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Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis

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Science  21 Jun 2019:
Vol. 364, Issue 6446, pp. 1179-1184
DOI: 10.1126/science.aaw7479
  • Fig. 1 Mice generate anticommensal 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. (C) IgG1 microbiota flow cytometric analysis with paired feces and sera from mice of the indicated genetic backgrounds and vivaria. Balb/c mice were from The Jackson Laboratory. Jax B6 and Tac B6 C57BL/6 mice were from the Jackson Laboratory and Taconic Biosciences, respectively. Swiss Webster (SW) mice were 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) and (C)] or an OTU (D). Error bars represent mean ± SD. Gates on flow cytometry plots show mean ± SEM. P values were calculated by Kruskal-Wallis test followed by Dunn’s multiple-comparisons test (B) or by paired-ratio Student’s t test followed by the Benjamini, Krieger, and Yekutieli two-stage false discovery rate (FDR) correction for multiple comparisons, with an FDR of 0.01 (D).

  • Fig. 2 A. 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. Geometric mean fluorescence intensity (gMFI) of the assay was quantified in (C) for multiple mice. (B) Quantification of A. muciniphila colonization by fecal 16S quantitative polymerase chain reaction (qPCR) for mice described in (C). LoD, limit of detection. (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. 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 colony-forming units. n = 6 mice. Data are representative of three independent experiments. (E and 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 (Akk-colonized) colonization. n = 5 mice. Data are representative of three independent experiments. (G) Quantification of A. muciniphila colonization by fecal 16S qPCR. ASF, n = 6 mice; ASF+Akk, n = 16 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. 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 test (B and C) or Mann-Whitney test (D, F, and G).

  • Fig. 3 A. muciniphila induces antigen-specific TFH cell responses during homeostasis.

    (A) Representative flow cytometric analysis depicting transferred T cells (Thy1.1+) as a percentage of all CD4+ T cells in the PPs of ASF and ASF+Akk mice 12 days after low-frequency adoptive transfer of Amuc124 (TCR-transgenic) T cells. (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 TFH markers (PD-1, Bcl6, and CXCR5) by endogenous and transferred T cells in the PPs 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 PPs (E) and SILP (F) of ASF+Akk mice. n = 9 mice. Data are representative of six independent experiments. (G) Representative Am3735-1 tetramer flow cytometric analysis of PPs from ASF and ASF+Akk mice. (H) Frequencies of Am3735-1- or Am3740-1 tetramer–positive endogenous T cells in ASF and ASF+Akk mice as a percentage of total CD4+ T cells. ASF, n = 4 mice; ASF+Akk, n = 5 to 7 mice. Data are representative of three (Am3740-1) or six (Am3735-1) independent experiments. (I) Frequencies of Am3735-1- or Am3740-1 tetramer–positive cells expressing TFH markers [PD-1 and CXCR5, as shown in (J)] in the PPs of ASF+Akk mice. n = 5 to 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–positive cells. (K) Numbers of Am3735-1- and Am3740-1 tetramer–positive cells in all intestinal tissues in ASF and ASF+Akk mice. ASF, n = 4 mice; ASF+Akk, n = 5 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 test (B and K) or Mann-Whitney test (H).

  • 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 Amuc124 T cells (Thy1.1+) as a percentage of all CD4+ T cells in the PPs of SPF Akk and SPF Akk+ mice. (B) Frequencies of transferred T cells as a percentage of all CD4+ T cells in intestinal tissues of conventional SPF A. muciniphila–negative (n = 4) and SPF A. muciniphila–positive (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 PPs (C) or SILP (D) of SPF A. muciniphila–positive 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–positive 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 test (B).

Supplementary Materials

  • Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis

    Eduard Ansaldo, Leianna C. Slayden, Krystal L. Ching, Meghan A. Koch, Natalie K. Wolf, Damian R. Plichta, Eric M. Brown, Daniel B. Graham, Ramnik J. Xavier, James J. Moon, Gregory M. Barton

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figs. S1 to S8
    • Table S1 
    • References 

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