Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease

Abstract

Specific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow-cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA-SEQ) and show that high IgA coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA-SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with predefined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.

Figure 1. IgA−based sorting and 16S sequencing of fecal bacteria from specific pathogen free (SPF) mice

(A) Overview of IgA−based cell sorting of fecal bacteria combined with 16S rRNA gene sequencing (IgA−SEQ). (B) Representative results and a cartoon of cell sorting of IgA+ and IgA− fecal bacteria from mice. (C) Heatmap depicting IgA Coating Index (ICI) scores and average relative abundance of bacterial genera in Total (Presort), IgA+ and IgA− fractions of fecal bacteria from C57Bl/6 SPF mice (n=17 samples). Relative abundance heatmaps are depicted on a logarithmic scale. Genera that are highly coated with IgA (significantly higher relative abundance in the IgA+ fraction as compared to the IgA− fraction by LEfSe; P < 0.05) are marked with an asterisk. Genera with ICI > 10 are labeled in red. UC, unclassified in the Greengenes reference database. Gen., classified as a distinct but unnamed genus in the Greengenes reference database. (D) Relative abundance of significantly coated bacterial genera in Presort, IgA+ and IgA− fractions. * P < 0.05; *** P < 0.001 (Wilcoxon rank-sum). Indicated are mean ± standard error of the mean. See also Figure S1 and Table S1.

Figure 2. IgA coating identifies colitogenic bacteria in mice with inflammasome-mediated intestinal dysbiosis (SPF^dys)

(A) Average relative abundance of bacterial genera of greater than 1% abundance in the intestinal microbiota of SPF and SPF^dys mice. UC Prevotellaceae is marked with an arrow. SPF^dys mice were co-housed with Asc^−/− mice to allow for the acquisition of dysbiosis. (B) Dextran Sodium Sulfate (DSS)-induced colitis in SPF (n=5) and SPF^dys (n=4) mice. * P < 0.05; ** P < 0.01; *** P < 0.001 (one-way ANOVA). (C) IgA coating of fecal bacteria from 10-16 week old SPF (n=8) and SPF^dys (n=9) mice at the steady state. *** P < 0.001 (unpaired Student's t-test). (D) Heatmap depicting IgA Coating Index (ICI) scores and average relative abundance of bacterial genera in Total (Presort), IgA+ and IgA− fractions of fecal bacteria from 10-16 week old SPF^dys mice (n=14 samples) sampled under steady state conditions. Relative abundance heatmaps are depicted on a logarithmic scale. Genera that are highly coated with IgA (significantly higher relative abundance in the IgA+ fraction as compared to the IgA− fraction by LEfSe; P < 0.05) are marked with an asterisk. Genera with ICI > 10 are labeled in red. (E) Relative abundance of significantly coated genera in Presort, IgA+ and IgA− fractions. * P < 0.05; *** P < 0.001 (Wilcoxon rank-sum). Indicated are mean ± standard error of the mean. UC, unclassified in the Greengenes reference database. Gen., classified as a distinct but unnamed genus in the Greengenes reference database. See also Figure S2 and Table S2.

Figure 3. IgA coating of fecal bacteria from healthy humans and inflammatory bowel disease patients

(A) IgA coating of fecal bacteria from 20 healthy subjects, 27 Crohn's disease patients (CD) and 8 Ulcerative colitis patients (UC). *P < 0.05; ***P < 0.001 (one-way ANOVA). (B) Venn-diagram depicting the distribution of highly coated bacterial species in healthy, UC and CD patients. Bacterial taxa that showed an ICI score greater than 10 in at least one subject were classified as highly coated within that group. (C) Heatmap depicting IgA coating index (ICI) scores for bacterial species that are uniquely highly coated (ICI > 10) in IBD and never highly coated or never present in healthy controls. Bars to the right of the heatmap correspond with the color-coding of the Venn-diagram in panel (B). Each column represents an individual human subject. UC, unclassified in the Greengenes reference database. Spp., classified as a distinct but unnamed species in the Greengenes reference database. See also Figure S3 and Table S3.

Figure 4. Isolation of personalized IBD-associated gut microbiota culture collections, assembly of IgA+ and IgA− consortia and colonization of germ-free mice

(A) Strategy for isolation of personalized IBD-associated gut microbiota culture collections. (B) Selection of individual bacterial isolates comprising IgA+ and IgA− consortia and colonization of germ-free mice. Specific isolates that were included in the consortia are boxed in green (IgA−) or red (IgA+). (C) Barplots depicting relative abundance of bacterial taxa in IgA+ and IgA− consortia pre-gavage (D0) and in the feces of IgA+ and IgA− colonized mice 2 weeks post-colonization. All members of the pre-gavage consortia were detectable in colonized mice except Peptinophilus spp. in the IgA− consortium and Streptococcus spp. in the IgA+ consortium. UC, unclassified in the Greengenes reference database. Spp., classified as a distinct but unnamed species in the Greengenes reference database. (D) IgA coating of fecal bacteria from germ-free mice colonized with IgA+ (n=5) or IgA−consortia (n=5) on days 7 and 24 post-colonization. Representative plots are shown. *** P < 0.005 (unpaired Student's t-test). Indicated are mean ± standard error of the mean. (E) Microbiota localization as visualized by 16S rRNA FISH (red) and DAPI (blue) staining. The mucus layer is demarked by two dotted lines. See also Figure S4.

Figure 5. IBD-associated IgA+ bacteria exacerbate DSS-induced colitis in gnotobiotic mice

(A) Timeline of colonization and DSS treatment in germ-free mice colonized with IgA+ and IgA− consortia. (B) Colon length after DSS. * P < 0.05 (unpaired Student's t-test). (C) Gross pathology of large bowels after DSS. Note the extensive bleeding and diarrhea in the IgA+ colonized mice. (D) Colon histopathology scores after DSS. Scores were assigned as follows: 0, Intact colonic architecture. No acute inflammation or epithelial injury; 1, Focal minimal acute inflammation; 2, Focal mild acute inflammation; 3, Severe acute inflammation with multiple crypt abscesses and/or focal ulceration; 4, Severe acute inflammation, multiple crypt abscesses, epithelial loss and extensive ulceration. *** P < 0.0001 (unpaired Student's t-test). (E) Representative histology pictures from hematoxylin and eosin stained colons after DSS. Note that IgA+ colonized mice exhibit extensive inflammation, crypt abscesses, epithelial loss, and ulceration, while all IgA− colonized mice showed either no inflammation or minimal/mild focal inflammation. Data are representative of 3 independent experiments. See also Figure S5.