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Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse models

  1. Sergio E. Baranzinia,5
  1. aDepartment of Neurology, University of California, San Francisco, CA 94158;
  2. bDivision of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
  3. cCenter for Microbiome Innovation, University of California, San Diego, La Jolla, CA 92093;
  4. dDepartment of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029;
  5. eAdvanced Science Research Center, City University of New York, New York, NY 10031
  1. Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved August 7, 2017 (received for review June 30, 2017)

  1. Fig. 2.

    Relative abundances of individual microbial genera differ between MS patients and controls. (A) Volcano plots of the relative abundance distribution of microbial genera (Left) and OTUs (Right). The x axes show log twofold of relative abundance ratio between MS patients (n = 71) and controls (n = 71) after variance-stabilizing transformation. The y axes show negative log10 of P value (negative binomial Wald test with Benjamini–Hochberg correction for multiple comparisons). (B) Summary of taxonomic differences between MS and control microbiomes. (C) Relative abundance plots of selected microbial genera (highlighted in A) that were found to be significantly different between MS and controls. Data are shown as mean ± SEM.

  2. Fig. 3.

    A. calcoaceticus inhibits Treg differentiation and stimulates Th1 differentiation in vitro. (A and B) Representative flow cytometry plots (A) and quantification (B) of CD25+FoxP3+ cell differentiation within the CD3+CD4+ population in response to A. calcoaceticus (A. calc) (n = 6 PBMC donors). (C and D) Representative flow cytometry plots (C) and quantification (D) of IFNγ+ Th1 lymphocytes within the CD3+CD4+ population in response to A. calcoaceticus (A. calc) (n = 11 PBMC donors). *P < 0.05, **P < 0.01, two-tailed repeated-measures t test. Data are shown as mean ± SEM.

  3. Fig. 4.

    A. muciniphila increases Th1 lymphocyte differentiation in vitro. (AD) Representative flow cytometry plots (A and B) and quantification (C and D) of IFNγ+ and of Tbet+ Th1 lymphocytes within the CD3+CD4+ population in response to A. muciniphila (A. muci). n = 6 PBMC donors for the IFNγ experiment; n = 7 PBMC donors for the Tbet experiment. *P < 0.05, two-tailed repeated-measures t test. (EH) Representative flow cytometry plots (E and F) and quantification (G and H) of IFNγ+ Th1 lymphocytes within the CD3+CD4+ population in response to nonself or self bacteria from subjects with or without detected A. muciniphila. n = 6 subjects without A. muciniphila; n = 12 subjects with A. muciniphila. *P < 0.05, two-tailed t test; **P < 0.01, two-way ANOVA for repeated measures. Data are shown as mean ± SEM.

  4. Fig. 5.

    P. distasonis stimulates IL-10+ Treg differentiation in vitro. (AD) Representative flow cytometry plots (A and B) and quantification (C and D) of CD25+ and CD25+IL-10+ lymphocytes within the CD3+CD4+ population in response to P. distasonis (P. dist). n = 6 PBMC donors. *P < 0.05, **P < 0.01, two-tailed repeated measures t test. Data are shown as mean ± SEM.

  5. Fig. 6.

    Monocolonization of GF mice with MS-associated bacteria mediates T lymphocyte differentiation. (AC) Representative flow cytometry plots (A) and quantification (B and C) of CD4+IFNγ+ lymphocytes (B) and CD4+IL-10+ lymphocytes (C) within the live cell population in GF mice colonized with A. calcoaceticus, A. muciniphila, and P. distasonis. GF mice and specific pathogen-free (SPF) mice are used as controls. n = 3–8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey adjustment for multiple comparisons. Data are shown as mean ± SEM. CLN, cervical lymph nodes; MLN, mesenteric lymph nodes.

  6. Fig. 7.

    Transfer of healthy control microbiota protects against EAE and mediates Treg induction in mouse mesenteric lymph nodes compared with transfer of MS patient microbiota. (A) Clinical EAE scores of mice that had been colonized with healthy control or MS patient microbiota for at least 5 wk or kept GF before the induction of EAE at 9–10 wk of age. Asterisks indicate significance between both the MS vs. control and the MS vs. GF groups. n = 6–8 mice per group. ****P < 0.0001, two-way ANOVA with Tukey adjustment for multiple comparisons. Data are shown as mean ± SEM. (B) PCoA of mouse microbiota at different time points after colonization with fecal microbiota from donor pair #1. n = 3–5 mice per group. EAE induction occurs at 35 d after transplantation. PC1, -2, -3, principal components 1, 2, and 3. (C and E) Representative flow cytometry plots of FoxP3+ lymphocytes within CD4+ populations and IL-10+ lymphocytes within CD4+FoxP3+ populations before EAE induction (C) and at peak of EAE disease (E). (D and F) Frequencies of IL-10+ lymphocytes within CD4+FoxP3+ populations in mesenteric lymph nodes of mice killed before EAE induction (D) and at peak of EAE progression (22 d after immunization) (F). *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey adjustment for multiple comparisons. Data are shown as mean ± SEM.

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