (3 weeks postinoculation) cecal-predominant inflammation that progressed to moderately severe to severe disease by 16 weeks. Both organisms induced bacterial species-antigen-specific interferon-γ (IFN-γ) production by mesenteric lymph node (MLN) CD4+ T cells from monoassociated mice. However, neither organism alone induced the level of aggressive disease that we observed in IL-10−/− mice colonized with the complex assortment of commensal bacterial species comprising SPF enteric flora. E. faecalis or E. coli are potentially important contributors within the complex SPF bacterial populations responsible for inducing colitis in this model. These 2 organisms may act additively or synergistically to induce intestinal inflammation, or they may work in conjunction with the other bacterial species found in the SPF environment to induce the rapid-onset, severe colitis that develops in IL-10−/− mice moved into SPF conditions.2, 3 Alternatively, they could have minor roles and the aggressive colitis in SPF IL-10−/− mice is due to other commensal bacterial species yet to be determined.
The goals of the current study were 1) to characterize the disease phenotype when GF IL-10−/− mice are simultaneously colonized with the previously studied nonpathogenic strains of E. faecalis and E. coli, and 2) to quantify immune responses in these dual-associated mice in comparison with WT dual-associated and IL-10−/− mice monoassociated with the same organisms. We hypothesize that dually associating gnotobiotic IL-10−/− mice with E. faecalis and E. coli will act additively or synergistically to potentiate the degree and alter the location of experimental colonic inflammation.
- Top of page
- MATERIALS AND METHODS
Our results demonstrate the ability of 2 common nonpathogenic commensal bacterial species that individually induce different phenotypes of enterocolitis in a single genetically defined host,7, 13 together potentiate the aggressiveness of intestinal inflammation. These data support and expand the results of our previous studies by showing that inoculating IL-10−/− mice with both organisms leads to more rapid progression of severe intestinal inflammation with wider regional distribution of disease. Proinflammatory cytokine-driven immune activation, manifested by spontaneous colonic IL-12/23p40 secretion and IFN-γ production from MLN CD4+ T cells stimulated with E. faecalis and E. coli lysate pulsed–APC, was significantly higher in dual-associated IL-10−/− compared with E. faecalis- and E. coli-monoassociated mice at an early phase of bacterial colonization (3 weeks). Interestingly, E. faecalis pulsed APC activated a lower IL-17 response in MLN CD4+ T cells from dual-associated compared to monoassociated mice at the earliest timepoint we evaluated. However, IL-17 production in response to components of both bacteria accompanies the subsequent development of intestinal inflammation evaluated at 5 and 7 weeks after dual association. These findings highlight the complex nature of bacterial-driven activation of immune responses with both Th1 and Th17 profiles in the IL-10−/− model of colonic inflammation.
Previously, investigators have studied the differential ability of several nonpathogenic bacterial species to variably induce or ameliorate disease in genetically susceptible rodent inflammatory bowel disease (IBD) models. IL-2 gene-deficient (IL-2−/−) mice develop severe colitis and progress to early death when raised in conventional housing16 but much milder, slow-onset, and nonlethal intestinal inflammation when raised in GF conditions.17 Waidmann et al18 showed that gnotobiotic IL-2−/− mice monoassociated with a nonpathogenic E. coli strain developed colitis, whereas IL-2−/− mice either monoassociated with a nonpathogenic B. vulgatus strain or dually associated with E. coli plus B. vulgatus did not develop colonic inflammation. These results indicate that B. vulgatus ameliorates colitis in IL-2−/− mice when co-inoculated with a colitogenic E. coli strain. This finding further emphasizes the importance of interactions between different components of the normal commensal microbiota in maintaining the tenuous balance between the induction, maintenance, and amelioration of intestinal inflammation.
It is possible that differences in disease progression are, in part, due to the number of bacteria present or the degree of mucosally adherent bacteria within a specific region of the intestinal tract. The bacterial concentrations quantified from the distal colon and cecum by routine culture and PCR did not explain the regional differences in histologic inflammation in mice monoassociated with E. faecalis or E. coli in our previous study of monoassociated IL-10−/− mice.7 These results are further supported by our current results, which show no significant differences in bacterial concentrations of E. faecalis versus E. coli in different colonic regions. Waidmann et al18 also showed that the anti-colitogenic effect of B. vulgatus on E. coli in IL-2−/− mice is not due to a reduction in E. coli numbers within the intestinal tract, suggesting that potentiating and/or ameliorating effects of different commensal organisms are due to their differential abilities to breach the mucosal barrier and/or to activate the resident macrophages and dendritic cells and to induce antigen-specific immune responses rather than altering luminal bacterial composition.
There are parallels between these observations in rodent colitis models and human IBD.19 Duchmann et al20, 21 have shown that peripheral blood CD4+ TCRαβ+ T cell clones from IBD patients respond to several commensal bacteria, including E. coli. In addition, invasive E. coli and Enterococci have been recovered from inflamed intestinal tissue immediately adjacent to ulcers and fistulae of patients with CD.22 Moreover, enteroadherent/invasive E. coli strains were found in greater frequency in the ileal mucosa of patients with postoperative recurrence of disease compared to either CD patients with chronic ileal lesions or healthy controls.23 Other investigators have isolated different microorganisms and their associated proteins from CD patients, including P. fluorescens,6 and documented serologic responses to defined bacterial antigens in the majority of CD and ulcerative colitis (UC) patients.24 Swidsinski et al25, 26 demonstrated mucosal association of coliforms and Bacteroides species in patients with CD, UC, and nonspecific intestinal inflammation.
The question arises whether different disease phenotypes observed in IBD patients may be driven in part by various combinations of distinct commensal bacteria that act in an additive manner. Several published reports show that patients with more severe disease phenotypes (i.e., intestinal perforating disease, fibrostenosis, small intestinal resection) had a high frequency of antibody responses to bacterial and fungal antigens.27, 28 In addition, patients with antibody responses to multiple microbial antigens appeared to have an increased likelihood of complicated small bowel disease phenotypes. These observations suggest that additive effects of immune responses to antigens from various commensal microorganisms intensify intestinal inflammation and complications of CD.
The clinical implications of understanding the relationship between bacterial antigen-driven immune responses in genetically susceptible individuals extend beyond the diagnostic arena. Antibiotics with different narrow spectra of bactericidal activity in colitic SPF IL-10−/− mice preferentially treat different colonic regions,29 suggesting the ability to tailor antimicrobial therapy in IBD patients, depending on which bacterial species are present. Metronidazole, which has predominant anaerobic antimicrobial activity, either alone or in combination with ciprofloxacin, is more active in colonic versus ileal CD,30, 31 suggesting that differential bacterial colonization in the colon versus the small intestine is at least partially responsible for different disease phenotypes. In pilot studies, Mow et al32 showed that CD patients with ileal +/− right-sided colonic disease whose sera contained antibodies reactive with both E. coli outer membrane porin C (OmpC) and I2 had the highest disease remission rate when treated with metronidazole + ciprofloxacin + budesonide compared to patients with antibody reactivity to either OmpC or I2 or to neither bacterial protein. Although not statistically significant, these findings are intriguing and warrant future study.
The results of our study demonstrate that 2 common nonpathogenic commensal organisms that individually induce phenotypically distinct colitis in the same genetically susceptible host act synergistically to induce aggressive, early-onset distal colonic and cecal inflammation accompanied by amplified bacterial antigen-specific immune responses. The clinical and pathogenic implications of this observation include the likelihood that various commensal enteric bacterial species interact to induce and perpetuate mucosal immune responses that accompany intestinal inflammation. Elucidating which specific components of the complex luminal microbiota drive pathogenic versus protective immune responses can guide therapeutic manipulation of the complex bacterial milieu of the distal intestine.