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The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue–resident regulatory T cells

An Erratum to this article was published on 21 April 2015

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Abstract

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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Figure 1: Blimp-1+ eTreg cells have a distinct transcriptional profile and localize outside the T cell area.
Figure 2: ST2 is specifically expressed by eTreg cells, in particular in the VAT.
Figure 3: ST2 is required for the differentiation of VAT-Treg cells.
Figure 4: IL-33 induces proliferation and maintains identity of VAT-Treg cells.
Figure 5: TCR signals induce the VAT-Treg cell transcriptional program in a BATF- and IRF4-dependent manner.
Figure 6: IRF4 and BATF bind the Pparg and Il1rl1 loci, and continuous IRF4 expression is required for maintaining VAT-Treg cell identity.
Figure 7: IL-33 administration increases VAT-Treg cell numbers in genetically obese mice.

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  • 08 April 2015

    In the version of this article initially published, the Acknowledgments section was incomplete. The correct text should begin "We thank P. O'Brien, M. Mochizuki and N. Takeno for assistance with tissue collection...." The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

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.

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Authors and Affiliations

Authors

Contributions

A.V. planned and performed most experiments; K.M. performed experiments related to the IL-33 and ST2-deficient mice; A.X., S.A.-S., Y.L., P.L., W.L., W.S., W.J.L. and G.K.S. did or analyzed the RNA and ChIP sequencing experiments; S. Kawamoto and S.F. did the immunofluorescence; L.A.M. and R.G. performed additional experiments; S.N. and H.S. contributed reagents; S.L.M. and J.M.W. contributed to the scientific planning and organization of experiments; S.L.N. and S. Koyasu designed experiments; A.K. oversaw and designed the study; A.K. and A.V. wrote the manuscript.

Corresponding author

Correspondence to Axel Kallies.

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Competing interests

S.K. is a consultant for Medical and Biological Laboratories (MBL).

Integrated supplementary information

Supplementary Figure 1 eTreg cells are a transcriptionally distinct Treg cell population.

(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.

Supplementary Figure 2 ST2 expression correlates with other VAT-Treg cell markers.

(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).

Supplementary Figure 4 IL-33 drives proliferation of VAT-Treg cells.

(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).

Supplementary Figure 5 IL-33 signaling through MyD88 is required for VAT-Treg cell differentiation.

(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).

Supplementary Figure 6 BATF and IRF4 are required for VAT-Treg cell development.

(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 information

Supplementary Text and Figures

Supplementary Figures 1–8, and Supplementary Tables 4 and 5 (PDF 5066 kb)

Supplementary Table 1

Top 200 DE genes_cTregs Vs eTregs (CSV 13 kb)

Supplementary Table 2

DE genes encoding transcription factors_cTregs Vs eTregs (CSV 2 kb)

Supplementary Table 3

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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  • DOI: https://doi.org/10.1038/ni.3085

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