Bioactive MMP-9 is present within the colonic epithelium and becomes localized primarily near the apical surface of the intestinal epithelium when associated with C. rodentium infection. Studies have shown that MMP-9 is upregulated in human IBD with acute inflammation [11, 24]. Deletion of MMP-9 in animal models has proven beneficial in attenuating S. typhimuium and DSS-induced colonic injury and inflammation [19, 25, 26]. The effect of MMP-9 on the gut microbiota has not been previously evaluated. This study shows the contribution of MMP-9 in the pathobiology of C. rodentium infection and an impact on the composition of the fecal microbiota. We demonstrate that despite similar C. rodentium-induced colonic epithelial responses between WT and MMP-9−/− mice, there is a different microbial composition between genotypes that results an altered microbial response following an infectious challenge. These differences were revealed by nonmetric multidimensional scaling of terminal restriction fragments. The findings indicate that a difference in genotypes plays a role in influencing the microbiome composition in uninfected mice.
A healthy gut microbiome is maintained through microbe-microbe and host-microbe interactions. An alteration in gut microbe homeostasis is associated with chronic IBD in humans  and with post-infectious IBS . A change in the microbiome also occurs in response to infection with the murine-specific pathogen Citrobacter rodentium. The importance of a healthy gut microbiome is also implicated in toxigenic Clostridium difficile infection, which is triggered by the loss of microbiota colonization resistance and the release of ecological niches previously unavailable following antibiotic treatment .
Infection with C. rodentium resulted in activation of MMP-9, as demonstrated by zymography of colonic tissue. The resulting pro-inflammatory response to infection, including colonic epithelial cell hyperplasia and barrier dysfunction, was similar irrespective of genotype. Taken together, these findings indicate that increased expression of colonic MMP-9 following infection with C. rodentium is not associated with the host pro-inflammatory immune responses to the enteric pathogen.
Elimination of various factors contributing to innate and humoral immunity can dramatically alter the gut microbiome. Specifically, TLR5-deficient mice develop a markedly different intestinal microbiome, which predisposes the animals to develop metabolic syndrome . Furthermore, impaired innate immune function in T-bet−/−Rag1−/− mice develop a microbiota which is colitogenic and transferable to WT mice by fecal transplantation .
MMP-9 deficiency is associated with altered goblet cell differentiation, leading to an enrichment of bactericidal mucins in the intestine of mice treated with dextran sodium sulphate and Salmonella typhimurium. This enrichment in mucus secretion in the lumen could prove important for reducing nutrients for pathogen growth and, in turn, lead to altered microbe-microbe interactions thereby disrupting gut microbe homeostasis in MMP-9−/− mice. Although there was no difference in the number of goblet cells present in the colonic crypts of WT and MMP-9−/− mice at the height of infection 10d PI, delayed changes to the goblet cell population were observed by 30d PI. This indicates a fundamentally different innate response to infection between WT and MMP-9−/− mice which may contribute to an atypical fecal microbiome in MMP-9−/− mice. Recent evidence also indicates that MMPs regulate the intercellular expression of several key mediators of cell-cell binding including claudin-5 and occludin . For instance, in the context of lung injury, the pore-forming cytotoxin α-hemolysin from Staphylococcus aureus upregulates the zinc-dependent metalloprotease ADAM10, resulting in cleavage of E-cadherin and disruption of intercellular tight junctions .
Most MMPs are secreted factors, but many of the proteases localize to cell surfaces where they associate with and regulate a variety of adhesion molecules, such as CD44 and β-integrins [32, 33]. This indicates that MMPs could alter the binding efficiency of intestinal bacteria to host colonocytes, thereby altering the pathobiology of an infectious colitis. MMP-7 also affects gut microbe homeostasis through cleavage of reduced cyptdin-4 (r-Crp4), a mouse Paneth cell-derived α-defensin. In an in vitro model, cleavage of the peptide resulted in increased survival of Salmonella enterica serovar Typhimurium, E. coli ML35, Staphylococcus aureus, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus casei
Bacteroides thetaiotaomicron, and Bacteroides vulgatus relative to undigested r-Crp4 . Therefore, the presence of MMPs in the colonic mucosa can mediate physiological parameters that impact on both gut homeostasis and host-microbe interactions. Disruption of these interactions leads to an altered microbial ecology and disease .
Segmented filamentous bacteria (SFB) "Arthromitus immunis” ; provides mucosal protection against C. rodentium infection, as well as mediates the production of the proinflammatory cytokines IL-17 and IL-22 . In the present study, qPCR analysis of the fecal microbiome revealed a larger population of SFB and higher mRNA levels of IL-17 in MMP-9−/− mice compared to WT controls, even under baseline conditions. “A. immunis” inhibits colonization of rabbit enteropathogenic Escherichia coli O103 and protects against subsequent disease development . In this study, electropherograms showed that C. rodentium became a dominant component of the detectable microbiota in WT, but not MMP-9−/− mice. As noted by others , this study shows that the presence of SFB may provide protection against C. rodentium colonization, although our results demonstrate that commensal SFB does not offer full protection against C. rodentium-induced colitis in C57BL/6 J mice. This observation emphasizes that a shift in the bacterial population does not have an all-or-none effect; rather, it produces a graded series of responses.
In previous studies, infection of C57BL/6 J mice with C. rodentium reduced fecal microbial diversity and evenness due to the dominance of C. rodentium in the gut microbiome . A similar pattern was observed in the current study in WT but not MMP-9−/− mice, as the fecal microbiota of the latter group had no changes in diversity following infection. Colonization of the cecal mucosa by the murine pathogen Helicobacter hepaticus also reduces microbial diversity .
The distinct and stable fecal microbiome in MMP-9−/− mice identified in this study emphasizes that the presence of MMP-9 in mouse colon supports a microbiome that is more susceptible to C. rodentium colonization and reductions in microbial diversity. Given that MMP-9−/− (B6.FVB(Cg)-Mmp9
/J) mice have a microbiota that is more resistant to C. rodentium colonization, this genotype should prove useful for future studies evaluating the contribution of microbe-microbe interactions to the pathogenesis of C. rodentium infection and the maintenance of microbial diversity. The role of other MMPs in maintaining the fecal microbiota upon infectious challenge will also prove to be of interest in future experimental studies.