MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism

doi:10.1016/j.immuni.2015.06.014

. 2015 Aug 18;43(2):289-303.

doi: 10.1016/j.immuni.2015.06.014. Epub 2015 Jul 28.

MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism

Sen Wang¹, Louis-Marie Charbonnier¹, Magali Noval Rivas¹, Peter Georgiev¹, Ning Li², Georg Gerber², Lynn Bry², Talal A Chatila³

Affiliations

¹ Division of Immunology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
² Center for Clinical and Translational Metagenomics, Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
³ Division of Immunology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: talal.chatila@childrens.harvard.edu.

PMID: 26231118
PMCID: PMC4545404
DOI: 10.1016/j.immuni.2015.06.014

MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism

Sen Wang et al. Immunity. 2015.

. 2015 Aug 18;43(2):289-303.

doi: 10.1016/j.immuni.2015.06.014. Epub 2015 Jul 28.

Authors

Sen Wang¹, Louis-Marie Charbonnier¹, Magali Noval Rivas¹, Peter Georgiev¹, Ning Li², Georg Gerber², Lynn Bry², Talal A Chatila³

Affiliations

¹ Division of Immunology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
² Center for Clinical and Translational Metagenomics, Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
³ Division of Immunology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: talal.chatila@childrens.harvard.edu.

PMID: 26231118
PMCID: PMC4545404
DOI: 10.1016/j.immuni.2015.06.014

Abstract

Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll-like receptors (TLRs). We report that Treg-cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper 17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer's patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA responses.

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Figures

**Figure 1. Treg cell-specific MyD88 deletion results in gut mucosa Treg cell deficiency**
A. Flow cytometric analysis of CD4⁺Foxp3⁺ Treg cells in the cLP of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. B. Frequencies and absolute numbers of Treg cells in different tissues of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. **C, D**. Flow cytometric analysis (C) and mean fluorescence intensity (MFI) **(D)** of CD25, CTLA-4, Helios, Nrp1, CD103 and Foxp3 cLP Treg cells of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. E. Flow cytometric analysis of Foxp3 expression in cLP CD45.2⁺CD4⁺ T cells in CD45.1⁺ WT mice that received naïve CD45.2⁺CD4⁺Foxp3⁻ T cells from OT-II⁺ *Foxp3^EGFPcre* or OT-II⁺ *Foxp3^EGFPcreMyd88*^Δ/Δ mice and were then fed OVA in the drinking water for 1 week. F. Frequencies (left panel) and absolute numbers (right panel) of donor CD45.2⁺CD4⁺EGFP⁺ iTreg in the cLP of recipient mice from panel (E). G. Absolute numbers of donor CD45.2⁺CD4⁺Foxp3^EGFP+ iTreg in the MLN of recipient mice. H. Methylation status of individual CpG motifs within the *Foxp3* CNS2 in cLP Treg cells of *Foxp3^EGFPcre* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. Individual CpG motifs are numbered with reference to the transcription initiation site of *Foxp3*. I. Global methylation status of Foxp3 CNS2 in splenic and cLP Treg cells shown in H. Results are representative of 4 independent experiments. N=3-8 mice/group. *p<0.05; **p<0.01; ***p<0.001 by Student’s unpaired two tailed t test and One-way ANOVA.

**Figure 2. Treg cell-specific MyD88 deletion results in colonic Th17 skewing**
**A, B**. Flow cytometric analysis (A) and enumerated cell frequencies and numbers (B) of cLP RORγ-t⁺CD4⁺Foxp3⁺Treg cells in *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. C. Real time PCR analysis of *Rorc* expression in cell-sorted cLP CD4⁺Foxp3⁺ Treg cells. **D-I**. Analysis of GATA3 expression and *Gata3* mRNA (**D-F**) and IL-10 expression and *Il10* mRNA (**G-I**) in cLP CD4⁺Foxp3⁺ Treg cells of the respective mouse strain, performed as in **A-C**. **J, K.** Expression of IL-17 and IFN-γ in cLP CD4⁺Foxp3⁻ T cells of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. Flow cytometric analysis (J), frequencies and absolute numbers (K) of cytokine expressing cells. L, M. Flow cytometric analysis (L), and frequencies and absolute numbers (M) of cLP CD4⁺Foxp3⁻RORγ-t⁺ T cells of the respective mouse strain. N. Frequencies (left panel) and total cell number (right panel) of cLP CD4⁺Foxp3⁻T-bet⁺ of *Foxp3^EGFPcreMyd88*^Δ/Δ mice compared with *Myd88^fl/fl* control mice. Results are representative of 4 independent experiments. N=4-7 mice/group. *p<0.05; **p<0.01; ***p<0.001 by Student’s unpaired two tailed t test.

**Figure 3. Treg cell-specific MyD88 deficiency aggravates the severity of experimental colitis**
A. Survival curves of the following mouse groups treated with 5% DSS for 5 days then followed thereafter: 1) *Myd88^fl/fl*, 2) *Foxp3^EGFPcreMyd88*^Δ/Δ and 3, 4) *Foxp3^EGFPcreMyd88*^Δ/Δ mice that were given cell-sorted CD4⁺EGFP⁺ splenic Treg cells from either *Foxp3^EGFPcre* (MyD88-sufficient) or *Foxp3^EGFPcreMyd88*^Δ/Δ (MyD88-defcient) mice. B. Body weight loss following treatment of mouse groups as in panel A with 3.5% DSS for 5 days. C. Colon length and histological scores of the respective mouse groups in (B) at day 9 post DSS treatment. **D-I.** Flow cytometric analysis and frequencies of total and IL-10⁺ CD4⁺Foxp3⁺ Treg cells (D-F) and IFN-γ and IL-17 secreting CD4⁺Foxp3⁻ Tconv cells (**G-I**) isolated from the cLP of the respective mouse groups in (**B, C**) at day 9 post DSS treatment. **J-M.** Groups of *Foxp3^EGFPcre* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice were subjected to DSS-induced colitis and treated with either isotype control or neutralizing anti-IL-17A mAbs at days 0, 2, and 4 of DSS treatment. J. Colon histology (Hematoxylin and eosin staining; 40× magnification). Body weight loss (K), colon length and clinical score (L) of the respective mouse groups. M. Frequencies and absolute numbers of IL-17 and IFN-γ secreting cLP CD4⁺Foxp3⁻ Tconv cells of the respective mouse groups. Results are representative of 3 independent experiments. N =9-10 mice/group for panel A; **p<0.01 by log rank test. N= 4-7 mice/group for panels B-M; *p<0.05; **p<0.01; ***p<0.001 by One-way ANOVA.

**Figure 4. Treg cell-specific MyD88 deficiency impairs PP Tfr and Tfh cell differentiation and intestinal IgA production**
**A-D.** Flow cytometric analysis, frequencies and absolute numbers of Tfh cells (CD4⁺CXCR5⁺PD-1^high) (A), CXCR5⁺IL-21⁺ cells within the Tfh populations (B), Tfr cells (CD4⁺Foxp3⁺CXCR5⁺PD-1^high) (C) and IgA⁺B220⁺ B cells (D) in PP of *Foxp3^EGFPcreMyd88*^Δ/Δ and *Myd88^fl/fl* mice. E. Serum and intestinal lavage fluid concentrations of IgA (I) and IgG, IgG1 and IgG2a (J) in *Foxp3^EGFPcreMyd88*^Δ/Δ and *Myd88^fl/fl* mice. **F-H.** Flow cytometric analysis, and frequencies and absolute numbers of Tfh (F), Tfr (G) and IgA⁺B220⁺ (H) cells isolated from the PP of *Tcra^−/−* mice 2 weeks following the transfer of CD4⁺EGFP⁺Treg cells isolated from either *Foxp3^EGFPcre* or *Foxp3^EGFPcreMyd88*^Δ/Δ. I. Luminal concentration of IgA and IgG1 in the mouse groups from panel (H). Results are representative of 3 independent experiments. N=3-5 mice/group. *p<0.05; **p<0.01; by Student’s unpaired two tailed t test.

**Figure 5. T_R-cell specific MyD88 deficiency promotes dysbiosis and and bacterial translocation to deep tissues**
A. Flow cytometric analysis and frequencies of IgA- and IgG1-coated bacteria in the fecal pellets of *Rag1^−/−*, *Foxp3^EGFPcreMyd88*^Δ/Δ and *MyD88^fl/fl* mice. B. OVA-specific luminal IgA and IgG1 concentrations in mice (panel A) gavaged with OVA-DH5α bacteria. C. Weighted Unifrac plot of ileal mucosa-associated microbial communities, visualized with principal coordinate analysis (PCoA), in *Foxp3^EGFPcreMyd88*^Δ/Δ versus *Myd88^fl/fl* mice (p-value = 0.001 by AMOVA). D. Relative abundance of different microbial phyla in ileal mucosa-associated microbiota in *Foxp3^EGFPcreMyd88*^Δ/Δ and *Myd88^fl/fl* mice (*false discovery rate<0.05)**. E.** Relative abundance of SFB taxa in illeal mucosa-associated microbiota in *Foxp3^EGFPcreMyd88*^Δ/Δ and *Myd88^fl/fl* mice. F. Real time PCR analysis of SFB 16S rRNA in IgA⁺ and IgA⁻ fecal bacterial fractions of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. G. Stool (left panel) and liver (right panel) OVA-DH5α bacterial load [expressed as log colony forming units (lg CFU)/g] in *Foxp3^EGFPcreMyd88*^Δ/Δ and *Myd88^fl/fl* mice treated with the OVA-DH5α bacteria by oral gavage. H. Loads of total bacteria, *Lactobacilli* and *Pasteurella* (all expressed as CFU/g tissue) in the lung and liver of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice. Results are representative of 3 independent experiments. Results representative of 3 independent experiments. N=5 mice/group. *p<0.05; **p<0.01; by Student’s unpaired two tailed t test.

**Figure 6. Adoptive transfer of IgA⁺ PP B cells restores intestinal IgA production and inhibits Th17 skewing in *Foxp3^EGFPcreMyd88*^Δ/Δ recipients**
A. Flow cytometric analysis and frequencies of CD45.1⁺B220⁺IgA⁺ and CD45.1⁺B220⁺IgG⁺ B cells found in the PP of *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ recipients 2 weeks following their adoptive transfer. B. Serum and intestinal IgA (upper panels) and IgG1 (lower panels) concentrations in sham treated *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ mice and in *Foxp3^EGFPcreMyd88*^Δ/Δ mice that received CD45.1⁺B220⁺IgA⁺ and CD45.1⁺B220⁺IgG⁺ B cells. **C, D.** Flow cytometric analysis (C), frequencies and absolute numbers (D) of IL-17 and IFN-γ -expressing cLP CD4⁺Foxp3⁻ Tconv cells in the mouse groups described in (B). **E, F.** Absolute numbers of Treg cells in the cLP (E) and Tfr cells in the PP (F) in mouse groups outlined in (B). G. Real time PCR analysis of SFB 16S rRNA in the fecal pellets of the respective mouse groups. H. Translocation of gavaged DH5α bacteria into livers of mice of the respective mouse groups. Results representative of 3 independent experiments. N=3-5 mice/group. *p<0.05; **p<0.01; ***p<0.001 by Student’s unpaired two tailed t test and one-way ANOVA.

**Figure 7. Treg cell-specific MyD88 signaling activates Stat3 and its deficiency is phenocopied by T_R-cell specific Stat3 deficiency**
**A-I**. TLR1-2-mediated activation of p65-NF-κB, Stat3 and Stat5 in *Myd88^fl/fl* and *Foxp3^EGFPcreMyd88*^Δ/Δ cLP CD4⁺Foxp3⁺Treg cells. **A, D, G**. Flow cytometric analysis of p-p65-NF-κB (A), pStat3 (D) and pStat5 (G) in cLP CD4⁺Foxp3⁺Treg cells treated with either PBS or the TLR1-2 ligand (TLR1-2-L) Pam3SCK4. (**B, E, H**) MFI of the respective phosphoprotein. (**C, F, I)**. Chromatin immunoprecipitation (ChIP) assay of NF-κB p65, Stat3 and Stat5 bound to the Foxp3-CNS2 in the respective cLP CD4⁺Foxp3⁺Treg cells treated with either PBS, TLR1-2-L (for p65-NF-kB and Stat3 ChIP) or IL-2 (for Stat5 ChIP). **J-O**. WT and *Foxp3^EGFPcreStat3*^Δ/Δ mice were either untreated or received adoptively transferred CD45.1⁺B220⁺IgA⁺ or CD45.1⁺B220⁺IgG⁺ PP B cell derived from CD45.1⁺ WT donor mice. Analysis was carried out 2 weeks following the B cell transfer. J. Frequencies of total B220⁺IgA⁺ in the PP. K. Intestinal lavage fluid IgA and IgG concentrations. **L, M**. Frequencies of PP Tfr and cLP Treg cells, respectively. N. Frequencies of CD4⁺IL-17⁺ and CD4⁺IFN-γ⁺ T_conv cells. O. Real time PCR analysis of SFB 16S rDNA in the stool. Similar results were obtained in 2 independent experiments. N= 4-7 /group *P<0.05; **P<0.01, ***P<0.001 by One-way ANOVA.

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References

1. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 2011;331:337–341. - PMC - PubMed
1. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–1920. - PubMed
1. Caramalho I, Lopes-Carvalho T, Ostler D, Zelenay S, Haury M, Demengeot J. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med. 2003;197:403–411. - PMC - PubMed
1. Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, Rudensky AY. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science. 2009;326:986–991. - PMC - PubMed
1. Chaudhry A, Samstein RM, Treuting P, Liang Y, Pils MC, Heinrich JM, Jack RS, Wunderlich FT, Bruning JC, Muller W, Rudensky AY. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity. 2011;34:566–578. - PMC - PubMed

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[1] Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 2011;331:337–341. - PMC - PubMed

[2] Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 2011;331:337–341. - PMC - PubMed

[3] Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–1920. - PubMed

[4] Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–1920. - PubMed

[5] Caramalho I, Lopes-Carvalho T, Ostler D, Zelenay S, Haury M, Demengeot J. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med. 2003;197:403–411. - PMC - PubMed

[6] Caramalho I, Lopes-Carvalho T, Ostler D, Zelenay S, Haury M, Demengeot J. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med. 2003;197:403–411. - PMC - PubMed

[7] Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, Rudensky AY. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science. 2009;326:986–991. - PMC - PubMed

[8] Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, Rudensky AY. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science. 2009;326:986–991. - PMC - PubMed

[9] Chaudhry A, Samstein RM, Treuting P, Liang Y, Pils MC, Heinrich JM, Jack RS, Wunderlich FT, Bruning JC, Muller W, Rudensky AY. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity. 2011;34:566–578. - PMC - PubMed

[10] Chaudhry A, Samstein RM, Treuting P, Liang Y, Pils MC, Heinrich JM, Jack RS, Wunderlich FT, Bruning JC, Muller W, Rudensky AY. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity. 2011;34:566–578. - PMC - PubMed

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MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism

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MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism

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