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Depletion of fat-resident Treg cells prevents age-associated insulin resistance

Abstract

Age-associated insulin resistance (IR) and obesity-associated IR are two physiologically distinct forms of adult-onset diabetes. While macrophage-driven inflammation is a core driver of obesity-associated IR1,2,3,4,5,6, the underlying mechanisms of the obesity-independent yet highly prevalent age-associated IR7 are largely unexplored. Here we show, using comparative adipo-immune profiling in mice, that fat-resident regulatory T cells, termed fTreg cells, accumulate in adipose tissue as a function of age, but not obesity. Supporting the existence of two distinct mechanisms underlying IR, mice deficient in fTreg cells are protected against age-associated IR, yet remain susceptible to obesity-associated IR and metabolic disease. By contrast, selective depletion of fTreg cells via anti-ST2 antibody treatment increases adipose tissue insulin sensitivity. These findings establish that distinct immune cell populations within adipose tissue underlie ageing- and obesity-associated IR, and implicate fTreg cells as adipo-immune drivers and potential therapeutic targets in the treatment of age-associated IR.

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Figure 1: fTreg cells are selectively enriched in aged mice.
Figure 2: fTreg knockout mice are protected from general hallmarks of metabolic ageing.
Figure 3: Loss of fTreg cells protects against the clinical hallmarks of age-associated IR.
Figure 4: fTreg cell depletion improves adipose glucose uptake.

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Sequence Read Archive

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RNA-Seq data can be accessed in the NCBI Sequence Read Archive under the accession SRP053799.

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Acknowledgements

We would like to thank L. Chong, J. Alvarez, Y. Dai, S. Kaufman and B. Collins for technical assistance, L. Ong and C. Brondos for administrative assistance, and J. Simon for assistance with graphics. S.P.B. is supported by National Institutes of Health (NIH) grants F30 DK096828 and T32 GM007198. C.L. and M.D. are funded by grants from the National Health and Medical Research Council of Australia Project grants 512354, 632886 and 1043199. R.M.E. is an Investigator of the Howard Hughes Medical Institute (HHMI) at the Salk Institute and March of Dimes Chair in Molecular and Developmental Biology, and is supported by NIH grants DK057978, DK090962, HL088093, HL105278 and ES010337, the Glenn Foundation for Medical Research, the Leona M. and Harry B. Helmsley Charitable Trust, Ipsen/Biomeasure, California Institute for Regenerative Medicine and The Ellison Medical Foundation. Y. Zheng is supported by the Nomis Foundation, the Rita Allen Foundation, the Emerald Foundation, the Hearst Foundation, the National Multiple Sclerosis Society, and National Institutes of Health (AI099295 and AI107027). This work was also supported by National Cancer Institute funded Salk Institute Cancer Center core facilities (CA014195) and the James B. Pendleton Charitable Trust.

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Authors and Affiliations

Authors

Contributions

S.P.B., J.M.S., M.D., R.M.E. and Y. Zheng designed and supervised the research. S.P.B., J.M.S., S.F., S.L., Y. Zhang, A.C., C.Z. and Y.L. performed research. S.P.B., J.M.S., M.L., C.L., A.R.A., R.T.Y., M.D., R.M.E. and Y. Zheng analysed data. S.P.B., J.M.S., S.L., A.R.A., R.T.Y., M.D., R.M.E. and Y. Zheng wrote the manuscript.

Corresponding authors

Correspondence to Ronald M. Evans or Ye Zheng.

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Competing interests

S.B., R.M.E., Y. Zheng and M.D. are co-inventors of technologies related to methods of targeting fat Treg cells to prevent insulin resistance.

Extended data figures and tables

Extended Data Figure 1 Schematic outlining study premise and selected gating strategies used to generate AIPs.

a, Schematic outlining study premise. bd, AIPs were generated through the use of several distinct antibody cocktails. Here, using Foxp3Cre (Foxp3-IRES-YFP-Cre) reporter mice, we show how the stromal vascular fraction of VAT was analysed by flow cytometry to identify several T cell subtypes (b), macrophage subsets (c) and eosinophils and neutrophils (d).

Extended Data Figure 2 AIPs of aged fTreg knockout and control mice in VAT, SAT and spleen.

a, AIPs of aged (~14 months) fTreg knockout and control male mice depicting immune cell abundance, expressed as percentage of CD45.2+ cells. Entirety of immune compartment (top) is further divided into pan-macrophage (middle) and non-macrophage (bottom) pie charts (n = 9 mice per group). bd, Immune cell abundance between fTreg knockout and control mice, expressed as cells per gram of VAT (b), SAT (c) and spleen (d) (n = 9 mice per group). Data are mean ± s.e.m. #, false discovery rate < 2%.

Extended Data Figure 3 Aged fTreg knockout mice do not show signs of systemic autoimmunity or breakdown in peripheral tolerance.

a, Percentage of splenic naive CD4+ T cells as defined by CD62hi CD44lo relative to total CD4+ CD25 Foxp3Cre population (n = 9 mice per group). b, Representative histology of gastrointestinal tract—duodenum, jejunum, ileum and colon (left to right) (n = 3 mice per group). There were no significant lesions observed or differences in inflammation, epithelial changes, or mucosal architecture between the two groups (H&E, original magnification, ×100). Scale bar, 50 μm. c, Histopathology score in the small intestine and colon of fTreg knockout and control mice. The severity and extent of inflammation and epithelial changes as well as mucosal architecture were each graded on a score of 1 (minimal) to 5 (severe) and added to obtain an overall score over 20. There were minimal inflammatory changes with no significant differences between groups. d, Multiplex inflammation panel of serum from fTreg knockout and control mice (n = 4 pooled samples (3 mice per sample) per group). Data are mean ± s.e.m. *P < 0.05, ***P < 0.001, Student’s t-test.

Extended Data Figure 4 Weight-matched cohorts’ body weights and adipocyte size frequency in VAT of aged control and fTreg knockout mice.

a, Body weights of fTreg knockout and control male mice used in weight-matched metabolic studies in young (12 week; control n = 9; fTreg KO n = 9), aged (36 week; control, n = 9 mice; fTreg KO, n = 11 mice) and obese (diet-induced obese, 12 weeks of HFD starting at 12 weeks; control n = 10; fTreg KO n = 10) settings. b, Frequency of small (0–5,000 μm2), medium (5,000–10,000 μm2) and large (>10,000 μm2) adipocytes in VAT of aged control and fTreg knockout mice (n = 3 mice per group, 850 adipocytes counted from control mice, 269 adipocytes counted from fTreg knockout adipose). Data are mean ± s.e.m.

Extended Data Figure 5 VAT AIPs of obese fTreg knockout and control mice.

a, AIPs of diet-induced obese (16 weeks high fat diet started at 12 weeks) control (n = 6 mice) and fTreg knockout (n = 8 mice) male mice depicting immune cell abundance, expressed as percentage of CD45.2+ cells. Entirety of immune compartment (top) is further divided into pan-macrophage (middle) and non-macrophage (bottom) pie charts. b, Immune cell abundance between fTreg knockout and control mice, expressed as cells per gram of VAT (n = 9 mice per group). Data are mean ± s.e.m.

Extended Data Figure 6 fTreg cells are dispensable for TZDs to exert their therapeutic insulin-sensitizing effect.

a, Scheme used for longitudinal interventional study of control and fTreg knockout mice which indicates when particular assays were conducted and whose results are described in bg, in which rosiglitazone (Rosi) was introduced in diet after firmly establishing obesity with a HFD alone for 12 weeks (n = 8 mice per group). b, Cohort weights during course of study. Black arrow indicates introduction of rosiglitazone to the diet. c, Homeostatic model assessment of IR (HOMA-IR). d, e, Glucose tolerance test (d) and glucose excursions of glucose tolerance test (e) described as area under curve (AUC). f, g, Insulin tolerance test (f) and bar-graph quantitation of relative serum glucose decrease during insulin tolerance test (g) described as area above curve (AAC). h, Scheme used for parallel prophylactic study of control and fTreg knockout mice, the results of which are described in il, in which mice were placed on a HFD or HFD with rosiglitazone for 12 weeks (n = 8 mice per group). i, Cohort weights at end of study. j, HOMA-IR. k, l, Glucose and insulin tolerance tests of control (k) or fTreg knockout (l) mice fed HFD or HFD with rosiglitazone. m, Scheme used to determine temporal relationship of TZD-induced fTreg expansion and TZD-induced insulin-sensitization in wild-type mice, the results of which are described in nq, where mice were fed HFD or HFD with rosiglitazone for up to 11 weeks (n = 10 mice per group, 5 mice of each group were euthanized at 5 weeks after diet introduction and remaining 5 mice were euthanized at 11 weeks). n, HOMA-IR at 4 weeks. o, p, Glucose (o) and insulin (p) tolerance tests at 5 weeks. q, Relative fTreg cell enrichment of mice fed HFD with rosiglitazone versus mice fed HFD alone at 5 and at 11 weeks. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test.

Extended Data Figure 7 Increased TNFα levels and gene expression pattern of aged fTreg knockout adipose tissue is consistent with an improved adipose remodelling capacity.

a, TNFα levels quantified by ELISA of whole adipose lysate (~40 weeks, n = 6 per group). bd, FPKM values of all differentially expressed genes (b), differentially expressed collagens (c) and differentially expressed extracellular matrix proteases (d) in VAT from aged fTreg knockout and control mice (~40 weeks, n = 3 mice per group). Data are mean ± s.e.m. ***P < 0.001, Student’s t-test.

Extended Data Figure 8 fTreg cell gene expression and depletion with anti-ST2 antibody treatment.

a, Expression of several canonical Treg cell genes across fat and splenic Treg and fat and splenic Tconv cells. Cells were pooled from 3 and 4 mice before isolating RNA for subsequent RNA-Seq analysis. b, Expression of St2 across all haematopoietic cells catalogued in the ImmGen database. Position of adipose CD4+ CD25+ T cells is marked. c, Total weight before beginning course of anti-ST2 or isotype control antibodies (day 0) and after terminal analysis (day 3) (n = 4 mice per group). d, e, Spleen weight (d) and percentage of splenic naive CD4+ T cells as defined by CD62hi CD44lo relative to total splenic CD45+ CD4+ CD25 T cell population (e) of mice after terminal analysis (day 3, n = 4 mice per group). Data are mean ± s.e.m.

Extended Data Figure 9 Aged fTreg knockout mice are resistant to short-term, but not persistent, HFD-induced weight gain and IR.

ae, Aged control and fTreg knockout mice were placed on HFD and monitored throughout course of diet for weight (a), fasting glucose levels (b), fasting serum insulin levels (c), performance on glucose tolerance test (d), and on insulin tolerance test (e). (Control, n = 10; fTreg KO, n = 11; mice were aged 27–29 weeks and weight-matched before HFD was introduced.) Data are mean ± s.e.m. ns, non-significant, *P < 0.05, **P < 0.01, Student’s t-test.

Extended Data Table 1 Antibodies used to identify the given immune cell type molecularly

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Bapat, S., Myoung Suh, J., Fang, S. et al. Depletion of fat-resident Treg cells prevents age-associated insulin resistance. Nature 528, 137–141 (2015). https://doi.org/10.1038/nature16151

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