Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice

Abstract

Th17 cells accrue in the intestine in response to particular microbes. In rodents, segmented filamentous bacteria (SFB) induce intestinal Th17 cells, but analogously functioning microbes in humans remain undefined. Here, we identified human symbiont bacterial species, in particular Bifidobacterium adolescentis, that could, alone, induce Th17 cells in the murine intestine. Similar to SFB, B. adolescentis was closely associated with the gut epithelium and engendered cognate Th17 cells without attendant inflammation. However, B. adolescentis elicited a transcriptional program clearly distinct from that of SFB, suggesting an alternative mechanism of promoting Th17 cell accumulation. Inoculation of mice with B. adolescentis exacerbated autoimmune arthritis in the K/BxN mouse model. Several off-the-shelf probiotic preparations that include Bifidobacterium strains also drove intestinal Th17 cell accumulation.

Keywords: Th17 cells; intestine; microbiota; mucosal immunology; probiotic.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

B. adolescentis (BA) induces a robust intestinal Th17 population. (A) Frequency of SI-LP Th17 cells in GF mice monocolonized with individual symbiont bacteria as described in Materials and Methods. White and gray symbols represent GF and SFB-monocolonized mice, respectively. Arrow indicates BA-monocolonized mice. (B) Inflammatory cytokine production by SI-LP CD4+ T cells in mice colonized as indicated. (Left) Representative flow cytometric dot plot. (Right) Summary data. (C) Frequencies of (Upper) Th17 and (Lower) Th1 cells in various tissues of mice colonized as indicated. (D) Frequencies of (Left) Th17 and (Right) Th1 cells in the SI-LP of SPF mice gavaged as described in Materials and Methods with the indicated microbes. SFB+ SPF mice were bred at Harvard Medical School and naturally colonized with SFB. (E) Induction of intestinal RORγt expressers. (Left) Representative flow cytometric dot plot of SI-LP CD4+ T cells; summary data for frequencies of (Center) RORγt+ Foxp3− cells and (Right) RORγt+ Helios− Treg cells. Numbers in B and E refer to the fractions of cells in the identical gates. (B–E) Mean ± SEM pooled from two to four independent experiments. BF, B. fragilis; Ce, cecum; CH, C. histolyticum; Co, colon; IEL, intraepithelial lymphocyte layer; ILN, inguinal lymph node; MLN, mesenteric lymph nodes; PP, Peyer’s patches; SI, small intestine. *P < 0.05 (Mann–Whitney u test); **P < 0.01 (Mann–Whitney u test); ***P < 0.001 (Mann–Whitney u test).

Fig. S1.

Effects of BA monocolonization on other intestinal immunocyte populations. (A) No correlation between induction of intestinal Th17 and RORγt+ Treg cells after monocolonization with the panel of bacteria detailed in Fig. 1A. Lines represent linear regressions performed on the two immunocyte populations in the respective tissues. Pearson correlations are found to be not significant. (B) Data in A plotted as SI-LP Th17 vs. colonic RORγt+ Treg frequencies. Linear regression lines and correlations were calculated as in A (not significant). (C) Frequencies of IL-17A+ cells within the RORγt+ CD4+ T-cell population in various intestinal tissues in BA-monocolonized mice. (D) Frequencies of intestinal Foxp3+ CD4+ T cells in mice colonized as indicated. (E–G) Frequencies of (E) IL-17A–producing γδ T cells, (F) IL-22–producing Thy1+ RORγt+ type 3 innate lymphoid cells (ILC3s), and (G) IL-17A–producing ILC3s in various tissues of mice colonized as indicated. (H) Frequencies of GC B cells in the Peyer’s patches (PPs) of mice colonized as indicated. (I) Frequencies of IgA+ B220− plasma cells in the SI-LP of mice colonized as indicated. SPF mice from Jax were SFB− or colonized with SFB. Each symbol represents one mouse, and data were pooled from at least two independent experiments. Mean ± SEM. Ce, cecum; CH, Clostridium histolyticum; Co, colon; Duo, duodenum; Il, ileum; ILN, inguinal lymph node; Jej, jejunum; MLN, mesenteric lymph nodes; SI, small intestine. *P < 0.05 (Mann–Whitney u test); **P < 0.01 (Mann–Whitney u test); ***P < 0.001 (Mann–Whitney u test).

Fig. 2.

B. adolescentis (BA) does not provoke intestinal inflammation. (A–D) Myeloid cells. Frequencies of the indicated myeloid cell populations in the intestines of mice colonized as indicated. Each symbol represents one mouse. Mean ± SEM. Data for GF and BA are pooled from at least two independent experiments. P value was not significant for all comparisons (Kruskal–Wallis test and Dunn’s multiple comparisons test). (E and F) Histopathology. H&E staining of representative sections of (E) the small intestine (SI) and (F) the colon. (Scale bar: 50 μm.) (G) Th17 cell phenotype. Fold change (FC)/FC plots comparing transcripts induced by BA vs. SFB in SI-LP CD4+ T cells. Red indicates transcripts up-regulated in (Upper) pathogenic Th17 cells or (Lower) canonical Th17 cells. Example genes induced for each Th17 phenotype are indicated (n = 3–4 per group). BF, B. fragilis; CH, C. histolyticum.

Fig. S2.

BA does not provoke intestinal inflammation. (A) Numbers of CD45+ cells and (B) frequencies of CD11b+F4/80+CD103− macrophages in the intestines of mice colonized as indicated. Each symbol represents one mouse. Data for GF and BA pooled from at least two independent experiments. *P < 0.05 (Kruskal–Wallis test and Dunn’s multiple comparisons test). (C) Pathology scores of ileum and colon sections from GF and BA-monocolonized mice. Scoring method is detailed in SI Materials and Methods. Each symbol represents one mouse, and data were pooled from two independent experiments. BF, Bacteroides fragilis; CH, Clostridium histolyticum; SI, small intestine.

Fig. 3.

B. adolescentis (BA)-driven intestinal Th17 responses are cognate. (A and B) BA-specific Th17 cells. Frequencies of SI-LP CD4+ T cells producing (A) IL-17A or (B) IFN-γ on overnight stimulation by splenic dendritic cells incubated with media alone or lysates prepared from the indicated bacterial species. +Anti–MHC-II (the blocking antibody M5/114.15.2) was added. (C) Frequency of SI-LP CD4+ T cells producing IL-17A on stimulation with media alone or BA lysate when T cells were isolated from mice colonized as indicated. (D) The degree of TCRVβ14 enrichment in SI-LP CD4+ T cells from SPF mice colonized as indicated, calculated as (percent of Vβ14+ cells in Th17 fraction)/(percent of TCRVβ14+ cells in non-Th17 fraction). Each symbol represents one mouse. Data are pooled from two to four independent experiments. Mean ± SEM. BF, B. fragilis; P + I, PMA and ionomycin. *P < 0.05 (Kruskal–Wallis test and Dunn’s multiple comparisons test); **P < 0.01 (Kruskal–Wallis test and Dunn’s multiple comparisons test); ***P < 0.001 (Kruskal–Wallis test and Dunn’s multiple comparisons test).

Fig. 4.

B. adolescentis (BA) colonizes the entire length of the intestines, closely associating with the ileal epithelium. (A) Titers of bacteria associated with various segments of the intestinal mucosa or shed into the corresponding lumen or the stool (St). Normalized to tissue or St weight or the volume of luminal wash. Each symbol represents one mouse. Data are pooled from three to four independent experiments. Mean ± SEM. (B) Frequencies of Th17 cells along the length of the intestinal LP in mice colonized as indicated. Each line represents frequencies from one mouse. Data are pooled from three independent experiments. (C) FISH quantification. Normalized bacterial fluorescence vs. distance from the epithelial surface of the terminal ileum (Il) from mice monocolonized as indicated. Data are plotted as the average fluorescence from six to eight total images from two to three fields of view per section from two to four mice per microbe. (D) Representative SEM photograph of the ileal surface in mice colonized as indicated. Arrows indicate sites of bacterial association with the intestinal epithelium. Ce, cecum; CH, C. histolyticum; Co, colon; Duo, duodenum; Jej, jejunum.

Fig. 5.

B. adolescentis (BA) and SFB both induce B-cell transcripts, but otherwise, they trigger distinct transcriptional programs. (A and B) Whole-tissue transcriptomes. Fold change (FC)/FC plots comparing ileum tissue transcripts induced by (A) BA vs. SFB (Th17 inducing) or (B) BA vs. C. histolyticum (CH; Th17 noninducing). In A, blue indicates genes up-regulated primarily by SFB, black indicates genes up-regulated primarily by BA, and red indicates genes up-regulated by both bacteria. Certain transcripts previously associated with SFB-mediated induction of Th17 cells or encoding Igs are indicated. In B, the genes highlighted in A are again highlighted in the same colors. (C) Statistics for A and B. FC distribution of (Upper) SFB-induced Ig or (Lower) non-Ig RNAs in the ileal transcriptomes of mice colonized as indicated. Only transcripts increased by SFB by at least ≥1.6-fold with a P value of <0.05 were plotted. ***P < 0.001 (Kolmogorov–Smirnov test). (D) Pathways (from the two databases as indicated) enriched in transcripts specifically up-regulated in the ileum by BA relative to GF mice were determined using Enrichr. P values are indicated to the right. (E and F) S-IEC transcriptomes. As per A and B, except that transcripts from isolated S-IECs were examined. In E, orange indicates genes up-regulated primarily by SFB, cyan indicates genes up-regulated primarily by BA, and purple indicates genes up-regulated by both bacteria. In F, the genes highlighted in E are again highlighted in the same colors. (G) Statistics for E and F. Calculated as per C. n = 3 (A–D) or 2–4 (E and F) per group. ***P < 0.001.

Fig. 6.

B. adolescentis (BA) exacerbates K/BxN arthritis. K/BxN mice were pretreated with antibiotics from 22 to 32 d of age and subsequently gavaged with PBS, C. histolyticum (CH), or BA over 12–13 d. Arrows indicate gavage time points. (A) Ankle-thickening values over the course of bacterial gavage. (B, Left) Representative flow cytometric plot of IL-17A production from SI-LP CD4+ T cells 11–13 d after the initial bacterial gavage. Summary data of (B, Center) frequencies and (B, Right) numbers of SI-LP Th17 cells at the same time point. (C) Antiglucose-6-phosphate isomerase titers 11–13 d after initial bacterial gavage. Each symbol represents one mouse [n = 3 (PBS), 8 (CH), 11 (BA), and 2–5 (SFB)]. Data for CH and BA are pooled from two to three independent experiments. Mean ± SEM. GPI, glucose-6-phosphate isomerase. *P < 0.05 (Kruskal–Wallis test with Dunn’s multiple comparisons test); **P < 0.01 (Kruskal–Wallis test with Dunn’s multiple comparisons test); ***P < 0.001; (Kruskal–Wallis test with Dunn’s multiple comparisons test).

Fig. 7.

Some probiotic formulations containing bifidobacterial species also elicit Th17 populations in the SI-LP. (A) Frequencies of SI-LP (Left) Th17 or (Right) Th1 cells in GF mice colonized with the indicated probiotic mixes. (B) Th17 cell phenotype. Fold change (FC)/FC plots comparing transcripts induced by B. adolescentis (BA) vs. the probiotic mix, Nexabiotic, in SI-LP CD4+ T cells. Symbols in red and labels are as per Fig. 2G (n = 2–3 per group). (C) Frequencies of SI-LP (Left) Th17 and (Right) Th1 cells in SPF (SFB−) mice gavaged with PBS or one of three other probiotic preparations in A. Full names and corresponding abbreviations of probiotics can be found in SI Materials and Methods. Each symbol represents one mouse. Data are pooled from two to three independent experiments. Mean ± SEM. *P < 0.05 (Mann–Whitney u test); **P < 0.01 (Mann–Whitney u test).

Fig. S3.

BA and related species are enriched in the microbiotas of IBD patients. Comparison of the microbiotas of 85 healthy vs. 39 IBD subjects in the MetaHIT database using LEfSe as outlined in SI Materials and Methods. Microbial clades with the largest effect sizes (>3.5) with a false discovery rate q < 0.05 (Kruskal–Wallis test with Benjamini–Hochberg correction) are shown. (Left) Negative and (Right) positive values correspond to healthy controls (HCs) and IBD patients, respectively. BA and other bifidobacterial species are represented as white bars.