Interspecies commensal interactions have nonlinear impacts on host immunity

Abstract

The impacts of individual commensal microbes on immunity and disease can differ dramatically depending on the surrounding microbial context; however, the specific bacterial combinations that dictate divergent immunological outcomes remain largely undefined. Here, we characterize an immunostimulatory Allobaculum species from an inflammatory bowel disease patient that exacerbates colitis in gnotobiotic mice. Allobaculum inversely associates with the taxonomically divergent immunostimulatory species Akkermansia muciniphila in human-microbiota-associated mice and human cohorts. Co-colonization with A. muciniphila ameliorates Allobaculum-induced intestinal epithelial cell activation and colitis in mice, whereas Allobaculum blunts the A.muciniphila-specific systemic antibody response and reprograms the immunological milieu in mesenteric lymph nodes by blocking A.muciniphila-induced dendritic cell activation and T cell expansion. These studies thus identify a pairwise reciprocal interaction between human gut bacteria that dictates divergent immunological outcomes. Furthermore, they establish a generalizable framework to define the contextual cues contributing to the "incomplete penetrance" of microbial impacts on human disease.

Keywords: IgA; IgA-Seq; gut microbiota; human gut bacteria; human microbiota-associated gnotobiotic mice; immunoglobulin A; immunostimulatory commensals; inflammatory bowel disease; mucosal immunity; reciprocal epistasis.

Figure 1.. An Allobaculum species from an ulcerative colitis patient exacerbates acute and chronic colitis in gnotobiotic mice.

(A) Identification and Isolation of IgA-coated Allobaculum sp. 128 from an ulcerative colitis patient. (B) Scanning electron micrographs of Allobaculum sp. 128 in vitro. Scale bars, 2μm (top), 10μm (bottom). (C-H) Germ-free WT mice were gavaged and colonized for seven days before treatment with 2% DSS-H₂O ad libitum. (C) Fecal microbiota on d0 (first bar) and d7 (bars 2–5) of DSS. (D-E) Colons on d7 and representative H&E sections. Scale bars, 1mm. (F) Colon length on d7. (G) Fecal lipocalin (LCN2) on d2. (H) Histopathology scores. (I-L) Acute DSS colitis in Rag1^−/− gnotobiotic mice: colon length (I), d2 fecal lipocalin (J), lamina propria CD45⁺Ly6G⁺ neutrophils (K), and colon explant cytokines (L). (M-N) Spontaneous colitis in Il10^−/− gnotobiotic mice: fecal LCN2 (M) and colon histopathology scores (N). Welch’s t-test was used to compare microbiota groups. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Error bars show mean ± SEM. (C-H) show one of N=6 independent experiments, n=5–6 mice per group. (I-L) show one of N=4 independent experiments, n=4–9 mice/group. (M-N) show one of N=3 independent experiments, n=5 mice/group.

Figure 2.. Allobaculum sp. 128 elicits mucosal and systemic immunity at steady state.

(A-B) GF WT mice colonized with MC or MC+Allobaculum sp. 128 were analyzed for fecal bacteria IgA coating (n=4 per group). (C) Allobaculum sp. 128-specific fecal water IgA and (D-E) serum IgA & IgG. (F) MC-specific serum IgG (n=3–6 mice per group). Welch’s t-test was used to compare microbiota groups at week 7. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Data shown in (A-F) are representative of N=2 independent experiments. (G) Colon sections stained with bacterial FISH probe EUB338 and DAPI. Scale bars, 25μm. Solid yellow line: epithelial brush border; white dashed line: inner mucus layer. N=2 independent experiments with n=2 mice/group. (H) Bulk colon RNAseq at 4wks colonization (n=2–3 per group). Volcano plot of significantly differentially expressed genes and (I) gene ontology pathway enrichment for MC+Allo vs MC. Data shown in (H-I) is from N=1 experiment.

Figure 3.. Allobaculum sp. 128 is inversely correlated with Akkermansia muciniphila in human microbiota-associated gnotobiotic mice.

(A) Experimental workflow. (B) GF WT mice were gavaged with Allobaculum sp. 128, then with healthy human stool 24h later. Fecal pellets were collected for microbiota profiling (n=19). Bacterial OTU legend shown in Figure S4B. (C) Spearman correlation of each genus OTU to Allobaculum sp. 128 abundance, and log-likelihood ratio P-values. (D) XY plot of data in (B). (E-F) Meta-analysis of human microbiome datasets (see Table S1): American Gut Project (McDonald, et al. 2018) and pediatric ulcerative colitis (UC) (Schirmer, et al. 2018). Data shown in (A-D) are from N=1 experiment.

Figure 4.. A. muciniphila attenuates Allobaculum sp. 128-mediated intestinal epithelial cell activation and colitis.

(A) Experimental schematic. (B) Fecal microbiota composition profiled over four weeks (n=3–4 mice/group). (C) Experimental schematic for DSS colitis. (D) d2 fecal lipocalin and (E-F) colon length at euthanasia. (G-K) Absolute quantitative microbial profiling. (L) Principal component analysis (PCA) of gene expression in intestinal epithelial cells (IEC) after 2 weeks of colonization. (M-N) Log2 fold change of the top differentially expressed genes. (O) Experimental schematic: colonization with live commensal microbes or daily gavage with sterile culture supernatant for 10 days before RNA harvest from IEC. (P-R) Ileal IEC expression of key Allobaculum-induced genes. Error bars show mean ± SEM. Welch’s t-test was used to compare across MC+Allo+A.m.supe vs MC+Allo; * P<0.05, ** P<0.01. Data shown in (D-F) are one representative of N=2 experiments. Data shown in (A), (G-K), (L-N), and (O-R) are each from separate N=1 experiments.

Figure 5.. A. muciniphila colonization protects against Allobaculum sp. 128-induced colitis in the context of a complex human microbiota.

(A) Experimental schematic using human fecal sample HC19. (B) Fecal microbiota composition on d3 of colitis. (C) Fecal lipocalin assessed on d2–3. (D-E) Colon length at d7 euthanasia. Error bars show mean ± SEM. Welch’s t-test was used to compare microbiota groups. * P<0.05, ** P<0.01. Data shown are from N=1 experiment.

Figure 6.. Allobaculum sp. 128 blunts antigen-specific serum antibody responses to A. muciniphila and oral vaccination.

(A) Experimental schematic. (B) Dilution curves of A. muciniphila-reactive and Allobaculum sp. 128-reactive serum IgA & IgG1 (n=3–5). (C-D) Cholera toxin (CT)-specific serum IgG at 5 weeks. Error bars show mean ± SEM. Welch’s t-test was used to compare MC+A.m. to MC+Both at each dilution. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Data shown in (B) are representative from one of N=4 independent experiments. Data shown in (D) are from N=1 experiment.

Figure 7.. Allobaculum sp. 128 and A. muciniphila induce context-dependent transcriptomic reprogramming in mucosal lymphoid tissues.

(A-B) Annotated dimensionality reduction plots of single-cell gene expression libraries, pooled by tissue (A, MLN; B, PP). Right, heatmap of cell lineage relative frequency normalized to MC. (C) TCR repertoire diversity. (D) Top 12 most expanded clonotypes in MC+A.m. mice. (E,H) MLN Tfh+Tfr and MigDC were re-clustered and highlighted by microbiome. (F) Expression of key genes within Tfh+Tfr. (G) Prominent TCR clonotypes within MLN Tfh+Tfr induced by MC+A.m. (H) MLN MigDC UMAP clustering. (I) Expression of key MigDC antigen presentation genes. (J) Top 100 differentially expressed genes within MLN MigDC transcriptomes. Error bars show mean ± SEM. Welch’s t-test was used to compare gene expression across microbiome groups. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Data shown from N=1 experiment with n=3 mice/group, pooled.