Foxp3+ regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.
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IL-37 isoform D acts as an inhibitor of soluble ST2 to boost type 2 immune homeostasis in white adipose tissue
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Josefowicz, S.Z., Lu, L.F. & Rudensky, A.Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).
Ohkura, N., Kitagawa, Y. & Sakaguchi, S. Development and maintenance of regulatory T cells. Immunity 38, 414–423 (2013).
Feuerer, M., Hill, J.A., Mathis, D. & Benoist, C. Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nat. Immunol. 10, 689–695 (2009).
Campbell, D.J. & Koch, M.A. Phenotypical and functional specialization of FOXP3(+) regulatory T cells. Nat. Rev. Immunol. 11, 119–130 (2011).
Cretney, E., Kallies, A. & Nutt, S.L. Differentiation and function of Foxp3(+) effector regulatory T cells. Trends Immunol. 34, 74–80 (2013).
Burzyn, D., Benoist, C. & Mathis, D. Regulatory T cells in nonlymphoid tissues. Nat. Immunol. 14, 1007–1013 (2013).
Chaudhry, A. & Rudensky, A.Y. Control of inflammation by integration of environmental cues by regulatory T cells. J. Clin. Invest. 123, 939–944 (2013).
Liston, A. & Gray, D.H. Homeostatic control of regulatory T cell diversity. Nat. Rev. Immunol. 14, 154–165 (2014).
Koch, M.A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat. Immunol. 10, 595–602 (2009).
Linterman, M.A. et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 17, 975–982 (2011).
Feuerer, M. et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat. Med. 15, 930–939 (2009).
Feuerer, M. et al. Genomic definition of multiple ex vivo regulatory T cell subphenotypes. Proc. Natl. Acad. Sci. USA 107, 5919–5924 (2010).
Cipolletta, D. et al. PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature 486, 549–553 (2012).
Fontenot, J.D., Rasmussen, J.P., Gavin, M.A. & Rudensky, A.Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6, 1142–1151 (2005).
Pierson, W. et al. Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3(+) regulatory T cells. Nat. Immunol. 14, 959–965 (2013).
Smigiel, K.S. et al. CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets. J. Exp. Med. 211, 121–136 (2014).
Hall, A.O. et al. The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37, 511–523 (2012).
Gratz, I.K. & Campbell, D.J. Organ-specific and memory Treg cells: specificity, development, function, and maintenance. Front. Immunol. 5, 333 (2014).
Cretney, E. et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat. Immunol. 12, 304–311 (2011).
Weiss, J.M. et al. Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells. J. Exp. Med. 209, 1723–1742 (2012).
Schmitz, J. et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor–related protein ST2 and induces T helper type 2–associated cytokines. Immunity 23, 479–490 (2005).
Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463, 540–544 (2010).
Molofsky, A.B. et al. Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J. Exp. Med. 210, 535–549 (2013).
Licona-Limon, P., Kim, L.K., Palm, N.W. & Flavell, R.A. TH2, allergy and group 2 innate lymphoid cells. Nat. Immunol. 14, 536–542 (2013).
Bonilla, W.V. et al. The alarmin interleukin-33 drives protective antiviral CD8+ T cell responses. Science 335, 984–989 (2012).
Miller, A.M. et al. Interleukin-33 induces protective effects in adipose tissue inflammation during obesity in mice. Circ. Res. 107, 650–658 (2010).
Kakkar, R. & Lee, R.T. The IL-33/ST2 pathway: therapeutic target and novel biomarker. Nat. Rev. Drug Discov. 7, 827–840 (2008).
Boyman, O., Kovar, M., Rubinstein, M.P., Surh, C.D. & Sprent, J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311, 1924–1927 (2006).
Turnquist, H.R. et al. IL-33 expands suppressive CD11b+ Gr-1int and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival. J. Immunol. 187, 4598–4610 (2011).
Martin, M.U. Special aspects of interleukin-33 and the IL-33 receptor complex. Semin. Immunol. 25, 449–457 (2013).
Murphy, T.L., Tussiwand, R. & Murphy, K.M. Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks. Nat. Rev. Immunol. 13, 499–509 (2013).
Glasmacher, E. et al. A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes. Science 338, 975–980 (2012).
Ciofani, M. et al. A validated regulatory network for Th17 cell specification. Cell 151, 289–303 (2012).
Li, P. et al. BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature 490, 543–546 (2012).
Man, K. et al. The transcription factor IRF4 is essential for TCR affinity-mediated metabolic programming and clonal expansion of T cells. Nat. Immunol. 14, 1155–1165 (2013).
Grusdat, M. et al. IRF4 and BATF are critical for CD8 T-cell function following infection with LCMV. Cell Death Differ. 7, 1050–1060 (2014).
Brint, E.K. et al. Characterization of signaling pathways activated by the interleukin 1 (IL-1) receptor homologue T1/ST2. A role for Jun N-terminal kinase in IL-4 induction. J. Biol. Chem. 277, 49205–49211 (2002).
Zheng, Y. et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control TH2 responses. Nature 458, 351–356 (2009).
Kluge, R., Scherneck, S., Schurmann, A. & Joost, H.G. Pathophysiology and genetics of obesity and diabetes in the New Zealand obese mouse: a model of the human metabolic syndrome. Methods Mol. Biol. 933, 59–73 (2012).
Wood, I.S., Wang, B. & Trayhurn, P. IL-33, a recently identified interleukin-1 gene family member, is expressed in human adipocytes. Biochem. Biophys. Res. Commun. 384, 105–109 (2009).
Zeyda, M. et al. Severe obesity increases adipose tissue expression of interleukin-33 and its receptor ST2, both predominantly detectable in endothelial cells of human adipose tissue. Int. J. Obes. 37, 658–665 (2013).
Boraschi, D. & Tagliabue, A. The interleukin-1 receptor family. Semin. Immunol. 25, 394–407 (2013).
Makrinioti, H., Toussaint, M., Jackson, D.J., Walton, R.P. & Johnston, S.L. Role of interleukin 33 in respiratory allergy and asthma. Lancet. Respir. Med. 2, 226–237 (2014).
Han, J.M. & Levings, M.K. Immune regulation in obesity-associated adipose inflammation. J. Immunol. 191, 527–532 (2013).
Williams, L.M. & Rudensky, A.Y. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat. Immunol. 8, 277–284 (2007).
Sakaguchi, S., Vignali, D.A., Rudensky, A.Y., Niec, R.E. & Waldmann, H. The plasticity and stability of regulatory T cells. Nat. Rev. Immunol. 13, 461–467 (2013).
Wang, Y., Su, M.A. & Wan, Y.Y. An essential role of the transcription factor GATA-3 for the function of regulatory T cells. Immunity 35, 337–348 (2011).
Wohlfert, E.A. et al. GATA3 controls Foxp3+ regulatory T cell fate during inflammation in mice. J. Clin. Invest. 121, 4503–4515 (2011).
Guo, L. et al. IL-1 family members and STAT activators induce cytokine production by Th2, Th17, and Th1 cells. Proc. Natl. Acad. Sci. USA 106, 13463–13468 (2009).
Kwon, H. et al. Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors. Immunity 31, 941–952 (2009).
Oboki, K. et al. IL-33 is a crucial amplifier of innate rather than acquired immunity. Proc. Natl. Acad. Sci. USA 107, 18581–18586 (2010).
Adachi, O. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, 143–150 (1998).
Jacob, J. & Baltimore, D. Modelling T-cell memory by genetic marking of memory T cells in vivo. Nature 399, 593–597 (1999).
Schraml, B.U. et al. The AP-1 transcription factor Batf controls TH17 differentiation. Nature 460, 405–409 (2009).
Kamanaka, M. et al. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity 25, 941–952 (2006).
Wan, Y.Y. & Flavell, R.A. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc. Natl. Acad. Sci. USA 102, 5126–5131 (2005).
Mittrucker, H.W. et al. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275, 540–543 (1997).
Klein, U. et al. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. Nat. Immunol. 7, 773–782 (2006).
Hoshino, K. et al. The absence of interleukin 1 receptor-related T1/ST2 does not affect T helper cell type 2 development and its effector function. J. Exp. Med. 190, 1541–1548 (1999).
Kallies, A. et al. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J. Exp. Med. 200, 967–977 (2004).
Kallies, A. et al. Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance. Nat. Immunol. 7, 466–474 (2006).
Kawamoto, S. et al. The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut. Science 336, 485–489 (2012).
Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5 (2004).
Lee, H.Y. et al. Blockade of IL-33/ST2 ameliorates airway inflammation in a murine model of allergic asthma. Exp. Lung Res. 40, 66–76 (2014).
We thank P. O'Brien, M. Mochizuki and N. Takeno for assistance with tissue collection, S. Wada for animal care, M. Febbraio and A. Lew for critical advice, E. Cretney for mice and E. Bandala-Sanchez, V. Bryant and J. Brady for reagents. We are grateful to K. Nakanishi (Hyogo College of Medicine), T. Mak (The Campbell Family Institute for Breast Cancer Research), and U. Klein (Columbia University) for mice. Supported by the National Health and Medical Research Council of Australia (A.K., S.L.N. and G.K.S.), the Sylvia and Charles Viertel Foundation (A.K.), the Australian Research Council (A.K. and S.L.N.), the Diabetes Australia Research Trust (J.M.W.), PRESTO from the Japan Science and Technology Agency (K.M.), and a Grant-in Aid for Scientific Research (B) (26293110 to K.M.) and a Grant-in-Aid for Scientific Research (S) (22229004 to S. Koyasu) from the Japan Society for the Promotion of Science. W.L., P.L. and W.J.L. are supported by the Division of Intramural Research, National Heart, Blood, and Lung Institute, US National Institutes of Health. This study was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support scheme.
S.K. is a consultant for Medical and Biological Laboratories (MBL).
Integrated supplementary information
(a) Gating strategy used to purify Blimp1- cTreg cells (blue gate) and Blimp1+ eTreg cell (red gate) from pooled spleen and lymph nodes (LNs) of Blimp1GFP mice. Representative of 6 experiments. (b) Heat map showing top 100 differentially expressed genes between cTreg cells and eTreg cells determined using likelihood ratio test. (c) RNAseq tracks showing the expression of Foxp3 in cTreg and eTreg cells. (d) Heat maps showing expression of transcriptional regulators (left) and genes related to migration and adhesion (right) that are differentially expressed between cTreg cells and eTreg cells. RNAseq for the indicated Treg cell populations performed in triplicate.
(a) Expression of ST2 against other surface molecules Ly6C, Ccr2, KLRG1, PD-1, CD69 and Tigit on Treg cells from spleen and VAT. Flow cytometric plots displaying CD4+Foxp3+ cells from a 35-week-old wild-type mouse, representative of 7 individual mice from two experiments.
Supplementary Figure 3 IL-33 is specifically required for VAT-Treg cells but dispensable for other Treg cell populations.
(a-c) Treg proportions and phenotype in selected lymphoid (a-b) and non-lymphoid (c) organs of wild-type (WT) and Il1rl1–/– mice as indicated. Values are mean ± S.D. of 9 individual mice from 3 experiments. LPL - Lamina propria lymphocytes of the small intestine. (d) Treg cell proportions in WT and Il33–/– mice and expression of KLRG1 in WT and Il33–/– Treg cells. Values are mean ± S.D. of 5 individual mice from two experiments. Numbers indicate percentages of cells. (e) Weight of VAT from 35-week old Il1rl1–/–, Il33–/– and WT mice. Values are mean ± S.D. from 8 individual mice from 3 experiments. (f-g) Glucose tolerance tests for Il1rl1–/– (f), Il33–/– (g) and corresponding WT control mice. The graphs are representative of at least two independent experiments with 3-5 mice per experiment. Two-way ANOVA test (P<0.0001), error bars denote S.E.M. (h) HOMA-IR calculated for Il33–/– and WT mice. (i) Flow cytometric analysis of adipose tissue from Il33–/– and WT mice. Graphs show VAT macrophages (TCRβ-, CD11b+, F4/80+ and CD11c+), pro-inflammatory monocytes (TCRβ-, CD11b+ Ly6C+) and CD8+ T cells. Panels representative of more than 5 mice analyzed in two independent experiments. (j) Serum leptin levels in Il33–/– mice. Values are mean ± S.D. *P<0.04; NS – not significant (unpaired, two tailed t-test).
(a-b) In vitro proliferation of VAT-Treg cells. Equal number of purified VAT lymphocytes from wild-type (WT) mice were CTV labeled and cultured for 3.5 days with (a) or without (b) plate bound CD3 and soluble CD28 antibodies, cytokines, and with or without IL-2 blocking antibodies as indicated. Bar graph shows relative numbers of Treg cells at the end of the culture. Figure representative of three experiments. (c) Relative Il33 mRNA expression in the VAT of young (8 weeks) versus old (35 weeks) mice. (d) IL-33 protein expression analyzed by immunoblotting of adipose tissue from young and old mice that were on a normal diet. Il33–/– mice were used as specificity control. Actin was used as loading control. Representative of 5 experiments. (e-f) ST2 expression on VAT-Treg cells isolated from WT mice of different ages as indicated. (e) and correlation of age and VAT-Treg prevalence (f). One way ANOVA for both panels, P<0.0001. (g) Frequency of Foxp3+ cells of CD4+ T cells in selected lymphoid and non-lymphoid organs from PBS, IL-33 and IL-2/anti-IL-2 Ab complex (IL-2c) treated mice. LPL - Lamina propria lymphocytes of the small intestine. (h) ST2 expression on KLRG1+ and KLRG1- Treg cells. (i) Flow cytometric analysis of splenic Foxp3+ cells showing expression of KLRG1 and ST2 in PBS and IL-33 treated mice (left). Graph showing proportion of KLRG1+ cells ± S.D. of total Treg cells in the spleen in control and IL-33 treated mice (right). (j) Proportion of Foxp3+ cells within CD4+ T cells in the VAT at different time points post IL-33 injection. One way ANOVA, P=0.0047. Symbols indicate data points for individual mice, values are mean ± S.D. Values in (a-c) are means ± S.D. from 3 experiments. Values in (e-g) are means ± S.D. from 5 individual mice from 2 experiments. *P=0.017; **P<0.008; ***P=0.0001; ****P<0.0001 (unpaired, two tailed t-test).
(a-b) Proportion of Treg cells in selected lymphoid (a) and non-lymphoid (b) organs of wild-type (WT) and Myd88–/– mice. LPL - lamina propria lymphocytes of the small intestine. (c) Flow cytometric analysis of Treg cells from the lymph nodes (LN) of wild-type and Myd88–/– mice assessed for eTreg cell markers ICOS and KLRG1 (left), frequency of KLRG1+ cells of lymph node Treg cells from WT and Myd88–/–mice (right). Numbers indicate percentages of cells. (d) VAT weight from WT and Myd88–/– mice. (e) Treg cells enriched from spleens of wild-type (WT) mice and cultured in the presence of plate bound CD3 and soluble CD28 antibodies, and cytokines for 3 days. Expression of ST2 and Foxp3 is shown in the flow cytometric plots. (f) Treg cells enriched from spleen of WT and Myd88–/– mice, CTV labeled and cultured as in (e). Expression of ST2 (dot plots, left) and proliferation measured by CTV dilution. Values in (a-d) are means ± S.D. from 8-9 mice. **P=0.003; NS – Not significant (unpaired, two tailed t-test).
(a-b) Proportion of Treg cells in the spleen and VAT of wild-type (WT) mice compared to Batf–/– (b) and Irf4–/– (b) mice. Values are means ± S.D. from 5-7 mice per group. (c-d) VAT mass (c) and body weight (d) of WT, Irf4–/– and Batf–/– mice. Values are the means from each 6-8 mice per group (one way ANOVA). (e) Bar graph showing proportions of WT and knock-out Foxp3+ cells as indicated from the spleens and VAT of Ly5.1 (WT) / Batf–/– (left) and Ly5.1 (WT) / Irf4–/– peripheral chimeric mice. (f) Flow cytometric analysis of ST2 expression on Treg cells from the VAT of mice of the indicated genotype. (g-h) MACS enriched CD4+CD25+ cells from WT (Ly5.1), Batf–/– (Ly5.2) and Irf4–/– (Ly5.2) mice as indicated were mixed as indicated, CTV labeled and cultured in conditions that induce ST2. Flow cytometric analysis of total Foxp3+ cells. Numbers indicate percentages of cells. Bar graphs show the proportion of Foxp3+ cells of the indicated genotype that express ST2. Histograms (gated on Foxp3+ cells) show CTV dilution profiles. Values are mean ± S.D. from 5 male 30-week-old mice per group. (i) RNAseq track showing expression of GzmB by cTreg cells and eTreg cells. (j) Weight of VAT from Irf4fl/flGzmBCre+ and control mice. Values are mean from 4 mice per group. *P<0.01; **P<0.002; ***P=0.0001; ****P<0.0001 (unpaired, two tailed t-test)..
Supplementary Figure 7 IL-33 administration can rescue VAT-Treg defects in genetically obese and HFD mice.
(a) Percentages of VAT and spleen Treg cells within the CD4+ compartments of C57BL/6 and NZO mice. Values are mean ± S.D. from 5 mice of each genotype. (b) Intraperitoneal glucose tolerance test (GTT) for NZO mice treated with PBS and IL-33. (c) Area under curve (AUC) for GTT performed on HFD and NZO mice as indicated. Values are mean from 4 and 5 mice per group. (d) Proportion of CD8+ T cells and VAT macrophages in the VAT of NZO mice treated with PBS or IL-33 as indicated. (e) Representative flow cytometric analysis of HFD mice. Plots show VAT macrophages (TCRβ-, CD11b+, F4/80+ and CD11c+) and pro-inflammatory monocytes (TCRβ-, CD11b+ Ly6C+). Numbers in boxes indicate percentages of cells. (f) Expression of Ccl2 (Mcp1), Ccl3 (Mip1α), Ccl5 (RANTES) and Il1β in the VAT of HFD and NZO mice treated with PBS or IL-33 analyzed by qPCR. Values are means ± S.D. For the GTT experiments in (b) a two way ANOVA test was performed (P<0.0001) and error bars denote S.E.M. P values for other graphs *P<0.05; **P=0.002; NS – not significant (unpaired, two tailed t-test).
Supplementary Figure 8 IL-33 treatment increases Treg cell numbers and improves metabolic parameters in NZO and HFD mice.
(a) Weight of VAT isolated from NZO and HFD mice treated with PBS or IL-33. Values are mean from 4 and 5 mice per group. (b) Hematoxylin and eosin staining of VAT sections. Numbers of adipocytes per field and adipocyte sizes from PBS and IL-33 treated NZO (upper panels) and HFD mice (lower panels) as indicated. Values are means ± S.E.M. of three sections each from 4-5 mice analyzed. (c) HOMA-IR calculated from PBS or IL-33 treated NZO and HFD mice as indicated. (d) Immunoblot showing Akt phosphorylation in VAT of PBS and IL-33 treated NZO or HFD mice after intravenous insulin injection. Representative of three experimets. (e) Analysis of ST2 expression on human Treg cells from peripheral blood mononuclear cells or omental fat as indicated; representative of three samples. * P<0.01 (a); ***P=0.0001 (b); ****P<0.0001 (b).
Supplementary Figures 1–8, and Supplementary Tables 4 and 5 (PDF 5066 kb)
Top 200 DE genes_cTregs Vs eTregs (CSV 13 kb)
DE genes encoding transcription factors_cTregs Vs eTregs (CSV 2 kb)
DE genes involved in migration_cTregs Vs eTregs (CSV 1 kb)
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Vasanthakumar, A., Moro, K., Xin, A. et al. The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue–resident regulatory T cells. Nat Immunol 16, 276–285 (2015). https://doi.org/10.1038/ni.3085
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