Altered enteric microbiota ecology in interleukin 10-deficient mice during development and progression of intestinal inflammation

Abstract

Inflammatory bowel diseases (IBD) result from dysregulated immune responses toward microbial and perhaps other luminal antigens in a genetically susceptible host, and are associated with altered composition and diversity of the intestinal microbiota. The interleukin 10-deficient (IL-10 (-/-) ) mouse has been widely used to model human IBD; however the specific alterations that occur in the intestinal microbiota of this mouse model during the onset of colonic inflammation have not yet been defined. The aim of our study was to define the changes in diversity and composition that occur in the intestinal microbiota of IL-10 (-/-) mice during the onset and progression of colonic inflammation. We used high throughput sequencing of the 16S rRNA gene to characterize the diversity and composition of formerly germ-free, wild-type and IL-10 (-/-) mice associated with the same intestinal microbiota over time. Following two weeks of colonization with a specific pathogen-free (SPF) microbiota we observed a significant increase in the diversity and richness of the intestinal microbiota of wild-type mice. In contrast, a progressive decrease in diversity and richness was observed at three and four weeks in IL-10 (-/-) mice. This decrease in diversity and richness was mirrored by an increase in Proteobacteria and Escherichia coli in IL-10 (-/-) mice. An increase in E. coli was also observed in conventionally raised IL-10 (-/-) mice at the point of colonic inflammation. Our data reports the sequential changes in diversity and composition of the intestinal microbiota in an immune-mediated mouse model that may help provide insights into the primary vs. secondary role of dysbiosis in human IBD patients.

Keywords: Interleukin 10-deficient mouse; inflammatory bowel diseases; intestinal microbiota.

Figure 1. Inflammatory changes in the colons of formerly GF mice over time. Levels of IL-12 p40 secreted by colonic tissues from formerly GF WT and IL-10^−/− mice (A) and SPF raised WT and IL-10^−/− mice (C). The levels of IL-12 p40 in IL-10^−/− mice are significantly higher compared with WT mice in both formerly GF (p = 0.008) and SPF (p = 0.0001) environments. Histological scores for formerly GF WT and IL-10^−/− mice (B) and SPF WT (n = 7, as one tissue sample degraded during processing) and IL-10^−/− mice (D). The degree of histological inflammation is significantly higher in IL-10^−/− compared with WT mice in formerly GF (p = 0.008) and SPF (p = 0.001) environments.

Figure 2. Changes in weighted and un-weighted average UniFrac distances in formerly GF wild-type (WT) and IL-10^−/− mice over time. (A) Average weighted UniFrac distances of the intestinal microbiota significantly decrease in IL-10^−/− mice three and four weeks following colonization with a specific-pathogen free (SPF) microbiota. (B) Average un-weighted UniFrac distances of the intestinal microbiota do not significantly alter in the IL-10^−/− mice following colonization with an SPF microbiota. (C) Average weighted and un-weighted UniFrac distances significantly increase in WT mice between one and two weeks following colonization with an SPF microbiota.

Figure 3. Microbial richness of 16S rRNA data. (A) The number of observed bacterial species in formerly GF IL-10^−/− mice decreases between one and four weeks following colonization with an SPF microbiota. (B) The number of observed bacterial species in formerly GF WT mice significantly increases between one and two weeks following colonization with an SPF microbiota.

Figure 4. Bacterial taxa alterations over time in formerly GF WT and IL10^−/− mice.

Figure 5. Change in levels of Proteobacteria and Escherichia coli in formerly GF IL-10^−/− mice over time. Proteobacteria are expressed as the percentage of total 16S rRNA sequences. E. coli are expressed as fold increase with respect to baseline (WT at week 1). *The levels of Proteobacteria and E. coli are significantly higher at week 4 post colonization compared with week 1 (E. coli, p = 0.0001; Proteobacteria, FDR = 1.5 × 10⁻⁵).

Figure 6. Change in levels of E. coli(A),Akkermansia muciniphila(B) and Lactobacillus species (C) in SPF WT and IL-10^−/− mice over time.

Figure 7. Schematic outline of experimental design. Adult WT and IL-10^−/− 129 SvEv GF mice were inoculated with an SPF microbiota from a single donor (black arrow). Fresh fecal pellets were obtained from the WT group (n = 5) at 1 and 2 weeks following SPF inoculation. Fecal pellets were obtained from the IL-10^−/− group (n = 5) at 1, 2, 3 and 4 weeks following association with an SPF intestinal microbiota.