Abstract
Adipose tissue is an energy store and a dynamic endocrine organ1,2. In particular, visceral adipose tissue (VAT) is critical for the regulation of systemic metabolism3,4. Impaired VAT function—for example, in obesity—is associated with insulin resistance and type 2 diabetes5,6. Regulatory T (Treg) cells that express the transcription factor FOXP3 are critical for limiting immune responses and suppressing tissue inflammation, including in the VAT7,8,9. Here we uncover pronounced sexual dimorphism in Treg cells in the VAT. Male VAT was enriched for Treg cells compared with female VAT, and Treg cells from male VAT were markedly different from their female counterparts in phenotype, transcriptional landscape and chromatin accessibility. Heightened inflammation in the male VAT facilitated the recruitment of Treg cells via the CCL2–CCR2 axis. Androgen regulated the differentiation of a unique IL-33-producing stromal cell population specific to the male VAT, which paralleled the local expansion of Treg cells. Sex hormones also regulated VAT inflammation, which shaped the transcriptional landscape of VAT-resident Treg cells in a BLIMP1 transcription factor-dependent manner. Overall, we find that sex-specific differences in Treg cells from VAT are determined by the tissue niche in a sex-hormone-dependent manner to limit adipose tissue inflammation.
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Change history
05 March 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-2251-7
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Acknowledgements
This work was funded by the National Health and Medical Research Council (NHMRC, project grants 1106378 and 1149062 and fellowship 1139607 to A.K.), the Sylvia and Charles Viertel Foundation (fellowship to A.K.), the Diabetes Australia (grant Y18G-VASA to A.V.) and grants from the US NIH (R01DK092541) and JPB Foundation (to D.M.). W.S. is funded by a Walter and Eliza Hall Institute Centenary Fellowship funded by a donation from CSL. M.A.F. is a senior Principal Research Fellow of the NHMRC. P.A.B. was funded by an NBCF Career Development Fellowship. R.G.S. is an American Diabetes Association Postdoctoral fellow (1-17-PMF-005). J.D.Z. and R.A.D. are supported by funding from The Sir Edward Dunlop Medical Research Foundation, The Austin Health Research Foundation and a Les and Eva Erdi Research Grant. R.A.D. was funded by a fellowship from the Australian and New Zealand Bone and Mineral Society. We thank T. Korn for help with experiments, and G. Risbridger, K. S. Korach, M. Ernst, J. Silke and W. C. Boon for mice.
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Contributions
A.V. and A.K. designed the experiments, interpreted the results and wrote the paper. A.V. performed most of the experiments. D.C., Y.L. and W.S. analysed the sequencing data. J.B. and P.A.B. performed stromal cell analyses. R.G. and T.S. contributed to RNA-seq and ATAC-seq experiments. C.L., R.G.S. and D.M. performed adoptive transfer experiments using VAT TCR Tg mice as well as intracellular staining and flowcytometric analyses of IL-33 protein expression. S.L. and S.V.T. performed flow cytometry analyses. Y.Z. contributed to experiments using CCR2-deficient mice. S.L.N., J.D.Z., R.A.D. and P.A.B. contributed mice and scientific discussion. K.B. performed ICI 182-780 treatment and contributed to the hormone supplementation experiments. E.C. and N.T. performed the COX-inhibitor experiments. D.C.H. and M.A.F. did metabolic experiments, and T.G. and N.C. performed the parabiosis experiments.
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Extended data figures and tables
Extended Data Fig. 1 Multiple physiological and cellular parameters differ between male and female mice.
a, Weight gain of normal chow diet fed wild-type male and female mice with age (n = 6 mice of each sex). b–e, Multiple physiological parameters measured in age matched wild-type male and female mice, including fat mass (n = 7 mice of each sex) (b), lean mass (n = 7 mice of each sex) (c), serum insulin levels (n = 5 mice of each sex) (d) and serum adipokine levels 6 h post fasting (n = 5 mice of each sex) (e). f, Numbers of ILC2s in male and female VAT from 12–15-week-old mice. n = 8 females, n = 10 males. g, Proportions of different VAT resident immune cells determined from male and female mice (n = 4–9). h, Expression of CD44 and CD62L in VAT Treg cells from male and female wild-type mice. Graph on the right shows quantification (n = 9 mice of each sex). i, Flow cytometry plots (left) showing Foxp3 (RFP) and Il10 (GFP) expression in spleens and small intestine lamina propria (SI-LP) and quantification (right) of Il10 (GFP)+ Treg cells in VAT, spleen and SI-LP resident CD4+ T cells of female and male Foxp3RFPIl10GFP double-reporter mice (n = 3 mice of each sex). Unpaired t-test was performed (two-tailed). Data are mean ± s.d. Data pooled or representative of two independent experiments.
Extended Data Fig. 2 VAT-specific sexual dimorphism in Treg cells is underpinned by unique transcriptional signatures and chromatin accessibility.
a, Percentages of FOXP3+ cells in spleens (n = 6 of each sex), small intestine lamina propria (SI-LP) (n = 7, females and males), colons (n = 4 of each sex), livers and lungs (n = 5 of each sex) from 25–30-week-old wild-type mice. b, Percentages of FOXP3+ cells in subcutaneous adipose tissue (SC-AT) (n = 6 of each sex), VAT (n = 5 of each sex) and perinephric adipose tissue (PN-AT) (n = 6 of each sex) from 25–30-week-old wild-type mice. c, Expression of ST2 and KLRG1 in Treg cells from the PN-AT (left) and SC-AT (right) of wild-type male and female mice. Treg cells from male VAT are shown in green as positive control. d–f, Volcano plots show genes differentially expressed between male and female VAT Treg cells (d), VAT CD4+FOXP3− T cells (e) and VAT-ILC2s (f). Each dot represents a gene. Differentially expressed genes are marked in blue (downregulated) or red (upregulated). g, Heat map shows chromatin accessibility of VAT Treg-signature genes assessed by ATAC-seq. Data displayed from male VAT Treg cells, female VAT Treg cells and male splenic Treg cells. h, ATAC-seq tracks show chromatin accessibility at the Pparg locus of male splenic Treg cells (green) and Treg cells from female (red) and male (blue) VAT. Arrows indicate regions of differential chromatin accessibility. Data in a, b are pooled or representative of two independent experiments; two-tailed unpaired t-test. Data are mean ± s.d. Sequencing experiments performed in duplicates. For each RNA-seq sample, VAT Treg cells were sorted from male (n = 5) and female (n = 12) Foxp3RFP mice. For ATAC-seq, each sample contained Treg cells from n = 4 males and n = 10 females. Experiments were performed with 25–32-week-old mice. Statistical methods and software packages for sequencing data are described in Methods.
Extended Data Fig. 3 Opposing functions of male and female sex hormones in regulating VAT inflammation, Treg cell recruitment and glucose tolerance.
a, b, Representative flow cytometry histograms showing expression of CCR2 in wild-type and Ar−/− VAT Treg cells (a) and wild-type and Era−/− VAT Treg cells (b). c, Frequency of VAT Treg cells in male wild-type and Era−/− mice (n = 4 of each genotype). d, Flow cytometry plots (left) show VAT Treg cells from control and ICI 182-780-treated female mice; graph (right) shows quantification (n = 4 for both conditions). e, Expression of indicated markers in control and ICI 182-780-treated female VAT Treg cells. f, Body mass (left) (n = 5 wild type; n = 6 Ar−/−) and VAT mass (right) (n = 7 wild type; n = 8 Ar−/−) from 20–25-week-old male wild-type and Ar−/− mice. g, Body mass (left) (n = 4 wild type; n = 5 Era−/−) and VAT mass (right) (n = 9 wild type; n = 8 Era−/−) from 20–25-week-old female WT and Era−/− mice. h, i, Oral glucose-tolerance test (left) and area under the curve (right) comparing age-matched male wild-type and Ar−/− mice (n = 4 wild type; n = 5 Ar−/−) (h), or female wild-type and Era−/− mice (n = 4, wild type and Era−/− of each genotype) (i). j, k, Fasting serum insulin levels in wild-type (n = 6) and Ar−/− (n = 5) male mice (j), and in WT (n = 8) and Era−/− (n = 5) female mice (k). Two-tailed unpaired t-test; data are mean ± s.d. Data are pooled or representative of two independent experiments.
Extended Data Fig. 4 VAT Treg cell extrinsic function of sex hormones.
a, Schematic shows the strategy used to make bone marrow chimeric mice using wild-type and Ar−/− recipients. b, Proportions of Treg cells from the VAT of irradiated wild-type (n = 5) and Ar−/− (n = 6) mice that received wild-type bone marrow. Quantification on the right. c, Expression of indicated cell surface markers on VAT Treg cells from wild-type and Ar−/− mice that were reconstituted with wild-type bone marrow (from b). d, Flow cytometry plots (left) show expression of FOXP3 and ST2 in VAT CD4+ T cells from male Arfl/flFoxp3cre (n = 6) and Foxp3cre (n = 4) control mice. Quantification on the right. e, Expression of CCR2 and KLRG1 in VAT Treg cells from Arfl/flFoxp3cre and control mice (from d). f, Percentages of wild-type and Era−/− Treg cells in the VAT of female bone marrow chimeric mice. Irradiated wild-type female Ly5.1 recipient mice were reconstituted with a mixture of female Ly5.2 wild-type (n = 4) and female Era−/− (n = 5) bone marrow cells. g, h, Percentages of splenic Treg cells in oestrogen-treated (n = 12) and untreated (n = 6) male wild-type mice (g) and in testosterone-treated (n = 9) and untreated (n = 5) female wild-type mice (h). i, Expression of FOXP3 and CD25 in VAT CD4+ T cells isolated from oestrogen-treated or untreated male wild-type mice. j, Flow cytometry histograms show expression of KLRG1 and ST2 in VAT Treg cells from oestrogen-treated or untreated male wild-type mice. k, Expression of FOXP3 and CD25 in VAT CD4+ T cells isolated from testosterone-treated or untreated female wild-type mice. l, Expression of KLRG1 and ST2 in VAT Treg cells from testosterone-treated or untreated female wild-type mice. Two-tailed unpaired t-test. Data are mean ± s.d. Data are pooled or representative of two independent experiments.
Extended Data Fig. 5 Sex-specific VAT inflammation, Treg cell recruitment and maintenance in VAT.
a, Heat map shows top-200 differentially expressed genes between male and female VAT and subcutaneous adipose tissue (SC-AT). Duplicate samples used for RNA-seq. For each sample, VAT or SC-AT from three mice was pooled for RNA extraction. b, Proportions of Treg cells in wild-type and Ccr2−/− compartments of mixed bone marrow chimeric mice. c, Expression of specified markers in wild-type and Ccr2−/− VAT Treg cells from male mixed bone marrow chimeric mice. d, Flow cytometry plots (left) and quantification (right) of wild-type and CCR2-deficient ILC2s in the VAT of male chimeric mice containing congenically marked wild-type (n = 8) and Ccr2−/− (n = 8) haematopoietic cells. e, Expression of FOXP3 and KLRG1 in wild-type (n = 5), Tnf−/− (n = 3), Ifng−/− (n = 5) and Il1b−/− (n = 4) mice. Graph on the right shows quantification. f, Expression of KLRG1 and ST2 (top) and KLRG1 and CCR2 (bottom) in splenic Treg cells from wild-type male mice. Graph (right) shows percentages of KLRG1+ cells of Treg cells in the spleen of wild-type male and female mice (n = 6 of each sex). g, ST2 and CCR2 expression in male and female KLRG1+ splenic Treg cells. h, Graph shows percentages of KLRG1+ cells of Treg cells in the spleens of female (n = 5) and male (n = 4) mice treated with PBS or IL-33. i, Schematic of parabiosis experiment. j, k, Flow cytometry plots (left) and quantification (right) show proportions of Treg cells (n = 9) (j) and ILC2s (n = 9) (k) in the VAT of parabiotic wild-type female mice that were paired for 12 weeks. Two-tailed unpaired t-test. Data are mean ± s.d. Data are pooled or representative of two independent experiments.
Extended Data Fig. 6 Sex-hormonal control of VAT inflammation and stromal cell differentiation.
a, Expression of indicated genes in the VAT of testosterone- or oestrogen-treated male and female wild-type mice measured by quantitative PCR. Untreated females and males, testosterone-treated females (n = 5); oestrogen-treated males (n = 6). b, VAT weight from untreated (n = 7) and oestrogen-treated (n = 11) wild-type male (left) and untreated (n = 6) and testosterone-treated (n = 10) wild-type female (right) mice. c, Concentrations of indicated proinflammatory cytokines in the mesenteric lymph nodes of celecoxib-treated or untreated wild-type male mice (n = 6 per condition) measured by cytokine bead array. d, Expression of FOXP3 and KLRG1 in CD4+ T cells isolated from the VAT of celecoxib-treated and untreated male wild-type mice. e, Percentages (left) and numbers (right) of FOXP3+ Treg cells in the VAT of celecoxib-treated and untreated wild-type male mice (n = 6 per condition). f, Gating strategy used to identify VAT CD31+ endothelial cells and Gp38+ stromal cells in the CD45- non-haematopoietic cell compartment of wild-type male and female mice. g, Il33 transcript levels in Gp38+ stromal and Gp38−CD31+ endothelial cells and in adipocytes from 25-week-old male mice (data from RNA-seq analysis, two samples per cell type). h, MA plot showing genes differentially expressed between male and female Gp38+ VAT stromal cells. RNA-seq performed in duplicate samples. For each sample, the respective VAT stromal cell population was sorted from wild-type male (n = 5) and female (n = 7) mice. i, Numbers of Gp38+ cells in female (n = 13) and male (n = 14) VAT (left) and CD31+ cells (right) in female (n = 11) and male (n = 14) VAT from 25-week-old mice. j, Proportions of CD73+ cells within the female (n = 8) and male (n = 9) Gp38+ stromal compartment of perinephric adipose tissue (PN-AT, left) and subcutaneous adipose tissue (right) (n = 12 females; n = 10 males). In a, one-way ANOVA was performed. Other data were analysed using two-tailed unpaired t-test. Data are mean ± s.d., except in a, where data are mean ± s.e.m. Data are pooled or representative of two independent experiments.
Extended Data Fig. 7 Sex-specific distribution of IL-33+ VAT stromal cells and VAT Treg cell response to IL-33 administration.
a, Percentages of IL-33+ cells within each VAT Gp38+ stromal cell compartment of female (n = 4) and male (n = 3) Il33GFP mice. b, IL-33 expression in CD45−CD31−Gp38+ stromal cells of wild-type mice as measured by intracellular staining. IgG was used as a control. c, Percentage of IL-33+ cells in the VAT Gp38+ stromal cell compartment of wild-type female (n = 4) and male (n = 6) mice (left) and percentages of IL-33+Gp38+ of live cells in VAT (right). d−h, IL-33 (n = 5) or PBS (mock) (n = 4) was administered to 12-week-old male and female wild-type mice. Expression of FOXP3 and KLRG1 in VAT CD4+ T cells (d), numbers of VAT Treg cells (e), ST2 expression in VAT Treg cells from IL-33- or PBS-treated (f), expression of KLRG1 and Ki67 in VAT Treg cells of male mice (g) and quantification of Ki67+ VAT Treg cells in female and male wild-type mice (n = 4 of each sex) (h). i, Treg cells were sorted from the spleens of transgenic mice expressing a VAT-specific T cell receptor25 and transferred into congenically marked female (n = 6) or male (n = 5) wild-type mice. Percentages of ST2+ TCR transgenic (Tg) Treg cells within the adipose tissue 12 weeks after adoptive transfer. Two-tailed unpaired t-test. Data are mean ± s.d. Data are pooled or representative of two independent experiments.
Extended Data Fig. 8 Sex-hormonal regulation of CD73+ VAT stromal cell differentiation and BLIMP1 regulation of VAT Treg cells and organismal metabolism.
a, Flow cytometry plots from testosterone-treated and untreated female wild-type mice showing expression of CD73 and CD90 in Gp38+ VAT stromal cells. b, Flow cytometry plots from oestrogen-treated and untreated male wild-type mice showing expression of CD73 and CD90 in Gp38+ VAT stromal cells. c, Percentages of VAT Treg cells in male Ar−/− mice treated with PBS (n = 4) or IL-33 (n = 4). d, Percentages of CD73+ stromal cells in celecoxib-treated (n = 6) or untreated (n = 5) male mice. e, Expression of Foxp3 (RFP) and Blimp1 (GFP) in male and female VAT Treg cells from Foxp3RFPBlimp1GFP double-reporter mice (n = 4 of each sex). Percentages of Blimp1 (GFP)+ cells among Foxp3 (RFP)+ Treg cells. f, Expression of indicated molecules in Foxp3cre and Blimp1fl/flFoxp3cre VAT Treg cells. g, Oral glucose-tolerance test in normal chow diet-fed 25-week-old male Blimp1fl/flFoxp3cre (n = 7) and Foxp3cre (n = 6) mice. Graph on the right shows AUC. Two-tailed unpaired t-test. Data are mean ± s.d. Data are pooled or representative of two independent experiments.
Extended Data Fig. 9 BLIMP1 establishes the VAT Treg cell transcriptional and chromatin landscapes.
a, Volcano plot shows genes differentially expressed between male Blimp1fl/flFoxp3cre and control VAT Treg cells. For each genotype, duplicate samples were used for RNA-seq. Each sample contains VAT Treg cells from n = 7 Blimp1fl/flFoxp3cre and n = 5 Foxp3cre mice. b, Heat map shows top-200 genes differentially expressed between wild-type male and female VAT Treg cells and Blimp1fl/flFoxp3cre male VAT Treg cells. c, MD plot shows expression of genes in Blimp1fl/flFoxp3cre and Foxp3cre VAT Treg cells. Each dot represents a gene; genes highlighted in red are up regulated, and in blue are downregulated, in Blimp1fl/flFoxp3cre VAT Treg cells. Larger dots with black outline indicate genes that are also bound by BLIMP1 in regions of open chromatin in VAT Treg cells. d, Venn diagram shows overlap between genes differentially expressed between male VAT Treg cells and male splenic Treg cells (VAT Treg cell signature), male Blimp1fl/flFoxp3cre and control VAT Treg cells and genes that show BLIMP1 ChIP binding in regions of open chromatin (peaks) of male VAT Treg cells. Statistical methods and software packages described in Methods.
Extended Data Fig. 10 Blimp1 regulates putative VAT Treg cell precursors, diverse functions of IL-6 in the VAT, and a model of the sex-hormone-mediated circuitry that mediates recruitment, expansion and function of VAT Treg cells.
a, Expression of Foxp3 (RFP) and Blimp1 (GFP) in splenic CD4+ T cells from Foxp3RFPBlimp1GFP mice. b, Expression of Blimp1 (GFP) and KLRG1 in splenic Treg cells. c, Pparg expression in Blimp1 (GFP)+ versus Blimp1 (GFP)− splenic Treg cells. Bar graph generated from RNA-seq read counts26. d, Expression of KLRG1 and CCR2 in splenic Treg cells from Foxp3cre and Blimp1fl/flFoxp3cre mice. e, Graphs on the right show percentages of KLRG1+ cells among splenic Treg cells of Foxp3cre (n = 5) and Blimp1fl/flFoxp3cre (n = 6) mice and percentages of CCR2+ cells within the KLRG1+ fraction of splenic Treg cells. f, Proportion of Blimp1 (GFP)+ Treg cells obtained after Blimp1 (GFP)− Treg cells were sorted from Foxp3RFPBlimp1GFP mice and cultured in the presence of indicated cytokines (n = 3–4). g, Expression of Il6 transcripts as measured by quantitative PCR in haematopoietic cell populations sorted from the male VAT (n = 6). h, Flow cytometry plots (left) and quantification (right) of ILC2s in the VAT of male wild-type and Il6−/− (n = 4 per genotype) mice. i, Flow cytometry histograms show expression of indicated markers in wild-type and Il6−/− VAT Treg cells. j, Expression of CD73 and CD90 in wild-type and Il6−/− (n = 4 per genotype) VAT Gp38+ cells (left). Percentages of CD73+CD90− and CD73+CD90+ stromal cells in the VAT of male wild-type and Il6−/− (n = 4 per genotype) mice (right). k, Percentages of VAT Treg cells in male Il6−/− mice treated with PBS or IL-33. Two-tailed unpaired t-test. Data are mean ± s.d. Data pooled or representative of two independent experiments. l, Model of the sex-hormone-mediated circuitry that mediates recruitment, expansion and function of VAT Treg cells. Treg cells are recruited to the VAT in a CCL2–CCR2-dependent manner. IL-6 induces the expression of transcription factor BLIMP1, which in turn activates expression of prototypical VAT Treg signature genes IL-33 receptor ST2, CCR2 and IL-10. IL-33 production by androgen-responsive stromal cells leads to local expansion of VAT Treg cells in the male VAT, which in turn mediate repression of VAT inflammation.
Supplementary information
Supplementary Table 1
| RNASeq Male, VAT-Treg vs Spleen-Treg (Treat-FC-1.5-DE-P0.1).
Supplementary Table 2
| RNASeq Female, VAT-Treg vs Spleen-Treg (Treat-FC-1.5-DE-P0.1).
Supplementary Table 3
| RNASeq VAT-Tregs, Male vs Female (Treat-FC-1.5-DE-P0.1).
Supplementary Table 4
| RNASeq Spleen-Treg, Male vs Female (Treat-FC-1.5-DE-P0.1).
Supplementary Table 5
| RNASeq VAT-ILC2, Male vs Female (log2rpkms).
Supplementary Table 6
| RNASeq VAT-CD4, Male vs Female (Treat-FC-1.5-DE-P0.1e).
Supplementary Table 7
| ATACseq-differentially accessible sites, VAT-Treg, Male vs Female.
Supplementary Table 8
| RNASeq Male, Visceral AT vs Subcutaneous AT (Treat-FC-1.2-DE-P0.1).
Supplementary Table 9
| RNASeq Visceral AT, Male vs Female (Treat-FC-1.2-DE-P0.1).
Supplementary Table 10
| RNASeq VAT-Stroma and VAT-Adipocytes, Male vs Female (Treat FC-1.2-DE-P0.1).
Supplementary Table 11
| RNASeq VAT-Treg, Male, Blimp1Foxp3Cre vs control (FC-1.5-DE-P0.1).
Supplementary Table 12
| Overlapping-peaks-ATACseq vs Blimp1ChIPseq.
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Vasanthakumar, A., Chisanga, D., Blume, J. et al. Sex-specific adipose tissue imprinting of regulatory T cells. Nature 579, 581–585 (2020). https://doi.org/10.1038/s41586-020-2040-3
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DOI: https://doi.org/10.1038/s41586-020-2040-3
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