Summary of beta diversity analysis.

<p>A non-parametric ANOSIM (Analysis of Similarity) method was applied to dataset presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152044#pone.0152044.g003" target="_blank">Fig 3D</a> (PBS or adoptive T cell transfer in Rag2^-/- or DKO mice, from day 0 to day 13). Among single variables, only the genotype of the T cell recipient mice was significantly different based on a UniFrac beta diversity metric. In multivariate analysis, genotype was the single contributor to differences in beta diversity. Each row is a comparison of microbial diversity done with one or more factors, indicated by the labels. Beta diversity was calculated using Analysis of Similarity (ANOSIM) and significance was considered reached when both a and b were true as follows: a) p-value < 0.05 and b) R value > 0.3.</p

After adoptive T cell transfer, NHE3^-/-Rag2^-/- DKO mice develop severe colitis, which is alleviated by broad-spectrum antibiotic treatment.

<p>(<b>A</b>) Changes in body weight (% change of initial value 13 days post-transfer; boxes extend from 25th to 75th percentiles of values, whiskers represent min and max value, line within the box represents the mean value); (<b>B</b>) Histological colitis scoring (dots represent individual mice and lines mean value ± SD); (<b>C</b>) Representative H&E images of distal colon from respective experimental groups; (<b>D</b>) qPCR analysis of mucosal expression of proinflammatory cytokines in Rag/PBS (N = 10), Rag/AT (N = 8) and DKO/PBS (N = 9) and DKO/AT (N = 8) mice (bar graphs represent mean ± SD values); (<b>E</b>) Intestinal mucosal permeability measured by FITC-labeled dextran tracer flux (N = 3–6). Statistical analysis was performed using one-way ANOVA (ANOVA P value is indicated in each panel) with Fisher’s LSD post-hoc test. Asterisks in all panels indicate statistical significance: * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001. Brackets point to differences between indicated groups. Asterisks without brackets indicate a statistical difference from all other groups within the panel. Each experimental groups is color coded in a way consistent in the remaining figures.</p

Changes in microbiota composition in Rag and DKO mice before and after adoptive T cell transfer.

<p>(<b>A</b>) Endpoint taxonomic analysis (day 13) of the relative abundance of bacterial families in fecal samples; (<b>B</b>) Endpoint qPCR analysis of selected clusters of <i>Clostridia</i>; (<b>C</b>) Endpoint analysis of alpha diversity (Margalef index and the number of observed OTUs); (<b>D</b>) Principal Coordinate Analysis (PCoA) of unweighted UniFrac distances between of microbiota samples (first two principal coordinate axes versus time shown) in Rag2^-/- and DKO mice with or without adoptive T cell transfer over the course of the experiment. Samples were collected every 2 days post injection. Prematurely ended lines (DKO+AT) represent mice sacrificed earlier due to a critical body weight loss (≥20%). Points represent individual mice. In <b>B</b>-<b>C</b>, statistical analyses were performed using one-way ANOVA (P value indicated in each panel) with Fisher’s LSD post-hoc test. Asterisks in all panels indicate statistical significance: * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001.</p

Mucosal expression of protective cytokine IL-22 and IL-22-regulated antimicrobial peptide, RegIIIβ.

<p>Bars represent mean values ± SD. Statistical analysis were performed using one-way ANOVA (P value indicated in each panel) with Fisher’s LSD post-hoc test. Asterics in all panels indicate statistical significance: ** P < 0.01, *** P< 0.001, **** P < 0.0001.</p

Cytokine expression and histological soring in distal colon in WT (n = 3), Rag2^-/- (n = 10), NHE3^-/- (n = 5) and Rag/NHE3 DKO mice (n = 9).

<p>Bar graphs represent mean with SD values; dots represent individual mice and horizontal lines are mean values ± SD. Statistical analysis were performed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Asterisks in all panels indicate statistical significance: * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001.</p

PICRUSt prediction of functional profiling of the microbial communities based on the 16S rRNA gene sequences.

<p>Extended error bar plot indicating differences in functional profiles of the Rag+AT and DKO+AT microbiota (at taxonomic Level 3). All unclassified reads were removed and categories with minimum of 20 reads, and P value greater than 0.01 is displayed. Categories are sorted by P value calculated using two-sided Welch’s t-test.</p

Increased T cell and neutrophil infiltration in the colonic mucosa of DKO/AT mice is accompanied by altered MLN Treg phenotype.

<p>(<b>A and B</b>) Representative images of immunohistochemical analysis of (<b>A</b>) CD4⁺ cell abundance and (<b>B</b>) neutrophil abundance in the distal colonic mucosa; (<b>C</b>) qPCR analysis of mucosal expression of neutrophil collagenase MMP8; (<b>D</b>) Flow cytometry analysis of regulatory T cells in mesenteric lymph nodes (MLN) in adoptively transferred mice and effect of antibiotics. Bar graphs represent mean with SD values. Statistical analysis were performed using one-way ANOVA (P value indicated in each panel) with Fisher’s LSD post-hoc test. Asterisks in all panels indicate statistical significance: * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001.</p