Bacteria and bacterial rRNA genes associated with the development of colitis in IL-10(-/-) mice

Abstract

Background: Microorganisms appear to play important yet ill-defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population-based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL-10(-/-) mice.

Methods: Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue.

Results: Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture-independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4).

Conclusions: The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease-associated immunity in murine immune colitis and human Crohn's disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host-microbe interactions underlying IBD.

FIGURE 1

Development of colitis in IL-10^–/– mice housed in 2 different environments: a conventional mouse facility (Room 3) and a limited access facility (Room 4). A: Disease activity index (DAI) was monitored on a monthly basis. Data were pooled from 2 separate experiments. In all there were 9 mice raised in Room 3 and 8 in Room 4. The trend lines in the 2 rooms were different (multiple regression, P = 0.001). DAI was significantly lower in Room 4 than Room 3 at the indicated timepoints (*) (2-tailed Student's t-test, P < 0.05). B: Average monthly weight of mice was significantly higher in Room 4 than Room 3 at the indicated timepoints (*) (2-tailed Student's t-test, P < 0.05). C,D: Histopathology of the proximal colon from IL-10^–/– mice. Sections were stained with H&E plus Alcian Blue, which stains the goblet cells blue. C: Histology of the proximal colon of a mouse from Room 3 showing increased inflammatory infiltrate, crypt elongation, and decreased goblet cells. In panel C, I₁ = inflammatory cells on the outside of the serosa. I₂ = inflammatory cells in lamina propria (mucosa), I₃ = inflammatory cells in region below crypts (mucosa), I₄ = inflammatory cells in submucosa, C = elongated crypt, M = thickened muscularis externa. D: Histology of the proximal colon of a mouse from Room 4.

FIGURE 2

Associations between bacterial rRNA genes and disease activity in IL-10^–/– mice. Mice were raised in 2 different environments: a conventional mouse facility (Room 3) and a limited access facility (Room 4). Two replicate experiments were performed (Experiments 1 and 2). rRNA gene levels were measured in monthly fecal samples and in tissue samples collected at the end of the experiments using sequence-selective qPCR assays for 8 phylotypes (horizontal axis, see Table 1 for more details on the phylotypes). Disease activity was monitored every month for 6 months. Correlation analyses were performed between rRNA gene levels and disease activity index values for the fecal and tissue analyses and between rRNA gene levels and time in the chow analyses. Associations with P < 0.05 are shown as colored blocks (positive are red, negative are green). The brightness of the colors indicates the Pearson correlation coefficient (r) values (see scale bar at bottom). Suffixes indicate whether the tissue samples were washed in buffer before being analyzed: U (unwashed), W (washed). For the tissue analyses, “Pooled” is a combined analysis of all intestinal regions. n = 56 (Experiment 1, feces), 70 (Experiment 2, feces), 64 (Room 3, feces), 62 (Room 4, feces), 220 (Pooled), 20 (Duodenum-U), 20 (Duodenum-W), 20 (Jejunum-U), 20 (Jejunum-W), 20 (Ileum-U), 20 (Ileum-W), 10 (Cecum-U), 10 (Cecum-W), 20 (Proximal colon-U), 20 (Proximal colon-W), 20 (Distal colon-U), 20 (Distal colon-W), 7 (Room 3, chow), and 7 (Room 4, chow). The heat map was created using Java Tree View, v. 1.1.1.