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. Author manuscript; available in PMC: 2018 Apr 29.
Published in final edited form as: Mucosal Immunol. 2017 Aug 16;11(2):357–368. doi: 10.1038/mi.2017.55

Figure 1. Bacteria in the class Bacteroides maintain the colonic IEL population.

Figure 1

(a–d) Groups of 3–9 C57Bl/6 male and female mice aged 8–12 weeks were untreated, treated with antibiotics for one week, or treated with antibiotics followed by recolonization for one week each. Data are from two independent experiments. (a) Immunofluorescence of methacarn-fixed, paraffin embedded colon tissue from mice was performed and representative images shown at 40X. Bar=20 µm. Dotted lines outline crypts and arrows point to IELs as identified as CD3+ (green) cells in the epithelial layer (β-catenin, red). Nuclei were stained with bis-benzimide (blue). Dashed lines outline crypts while arrows indicate IELs. (b) CD3+ epithelial cells were counted in four well-oriented high-powered fields (HPF) from immunofluorescence staining performed in 6 untreated, 7 antibiotic-treated, 6 recolonized, and 3 germ free mice and shown as the mean number of cells per HPF ± SEM. **, P<0.01 as determined by Kruskall-Wallis analysis with Dunn’s post-test. (c,d) The absolute number of epithelial CD3+ cells harvested from the colons of mice was determined by flow cytometry. Each dot represents an individual mouse and bars are the mean ± SEM. Statistical analysis for (c) was performed using a two-tailed Student’s t-test; **, P<0.01. In (d) statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple comparisons test; ***, P<0.001 and ****, P<0.0001. (e) 16S rRNA sequencing from fecal DNA extracted from 5 untreated and 5 antibiotic-treated mice was performed. Order level differences in relative abundances ± SEM are shown with Wilcoxon rank test performed for statistical analysis. *, P<0.05; **, P<0.01 (f) Germ-free male and female mice aged 8–12 weeks were gavaged with PBS, Alistipes onderdonkii, Bacteroides fragilis, or Bacteroides thetaiotamicron and allowed to colonize for two weeks. Epithelial cells were harvested and CD3+ cells evaluated by flow cytometry. Each dot represents an individual mouse and bars are the mean ± SEM. ***, P<0.001 as determined by one-way ANOVA with Dunnett’s post-test.