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Akkermansia muciniphila Induces Intestinal Adaptive Immune Responses During Homeostasis

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Akkermansia muciniphila Induces Intestinal Adaptive Immune Responses During Homeostasis

Eduard Ansaldo et al. Science.

Abstract

Intestinal adaptive immune responses influence host health, yet only a few intestinal bacteria species that induce cognate adaptive immune responses during homeostasis have been identified. Here, we show that Akkermansia muciniphila, an intestinal bacterium associated with systemic effects on host metabolism and PD-1 checkpoint immunotherapy, induces immunoglobulin G1 (IgG1) antibodies and antigen-specific T cell responses in mice. Unlike previously characterized mucosal responses, T cell responses to A. muciniphila are limited to T follicular helper cells in a gnotobiotic setting, without appreciable induction of other T helper fates or migration to the lamina propria. However, A. muciniphila-specific responses are context dependent and adopt other fates in conventional mice. These findings suggest that, during homeostasis, contextual signals influence T cell responses to the microbiota and modulate host immune function.

Conflict of interest statement

Competing interests: Authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Mice generate anti-commensal IgG1 antibodies during homeostasis
(A) Representative IgG1 flow cytometric analysis of fecal microbiota with sera from WT and T cell-deficient (Tcrb−/−) mice. Feces and sera originated from the same mouse (paired serum), except when using antibody-deficient (Ighm−/−) serum as a negative staining control. SYBR-green labels a fraction of the microbiota, ensuring that SYBRhi events are bacteria, whereas some of the SYBRlo events are also commensals that are less permeable to the dye (8). (B) IgG1 microbiota flow cytometric analysis, compiled from eight independent experiments. All mice were housed at UC Berkeley. WT n=63, Tcrb−/− n=35 in total. (C) IgG1 microbiota flow cytometric analysis with paired feces and sera from mice of the indicated genetic backgrounds and vivaria. Balb/c from Jackson Laboratories. Jax B6 and Tac B6: C57BL/6 from Jackson Laboratories or Taconic Biosciences, respectively. SW: Swiss Webster from Taconic Biosciences. n=5 mice per group. Data are representative of two independent experiments. (D) Results from sorting and 16S rDNA sequencing of IgG1-bound and unbound fractions (n=12 mice). Graph depicts the average log2 ratio of abundances between both fractions for each individual OTU and the corresponding q-value. Data are representative of two independent experiments. Each symbol represents a mouse (B, C) or an OTU (D). Error bars represent mean ± SD. Gates on flow cytometry plots show mean±SEM. p-values were calculated by a Kruskal–Wallis test followed by Dunn’s multiple comparisons (B) or by Paired ratio Student’s t-test followed by Benjamini, Krieger and Yekutieli’s two-stage false discovery rate (FDR) to correct for multiple comparisons, with an FDR (Q) of 0.01 (D).
Fig. 2
Fig. 2. Akkermansia muciniphila is necessary and sufficient to induce cognate A. muciniphila-specific IgG1 antibody responses
(A) Representative IgG1 bacterial flow cytometric analysis of A. muciniphila incubated with the indicated mouse sera. Applies to results shown in C. (B) Quantification of A. muciniphila colonization by fecal 16S qPCR for mice in (C). (C) A. muciniphila IgG1 bacterial flow cytometric analysis for mice of the indicated genotypes and indicated A. muciniphila colonization status. WT Akk n=25, WT Akk+ n=41, Tcrb−/− n=15. gMFI: geometric mean fluorescence intensity. Data are compiled from seven independent experiments. (D) Quantification of A. muciniphila colonization by fecal 16S qPCR before (WT Akk) and 5 weeks after (WT Akk-colonized) a single A. muciniphila oral gavage of 109 cfu. n=6 mice. Applies to results shown in F. Data are representative of three independent experiments. (E, F) Representative plot (E) and quantification (F) of A. muciniphila IgG1 bacterial flow cytometric analysis using sera from mice before (Akk) and 5 weeks after colonization (Akk-colonized). n=5 mice. Data are representative of three independent experiments. (G) Quantification of A. muciniphila colonization by fecal 16S qPCR. n=6 ASF, n=16 ASF+Akk mice. Data are representative of two independent experiments. (H and I) A. muciniphila bacterial flow cytometric analysis with serial dilution of serum in ASF and ASF+Akk mice. Each line represents one mouse. The x-axis denotes total serum IgG1 (H) or serum IgA (I) concentration in the assay. n=9 mice per group. Data are representative of two independent experiments. LoD: limit of detection. Each symbol represents a mouse, error bars represent mean ± SD. p-values were calculated with a Kruskal–Wallis test followed by Dunn’s multiple comparisons (B, C) or a Mann–Whitney test (D, F, G).
Fig. 3
Fig. 3. A. muciniphila induces antigen-specific T follicular helper cell responses during homeostasis
(A) Representative flow cytometric analysis depicting transferred T cells (Thy1.1+) as percentage of all CD4+ T cells in the Peyer’s patches of ASF and ASF+Akk mice 12 days after low-frequency adoptive transfer of Amuc124 TCR transgenic T cells. Quantified in (B). (B) Frequencies of transferred T cells in intestinal tissues of ASF and ASF+Akk mice. n=5 mice per group, data are representative of six independent experiments. (C and D) Representative flow cytometric analysis of expression of T follicular helper markers (PD-1, Bcl6, and CXCR5) by endogenous and transferred T cells in the Peyer’s patches of ASF+Akk mice. (E and F) Expression of TH1 (T-bet+ FOXP3), TH2 (GATA3+ FOXP3), TH17 (RORγt+ FOXP3), Treg (FOXP3+), or TFH (Bcl6+ PD-1+) markers by transferred T cells in the Peyer’s patches (E) and small intestine lamina propria (F) of ASF+Akk mice. n=9 mice. Data are representative of six independent experiments. (G) Representative Am3735–1 tetramer flow cytometric analysis of Peyer’s patches from ASF and ASF+Akk mice. (H) Frequencies of Am3735–1 or Am3740–1 tetramer+ endogenous T cells in ASF and ASF+Akk mice as a percentage of total CD4+ T cells. n=4 ASF, n=5–7 ASF+Akk mice. Data are representative of three (Am3740–1) or six (Am3735–1) independent experiments. (I) Frequencies of Am3735–1 or Am3740–1 tetramer+ cells expressing TFH markers (PD-1 and CXCR5, as shown in (J)) in the PP of ASF+Akk mice. n=5–7 mice, data are representative of three (Am3740–1) or six (Am3735–1) independent experiments. (J) Representative flow cytometric analysis of expression of TFH markers (PD-1 and CXCR5) by endogenous Am3735–1 or Am3740–1 tetramer+ cells. (K) Numbers of Am3735–1 and Am3740–1 tetramer+ cells in all intestinal tissues in ASF and ASF+Akk mice. n=4 ASF, n=5 ASF+Akk mice. Data are representative of two (Am3740–1) or three (Am3740–1) independent experiments. Each symbol represents a mouse, error bars represent mean ± SD. Gates on flow cytometry plots show mean±SEM. p-values were calculated with unpaired Student’s t-tests (B, K), or a Mann–Whitney test (H)
Fig. 4
Fig. 4. A. muciniphila-specific T cells also adopt other fates in the context of a complex microbiota
(A) Representative flow cytometric analysis depicting transferred T cells (Thy1.1+) as percentage of all CD4+ T cells in the Peyer’s patches of SPF Akk and SPF Akk+ mice. (B) Frequencies of transferred T cells as percentage of all CD4+ T cells in intestinal tissues of conventional specific pathogen-free (SPF) A. muciniphila (n=4) and SPF A. muciniphila + (n=5) mice. Data are representative of three independent experiments. (C and D) Expression of TH1 (T-bet+ FOXP3), TH2 (GATA3+ FOXP3), TH17 (RORγt+ FOXP3), Treg (FOXP3+) or TFH (Bcl6+ PD-1+) markers by transferred T cells in the Peyer’s patches (C) or small intestine lamina propria (D) of SPF A. muciniphila + mice. n=5 mice, data are representative of three independent experiments. (E) Representative flow cytometric analysis of expression of TH1 and TH17 markers by endogenous total CD4+ T cells and A. muciniphila-specific (transferred) T cells in the SILP of SPF A. muciniphila + mice. Each symbol represents a mouse, error bars represent mean ± SD. Gates on flow cytometry plots show mean±SEM. p-values were calculated with unpaired Student’s t-tests (B).

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