Additional file 2 of Utilizing a reductionist model to study host-microbe interactions in intestinal inflammation

Additional file 1: Fig. S1. WASP deficiency results in altered composition of the fecal microbiota. Fecal microbial composition of Was-/- (n=5) and WT (n=3) mice raised under SPF conditions with weekly bedding exchanges was analyzed monthly between 4 and 20 weeks of age by 16S rRNA gene sequencing. (A) Microbial relative abundances at the phylum level. (B) Relative abundance of the phylum Deferribacteres at each timepoint by genotype. Statistics performed using the DESeq2 R package and adjusted for multiple taxa comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. Fig. S2. Establishing a reductionist model to study the role of the microbiota in the development of intestinal inflammation. (A) Experimental design. (B) Fecal microbial composition of donor mice harboring the ASF consortium and recipient ex-germ-free mice after 2 months of co-housing (females) or bedding exchanges (males) with donors. Composition assessed based on 16S rRNA sequencing. (C) Quantitative histological colitis scores 20 weeks after gavage with H. bilis in the cecum, proximal colon, and distal colon. (D) Gating strategy for IL-17A+ CD4 T cells. (E) Gating strategy for IL-22+ ILC3s. Lin includes CD3, CD19, CD11b, CD11c, NK1.1, Ly6C, Ly6G. Fig. S3. Correlations between intestinal inflammation and fecal microbial composition. (A-B) Germ free WT/HET (n=11) and Was-/- (n=8) mice colonized with the ASF community but not gavaged with H. bilis served as a control group. Fecal LCN2 (A) and absence of H. bilis (B) were monitored serially. (C) In mice that received H. bilis, H. bilis relative abundance is shown based on whether the mouse was in a cage that contained both genotypes (co-housed) or only one genotype (not co-housed). (D-I) Correlations between log-transformed fecal LCN2 and relative abundances of the indicated ASF members in mice of the indicated genotype colonized with ASF and H. bilis for all timepoints. Tests for linear dependence of log-transformed LCN2 on the relative abundance of each bacterial species was done using a linear mixed effects model, taking into account a mouse-specific random effect. Wilcoxon rank sum test was used for (A) and (C). * p < 0.05, *** p < 0.001. Fig. S4. Correlations between intestinal inflammation and mucosal microbial composition. (A-H) Germ free WT/HET (n=17) and Was-/- (n=13) mice were colonized with the ASF community and then gavaged with H. bilis. The mucosal-associated microbiota was assessed at 20 weeks post infection. Pearson correlations between log-transformed fecal LCN2 and mucosal relative abundances of the indicated ASF members are shown within mice of the indicated genotypes. Fig. S5. Metatranscriptomic analysis of ASF members in inflamed and uninflamed states. Germ-free WT/HET and Was-/- mice were colonized with the ASF community and a subset were gavaged with H. bilis. Feces from 3 mice of each genotype/microbiota combination (WT/HET or Was-/- with or without H. bilis) at 1.5 weeks and 15 weeks after H. bilis gavage were subjected to bacterial metatranscriptomic sequencing. (A-J) Volcano plots of corrected p-values vs log2(FC) for genes expressed by the indicated bacteria at 15 weeks after H. bilis gavage, except for (A-C), which are from 1.5 weeks after H. bilis infection. Plots compare gene expression of the specified bacteria between host genotypes within the ASF-only group or the ASF + H. bilis group as indicated. Dots on the right side of each plot represent genes that are more highly expressed in Was-/-, and dots on the left side indicate genes that are more highly expressed in WT/HET. Red dots represent genes that are significantly differentially expressed as determined by the DESeq2 Wald test.