Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

doi:10.1016/j.ebiom.2020.102913

. 2020 Aug:58:102913.

doi: 10.1016/j.ebiom.2020.102913. Epub 2020 Jul 22.

Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

Daisuke Takahashi¹, Naomi Hoshina¹, Yuma Kabumoto¹, Yuichi Maeda², Akari Suzuki³, Hiyori Tanabe¹, Junya Isobe¹, Takahiro Yamada¹, Kisara Muroi¹, Yuto Yanagisawa¹, Atsuo Nakamura⁴, Yumiko Fujimura¹, Aiko Saeki¹, Mizuki Ueda⁵, Ryohtaroh Matsumoto¹, Hanako Asaoka¹, Julie M Clarke⁶, Yohsuke Harada⁷, Eiji Umemoto⁸, Noriko Komatsu⁹, Takaharu Okada¹⁰, Hiroshi Takayanagi⁸, Kiyoshi Takeda⁷, Michio Tomura⁵, Koji Hase¹¹

Affiliations

¹ Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan.
² Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka565-0871, Japan.
³ Laboratory for Autoimmune Diseases, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Kanagawa230-0045, Japan.
⁴ Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan; Dairy Science and Technology Institute, Kyodo Milk Industry Co. Ltd., Nishitama, Tokyo190-0182, Japan.
⁵ Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka584-8540, Japan.
⁶ Preventative Health National Research Flagship, CSIRO Food and Nutritional Sciences, Adelaide, South Australia5000, Australia.
⁷ Laboratory of Pharmaceutical Immunology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba278-8510, Japan.
⁸ Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center (IFReC), Osaka University, Suita, Osaka565-0871, Japan.
⁹ Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo113-0033, Japan.
¹⁰ Laboratory for Tissue Dynamics, RIKEN IMS, Yokohama, Kanagawa230-0045, Japan.
¹¹ Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan; International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo (IMSUT), Minato-ku, Tokyo108-8639, Japan. Electronic address: [email protected].

PMID: 32711255
PMCID: PMC7387783
DOI: 10.1016/j.ebiom.2020.102913

Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

Daisuke Takahashi et al. EBioMedicine. 2020 Aug.

. 2020 Aug:58:102913.

doi: 10.1016/j.ebiom.2020.102913. Epub 2020 Jul 22.

Authors

Affiliations

¹ Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan.
² Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka565-0871, Japan.
³ Laboratory for Autoimmune Diseases, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Kanagawa230-0045, Japan.
⁴ Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan; Dairy Science and Technology Institute, Kyodo Milk Industry Co. Ltd., Nishitama, Tokyo190-0182, Japan.
⁵ Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka584-8540, Japan.
⁶ Preventative Health National Research Flagship, CSIRO Food and Nutritional Sciences, Adelaide, South Australia5000, Australia.
⁷ Laboratory of Pharmaceutical Immunology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba278-8510, Japan.
⁸ Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center (IFReC), Osaka University, Suita, Osaka565-0871, Japan.
⁹ Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo113-0033, Japan.
¹⁰ Laboratory for Tissue Dynamics, RIKEN IMS, Yokohama, Kanagawa230-0045, Japan.
¹¹ Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan; International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo (IMSUT), Minato-ku, Tokyo108-8639, Japan. Electronic address: [email protected].

PMID: 32711255
PMCID: PMC7387783
DOI: 10.1016/j.ebiom.2020.102913

Abstract

Background: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (T_FR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating T_FR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling T_FR cell differentiation remains unknown.

Methods: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5⁺Bcl-6⁺Foxp3⁺ T_FR (iT_FR) cell culture system and examined whether butyrate promotes the differentiation of iT_FR cells.

Findings: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting T_FR cell differentiation. Butyrate directly induced the differentiation of functional T_FR cells in vitro by enhancing histone acetylation in T_FR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the T_FR-cell marker genes. The adoptive transfer of the butyrate-treated iT_FR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis.

Interpretation: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance T_FR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk.

Funding: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).

Keywords: Autoimmunity; Butyrate; Follicular regulatory T cells; Intestinal microbiota; Rheumatoid arthritis.

PubMed Disclaimer

Conflict of interest statement

Declaration of Competing Interest The authors declare no competing financial interests. Correspondence should be addressed to K.H. ([email protected]).

Figures

**Fig. 1**
Commensal bacteria-derived butyrate ameliorates autoimmune arthritis (a) The faecal concentration of acetate, propionate, and butyrate in healthy control and new-onset untreated rheumatoid arthritis (NORA) subjects (n= 41, 31). (b-e) CIA incidence (b, d) and arthritis scores after booster immunization (c, e) in CIA mice fed the control HAMS, or HAMSB diet (n= 13–15; mean ± s.e.m. b, c), and CIA mice fed the control HAMS, HAMSA or HAMSP diet (n= 12; mean ± s.e.m. d, e). AUC was calculated in each mice for c, e. (f) Arthritis scores for SKG mice fed the control HAMS or HAMSB diet after laminarin injection (n= 11; means ± s.e.m.). AUC was calculated in each mice. (g–i) Micro-computed tomography (mCT) analysis of the calcaneus in ankle joint four weeks after booster immunization in CIA mice fed the control HAMS or HAMSB diet (n= 5). Representative mCT images (g). The eroded area is shown in Red. Scale bars: 1 mm. Eroded surface per bone surface (h,%) and eroded volume per bone volume (i,%). Results show one representative experiment of at least two experiments for mouse study. *P< 0.05, **P< 0.01, ***P< 0.001 (A, Mann-Whitney test; c, f, h, i, Welch's t-test or unpaired two-tailed Student's t-test; b, d, Log-rank test; e, one-way ANOVA followed by Tukey's post-hoc test.

**Fig. 2.**
HAMSB diet does not alter the T_H17 response in CIA mice (a–c) IFN-γ⁺ and IL-17A⁺ producing CD4⁺T cells in the hind knee joints of CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Cells were restimulated with PMA and ionomycin for 6 h before staining. Representative flow cytometry contour plots of intracellular IFN-γ and IL-17A staining within CD4⁺TCRβ⁺ gate (a), and the frequency (b) and total number (c) of IL-17A⁺, IFN-γ⁺, and IFN-γ⁺IL-17A⁺ cells (n= 4). (d–f) RORγt⁺ and IL-17A⁺ cells in the draining lymph nodes (DLNs) of CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Cells were restimulated with PMA and ionomycin for 6 h. Representative flow cytometry contour plots of intracellular RORγt and IL-17A staining within CD4⁺TCRβ⁺ gate (d), and the frequency (e) and total number (f) of RORγt⁺IL-17A⁺ and RORγt⁻IL-17A⁺ cells (n= 4). (g, h) IL-17A concentration in the hind knee joint extract (g, n= 6) and serum (h, n= 12) of CIA mice fed the control HAMS or HAMSB diet three weeks after the booster immunization. (i, j) The frequency in CD4⁺T cells (i) and total number (j) of Nrp-1⁻Foxp3⁺ pT_REG and Nrp-1⁺Foxp3⁺ tT_REG cells in the spleen (SP), DLNs and colonic patches (CoPs) of CIA mice fed control HAMS or HAMSB diet two weeks after the initial immunization (n= 10). Gating strategy is depicted in Figure S4. Results show one representative experiment of at least two experiments. (Welch's t-test or unpaired two-tailed Student's t-test).

**Fig. 3**
Butyrate suppresses the GC response and autoantibody production in CIA mice (a, b) CII-specific IgG responses in CIA mice fed the control HAMS or HAMSB diet. The number of CII-specific IgG antibody-forming cells (AFC) cells per 5 × 10⁵ DLN cells three weeks after booster immunization (a, each dot indicates data from 1 well, n= 36 from 6 mice). Statistical analysis was performed on the mean of each mouse. Serum levels of CII-specific IgG₁ and IgG_2a upon or three weeks after booster immunization (b, n= 5–10). (c–e) Colonic patches (CoPs) and solitary intestinal lymphoid tissues (SILTs) of CIA mice fed the control HAMS or HAMSB diet. Representative whole-mount B220 immunostaining (brown) of colon tissues (c), and the number of CoPs (d) and SILTs (e) in unimmunized DBA1/J mice and CIA mice one week after the initial immunization (n= 5 or 6).Dashed line, CoP, arrowheads, SILT. Scale bars, 5 mm. (f–h) GCB cells in CoPs from CIA mice fed the control HAMS or HAMSB diet. Representative flow cytometry contour plots of GL7 and intracellular Bcl-6 staining within CD19⁺ gate (f), and the frequency (g) and total number (h) of Bcl-6⁺GL7⁺ GCB cellstwo weeks after the initial immunization (n= 12). (i) GC area (mm²) of DLNs from CIA mice fed the control HAMS, or HAMSB diet Data were obtained two weeks after the initial immunization. Each dot indicates a single GC area of DLNs on cross-sections (n= 43, 30). Statistical analysis was performed on the mean of each mouse. (j–l) GCB cells in DLNs from CIA mice fed control HAMS or HAMSB diet. Representative flow cytometry contour plots of GL7 and intracellular Bcl-6 staining within CD19⁺ gate (j), and the frequency (k) and total number (l) of Bcl-6⁺GL7⁺ GCB cellstwo weeks after the initial immunization (n= 12). (m) Arthritis scores after antibody cocktail injection of collagen antibody-induced arthritis (CAIA) mice fed control HAMS or HAMSB diet (n= 3; means ± s.e.m.). AUC was calculated for each mice. Results show one representative experiment of at least two experiments. *P< 0.05, **P< 0.01, ***P< 0.001 (a, b, g–i, k, l, Welch's t-test or unpaired two-tailed Student's t-test; d, e, one-way ANOVA followed by Tukey's posthoc test; m, Log-rank test).

**Fig. 4**
Butyrate increased T_FR cells in CoPs (a, b) Follicular T cells in the DLNs and CoPs from CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Representative flow cytometry contour plots of CXCR5 and intracellular Bcl-6 staining (a), and the frequency (b) of Bcl-6⁺CXCR5⁺ follicular T cells within CD4⁺ TCRβ⁺ gate (n= 11, 10). (c–g) T_FR and T_FH cells in the DLNs and CoPs from CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Representative flow cytometry contour plots of CD25 and intracellular Foxp3 staining (c), and the frequency of CD25⁻Foxp3⁺ T_FR (d) and Foxp3⁻ T_FH (e) cells within Bcl-6⁺CXCR5⁺ follicular T cell gate and the total number of CD25⁻Foxp3⁺ T_FR (f) and Foxp3⁻ T_FH (g) cells (n= 11, 10). Gating strategy is depicted in Figure S4. (h) T_FR/T_FH ratio calculated using the total number of CD25⁻Foxp3⁺ T_FR and Foxp3⁻ T_FH cells in F and G. (i, j) B cell class switching to IgG₁ by T_FH cells. IgG⁻CD19⁺B cells sort-purified from the DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-B or HAMSB-B respectively) two weeks after the initial immunization were cultured alone with 100 µg/ml type II collagen (CII),and IgG⁻CD19⁺B cells sort-purified from the DLNs of CIA mice fed the control HAMS diet were co-cultured with CXCR5⁺ICOS⁺CD4⁺ T_FH cells sort-purified from DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-T_FH or HAMSB-T_FH respectively) in the presence or absence of CII. Representative flow cytometry contour plots of GL7 and intracellular IgG₁ staining six days after cultivation (i, n= 6), and the frequency of GL7⁺IgG1⁺B cells within CD19⁺ gate (j, n= 6). (k–n) Differentiation of T_FR and T_FH cells in CoPs of *Tcrb*^−/−*Tcrd*^−/− mice transferred with Foxp3-hCD2⁺iT_REG cells. Sort-purified CD45.1⁺Foxp3-hCD2⁺T cells cultured for five days under iT_REG conditions were injected intravenously with CD45.2⁺CD4⁺T cells into *Tcrb*^−/−*Tcrd*^−/− mice fed the control HAMS or HAMSB diet. Representative flow cytometry contour plots of hCD2 (Foxp3) and CD25 staining (k), and the frequency of Foxp3⁻, CD25⁺Foxp3⁺ and CD25⁻Foxp3⁺ cells (L) within CD45.1⁺CD4⁺ TCRβ⁺ gate (n= 7). Representative flow cytometry contour plots of CXCR5 and PD-1 staining among Foxp3⁻, CD25⁺Foxp3⁺ and CD25⁻Foxp3⁺ gate (m), and the frequency of CXCR5⁺PD-1⁺ cells (N) among Foxp3⁻ (T_FH cells), CD25⁺Foxp3⁺ (CD25⁺T_FR cells) and CD25⁻Foxp3⁺(CD25⁻T_FR cells) gates (n, n= 7). Results show one representative experiment of at least two experiments. *P< 0.05, **P< 0.01, ***P< 0.001 (b, d–h, j, l, n, Welch's t-test or unpaired two-tailed Student's t-test).

**Fig. 5.**
*In vitro* T_FR (iT_FR) cell differentiation (a) Schematic of iT_FR and iT_REG cell differentiation culture. (b) Relative mRNA expression of *Pdcd1, Cxcr5, Bcl6*, and *Tcf7* in sort-purified TCRβ⁺CD4⁺Foxp3-hCD2⁺ cells fro, iT_FR or iT_REG cell culture conditions (n= 5). Sort-purified naïve CD4⁺T cells from *Foxp3*^hCD2reporter mice were used for the culture. (c, d) Expression of CD25 and Foxp3 by cells from iT_FR or iT_REG cultures. Representative flow cytometry contour plots of CD25 and Foxp3-hCD2 staining (c), and the frequency of CD25⁺Foxp3⁺ and CD25⁻Foxp3⁺ cells (d) within CD4⁺ TCRβ⁺ gate (n= 5). (e, f) Expression of T_FR cell phenotypic markers by cells from iT_FR or iT_REG cultures. Representative flow cytometry histograms of FSC, PD-1, CXCR5, Bcl-6, and TCF-1 expression (e), and the gMFI of PD-1, CXCR5, Bcl-6, and TCF-1 (f) within CD25⁺Foxp3⁺ and CD25⁻Foxp3⁺ gate (n= 5). (g, h) Expression of Bcl-6-tdTomato reporter by cells from iT_FR or iT_REG cultures. Sort-purified naïve CD4⁺T cells from Bcl-6-tdTomato *Foxp3*^hCD2 double reporter mice or *Foxp3*^hCD2reporter mice (described as control) were used for the culture. Representative flow cytometry histograms of Bcl-6-tdTomato reporter and control expression (g), and Bcl-6-tdTomato gMFI (h) within CD25⁺Foxp3⁺ and CD25⁻Foxp3⁺gate (n= 5). (i–l) B cell class switching to IgG₁ and IgG_2a in suppression assays. IgG⁻CD43⁻CD19⁺ resting B cells, Foxp3-hCD2⁻CXCR5⁺Bcl-6-Eyfp⁺ T_FH cells, and CD25⁻Foxp3-hCD2⁺CXCR5⁺Bcl-6-Eyfp⁺ iT_FR cells sort-purified from Bcl-6-Eyfp *Foxp3*^hCD2 double reporter mice were used.The resting B cells from were co-cultured with T_FH cells alone, T_FH and T_FR cells, or T_FH and sort-purified CD25⁻Foxp3-hCD2⁺CXCR5⁺Bcl-6-Eyfp⁺ iT_FR cells under the exsitance of 5 μg ml⁻¹ anti-IgM and 2 μg ml⁻1 anti-CD3ε Abs. Representative flow cytometry histograms of Fixable Viability Stain 780 (FVS780) staining (i), and the frequency of FVS780⁻ live cells (j) within GL7⁺CD19⁺B cells (n= 3–5). Representative flow cytometry histograms of intracellular IgG₁ and IgG_2a staining (k), and the frequency of IgG₁⁺ and IgG_2a⁺ cells (l) within GL7⁺ CD19⁺B gate (n= 3–5). Results show one representative experiment of at least two experiments.***P< 0.001 (b, d, Welch's t-test or unpaired two-tailed Student's t-test; j, l, one-way ANOVA followed by Dunnett's post-hoc test; g, h, two-way ANOVA followed by Sidak's post-hoc test).

**Fig. 6**
Butyrate induces iT_FR cell differentiation (a-d) iT_FR cell differentiation with SCFAs treatment. Sort-purified naïve CD4⁺T cells from Bcl-6-tdTomato *Foxp3*^hCD2 double reporter mice were cultivated under iT_FR-cell culture conditions in the presence of sodium acetate (SA), sodium propionate (SP), or sodium butyrate (SB) at 10 μM or 100 μM. Representative flow cytometry contour plots of hCD2 (Foxp3) and CD25 staining (a), and the frequency of CD25⁺Foxp3⁺ (upper panel) and CD25⁻Foxp3⁺ (lower panel) cells within CD45⁺CD4⁺ TCRβ⁺ gate (b, n= 5). Representative flow cytometry contour plots of Bcl-6-tdTomato reporter signal and CXCR5 staining among CD25⁺Foxp3⁺ and CD25⁻Foxp3⁺ gate (c), and the frequency of Bcl-6-tdTomato⁺CXCR5⁺ cells among CD25⁺Foxp3⁺ (upper panel) and CD25⁻Foxp3⁺(lower panel) gates in (b) (d, n= 5). (e) gMFI of Bcl-6-tdTomato reporter in CXCR5⁺ and CXCR5⁻ populations among CD25⁺Foxp3⁺(upper panel) and CD25⁻Foxp3⁺(lower panel) gates of iT_FRculture in (c). CXCR5⁺and CXCR5⁻ populations are shown as areas equal to or higher than, or less than the dotted lines in (c), respectively (n= 5). (f, g) Expression of TCF-1 among CD25⁺Foxp3⁺(upper panel) and CD25⁻Foxp3⁺(lower panel) gates of iT_FRculture in (c). iT_FRculture was treated with 100 μM SA, SP or SB. Representative flow cytometry histograms of TCF-1 expression (f), and the gMFI of TCF-1 (g, n= 5). (h, i) iT_FR cell differentiation with pan-HDAC inhibitor treatment. Sort-purified naïve CD4⁺T cells from Bcl-6-tdTomato *Foxp3*^hCD2 double reporter mice were cultivated under iT_FR-cell culture conditions with the treatment of suberoylanilide hydroxamic acid (SAHA, 50 or 200 nM), trichostatin A (TSA, 2.5 or 10 nM), or vehicle control 0.01% dimethyl sulfoxide (DMSO). The frequency of Bcl-6-tdTomato⁺CXCR5⁺ cells (h) and the gMFI of TCF-1 (i) among CD25⁺Foxp3⁺CD4⁺ TCRβ⁺ (upper panel) and CD25⁻Foxp3⁺CD4⁺TCRβ⁺(lower panel) gates (n= 5). (j, k) Differentiation of T_FR cells in the inguinal lymph node (InLN) of mice treated with SAHA. C57BL/6 J mice were subcutaneously immunized with human insulin and intravenously injected with SAHA (20 mg/kg body weight) or vehicle control DMSO every day after immunization. The frequency of Bcl-6⁺CXCR5⁺ follicular T cells within CD4⁺ TCRβ⁺ gate (j, n= 6), and CD25⁺Foxp3⁺ T_FR cells within Bcl-6⁺CXCR5⁺ gate (k, n= 6) from InLN 10 days after immunization. (l) Accumulation of H3K27 acetylation (ac) at the promoter region of T_FRcell-related genes in Foxp3⁺ cells from iT_FR or iT_REG cultures. ChIP quantitative RT-PCR (qPCR) analysis of H3K27ac levels in the promoters of *Bcl6, Cxcr5,* and *Tcf7* in sort purified Foxp3⁺T cells cultured for the total three days under iT_FR or iT_REG-cell polarizing conditions (see Fig. 5a) with or without the treatment of 100 µM SB. (m) iT_FR cell differentiation using G-protein coupled receptor-deficient mice. Sort-purified naïve CD4⁺T cells from *Ffar3*^−/−, *Ffar2*^−/−, or *Hcar2*^−/−mice were cultivated under iT_FR-cell culture conditions with or without the treatment of 100 µM S. The frequency of Bcl-6⁺CXCR5⁺ cells within CD25⁻Foxp3⁺CD4⁺ TCRβ⁺gate (n= 5). Results show one representative experiment of at least two experiments.*P< 0.05, **P< 0.01, ***P< 0.001 (b, c, d, f-h, one-way ANOVA followed by Dunnett's post-hoc test; l-m Welch's t-test or unpaired two-tailed Student's t-test).

**Fig. 7**
iT_FR cells prevent autoimmune responses and CIA development (a, b) CII-specific IgG responses in CIA mice inoculated with Foxp3-hCD2⁺ cells cultured under iT_FR-cell conditions in the absence or presence of 100 µM SB. Sort-purified 5 × 10⁶ of Foxp3-hCD2⁺CD4⁺T cells were injected intravenously into DBA/1 J mice one week before the initial CII immunization.All mice were fed the control HAMS diet (n= 10, 11). The number of CII-specific IgG AFC cells per 1 × 10⁵ DLN cells (a, each dot indicates data from 1 well, n= 44, 32, statistical analysis was performed on the mean of each mouse.), and serum levels of CII-specific IgG₁ and IgG_2a three weeks after booster immunization (b, n= 5, 6). (c) Arthritis scores after booster immunization of CIA mice inoculated with Foxp3-hCD2⁺ cells from iT_FR cell culture (n= 10, 11). AUC was calculated in each mice. Results show one representative experiment of at least two experiments.**P< 0.01, ***P< 0.001 (a-c, Welch's t-test or unpaired two-tailed Student's t-test).

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[1] McInnes I.B., Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365:2205–2219. doi: 10.1056/NEJMra1004965. - DOI - PubMed

[2] McInnes I.B., Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365:2205–2219. doi: 10.1056/NEJMra1004965. - DOI - PubMed

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Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

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Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

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