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γδ T cells and adipocyte IL-17RC control fat innervation and thermogenesis

Abstract

The sympathetic nervous system innervates peripheral organs to regulate their function and maintain homeostasis, whereas target cells also produce neurotrophic factors to promote sympathetic innervation1,2. The molecular basis of this bi-directional communication remains to be fully determined. Here we use thermogenic adipose tissue from mice as a model system to show that T cells, specifically γδ T cells, have a crucial role in promoting sympathetic innervation, at least in part by driving the expression of TGFβ1 in parenchymal cells via the IL-17 receptor C (IL-17RC). Ablation of IL-17RC specifically in adipose tissue reduces expression of TGFβ1 in adipocytes, impairs local sympathetic innervation and causes obesity and other metabolic phenotypes that are consistent with defective thermogenesis; innervation can be fully rescued by restoring TGFβ1 expression. Ablating γδ Τ cells and the IL-17RC signalling pathway also impairs sympathetic innervation in other tissues such as salivary glands. These findings demonstrate coordination between T cells and parenchymal cells to regulate sympathetic innervation.

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Fig. 1: γδ T cells and adipocyte IL-17RC control adaptive thermogenesis.
Fig. 2: IL-17RC signalling deficiency impairs innervation in brown fat.
Fig. 3: IL-17RC promotes sympathetic innervation in BAT through TGFβ1 signalling.

Data availability

Source Data for Figs. 13 and Extended Data Figs. 15 are provided with the paper. Proteomics raw data were deposited in the PRIDE archive and can be accessed via the ProteomeXchange under accession number PXD017424. RNA-seq data are available at the Gene Expression Omnibus (GEO) repository under accession number GSE144255. Any other relevant data are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank M. L. Mather, E. Maratos-Flier, R. Garrity, Z. Deng, A. Mina and D. Cabarkapa for help with CLAMS studies. We thank T. Jacks for his support and input for this project as C.J.’s postdoctoral advisor. We thank S. L. Gaffen and Y. Iwakura for sharing the Il17f−/− mice, J. K. Kolls for the Il17rcfl/fl mice, and Amgen for the Il17rc−/− and Il17ra−/− mice. We thank A. Mann, N. Asinovski and K. Hattori for the Vγ6Vδ1 transgenic mice and the 17D1 antibody. We thank C. Zhao for help with the immunofluorescence staining and confocal imaging analysis; L. Kazak and E. T. Chounchani for help with the EMG study; and R. J. Akhurst and S. Basu for sharing mice. We thank the Nikon Imaging Center at Harvard Medical School for all imaging studies; the Rodent Histology Core at Harvard Medical School for histology studies; the viral core at Children’s Hospital Boston for AAV production; and the Neurobiology Department and the Neurobiology Imaging Facility for consultation and instrument availability that supported this work. This facility is supported in part by the Neural Imaging Center as part of an NINDS P30 Core Center grant NS072030. We thank Z. Herbert, A. Caruso and members of the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute for RNA-seq analysis. We thank the Flow Cytometry Core Facilities at the Swanson Biotechnology Center at MIT and the Dana-Farber Cancer Institute. We thank Y. Chen, D. Bogoslavsk, J. Szpyt and all members of the Spiegelman and Mathis laboratories for help and input in this project. B.H. is a Cancer Research Institute/Leonard Kahn Foundation Fellow. C.J. is supported by a K99 Award (CA226400) from the National Cancer Institute (NCI). This work was supported by grants to B.M.S. from the NIH DK 31405 and from the JPB Foundation.

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Authors

Contributions

B.H. and B.M.S. conceived and directed the study. B.H., C.J., X.Z., J.M.R., Z.Y., B.B.L., D.M. and B.M.S designed and performed experiments and analysed the data. M.P.J. performed mass spectrometry analysis. J.R., Z.Y. and B.B.L. helped with chemogenetic experiments. B.N.D. and A.S.B. helped with CLAMS studies. B.H., C.J., X.Z. and B.M.S. wrote the manuscript with comments from all authors. All authors provided input and reviewed the manuscript.

Corresponding author

Correspondence to Bruce M. Spiegelman.

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The authors declare no competing interests.

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Peer review information Nature thanks Bruno Silva-Santos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 γδ T cells promote adaptive thermogenesis.

a, Reduced oxygen consumption (VO2) of Tcrd−/− mice during cold exposure measured by indirect calorimetry (n = 9 mice). b, Increased lipid accumulation in the BAT of Tcrd−/− mice as measured by haematoxylin and eosin (H&E) staining. Scale bars, 100 μm. c, No significant differences in food intake (left) or physical movement (right) in wild-type and Tcrd−/− mice, as measured by indirect calorimetry (n = 9 mice). Xtotal count, total number of x-axis infrared-beam breaks count. d, No significant change in electromyography (EMG) root mean square (RMS) muscle activity in wild-type and Tcrd−/− mice during cold exposure (n = 4 mice). e, A significant population of γδ T cells in BAT are Vγ6Vδ1+ (n = 6 mice). f, Flow cytometry analysis of γδ Τ cells in BAT. g, Cold exposure (6 h) upregulates Il17f mRNA expression in BAT (n = 5 mice). RT, room temperature. Cyclo, cyclophilin B (also known as Ppib). Data are representative of at least two or three independent experiments. Data are mean ± s.e.m. P values were determined by two-way ANOVA (a, c) or unpaired Student’s two-sided t-test (d, g).

Source data

Extended Data Fig. 2 IL-17RC deficiency predisposes mice to cold sensitivity and obesity.

a, b, Il17rc mRNA expression shown by translating ribosomal affinity purification (TRAP) from adiponectin-positive (n = 3 mice) (a) and UCP1-positive (b) cells (BAT, iWAT n = 3 mice; eWAT n = 2 mice). eWAT, epididymal white adipose tissue; FPKM, fragments per kilobase of transcript per million mapped reads; iWAT, inguinal white adipose tissue. c, Il17ra−/− mice are not sensitive to cold exposure (WT n = 13, Il17ra−/− n = 10 mice). d, Increased lipid accumulation in the BAT of AdIl17rc-KO mice as determined by H&E histology. Scale bars, 100 μm. e, Ucp1-cre;Il17rcfl/fl mice are sensitive to acute cold exposure (WT n = 7, Ucp1-cre;Il17rcfl/fl n = 6 mice). f, AdIl17rc-KO;Vγ6Vδ1 transgenic mice are sensitive to acute cold exposure (WT n = 8; AdIl17rc-KO;Vγ6Vδ1, AdIl17rc-KO n = 5 mice). g, Reduced oxygen consumption (VO2) induced by a high-fat diet (HFD) in AdIl17rc-KO mice as measured by indirect calorimetry (2 weeks after the start of high-fat diet feeding) (n = 8 mice). h, i, No significant differences in physical movement (h) or food intake (i) in wild-type and AdIl17rc-KO mice (n = 8). j, k, H&E histology of iWAT (j) and eWAT (k) of high-fat-diet-treated wild-type and AdIl17rc-KO mice (n = 8 mice). Scale bars, 100 μm. Data are mean ± s.e.m. and representative of at least two or three independent experiments. P values were determined by unpaired Student’s two-sided t-test (h, i) or two-way ANOVA (c, eg).

Source data

Extended Data Fig. 3 IL-17RC signalling deficiency impairs sympathetic innervation in adipose and multiple tissue.

a, Reduced sympathetic innervation of BAT in Rag2−/− mice as measured by TH (green) and TUBB3 (yellow) immunostaining (n = 6 mice). b, Reduced sympathetic innervation of BAT in Il17f−/− but not Il17a−/− mice by TH (green) and TUBB3 (yellow) immunostaining (WT n = 6, Il17f−/− n = 5, Il17a−/− n = 3 mice). c, Reduced sympathetic innervation of iWAT in AdIl17rc-KO mice by TH immunostaining (n = 6 mice). d, FOS immunoreactivity in response to CNO administration. e, Forced expression of S100B by AAV-S100B rescues the cold sensitivity in AdIl17rc-KO mice (n = 5 mice). f, Decreased lipid accumulation in the AAV-S100B-treated BAT, as measured by H&E histology. g, Forced expression of S100B by AAV-S100B increases adipose innervation in AdIl17rc-KO mice by TH (green) and TUBB3 (yellow) immunostaining (n = 6 mice). h, Forced expression of CLSTN3β by AAV-DIO-CLSTN3β rescues the cold sensitivity in AdIl17rc-KO mice (n = 5 mice). i, Decreased lipid accumulation in the AAV-DIO-CLSTN3β-treated BAT as measured by H&E histology. j, Forced expression of CLSTN3β by AAV-DIO-CLSTN3β increases adipose innervation in AdIl17rc-KO mice by TH (green) and TUBB3 (yellow) immunostaining (n = 6 mice). k, Reduced sympathetic innervation of salivary glands in Il17rc−/− mice by TH (green) and TUBB3 (yellow) immunostaining of WT and Il17rc KO SG (n = 6 mice). l, Reduced sympathetic innervation of salivary glands in Tcrd−/−, Il17f−/− but not Il17a−/− mice by TH (green) and TUBB3 (yellow) staining (WT n = 6, Tcrd−/− n = 6, Il17f−/− n = 6, Il17a−/− n = 3 mice). m, Reduced neuronal innervation of salivary glands in Rag2−/− mice by TH (green) and TUBB3 (yellow) immunostaining. (n = 4 mice). n, No significant difference in sympathetic innervation of salivary glands in AdIl17rc-KO mice by TH (green) and TUBB3 (yellow) immunostaining (n = 3 mice). o, Reduced sympathetic innervation of bronchi in Rag2−/− and Tcrd−/− mice by TH and TUBB3 staining (WT n = 8, Rag2−/− n = 3, Tcrd−/− n = 4, Il17rc−/− n = 3 mice). Data are mean ± s.e.m. and are representative of at least two or three independent experiments. P values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test (b, l), unpaired Student’s two-sided t-test (a, g, j, k, m, n) or two-way ANOVA (e, h). Scale bars, 50 μm (a, b, g, jo), 100 μm (c, d, f, i).

Source data

Extended Data Fig. 4 Reduced Tgfb1 and collagen genes in AdIl17rc-KO mice.

a, Pathway enrichment analysis of genes downregulated both at the RNA and the protein level in BAT from AdIl17rc-KO mice compared with littermate controls. b, Reduced mRNA expression of Tgfb1, Col1a1, Col3a1, Col5a1 and Ncor2 in the BAT of AdIl17rc-KO mice (n = 3 mice). Data are representative of at least two or three independent experiments. ce, Reduced Tgfb1 mRNA expression in BAT, salivary glands (SG) and lungs of Il17rc−/−, Tcrd−/−, Il17f−/− and Rag2−/− mice (BAT: WT n = 7, Il17rc−/− n = 6; WT n = 5, Tcrd−/− n = 6, Il17f−/− n = 6; WT, Rag2−/− n = 4 mice; SG: WT n = 4, Il17rc−/− n = 3; WT, Tcrd−/−, Il17f−/− n = 3; WT, Rag2−/− n = 3 mice; lung: WT n = 5, Il17rc−/− n = 4; WT n = 6, Tcrd−/− n = 4, Il17f−/− n = 6; WT, Rag2−/− n = 6 mice). Cyclo, cyclophilin B (also known as Ppib). Data are mean ± s.e.m. P values or FDR q values determined by unpaired Student’s two-sided t-test (b, c, e), as described in the Methods (a), or one-way ANOVA with Bonferroni’s multiple comparisons test (d).

Source data

Extended Data Fig. 5 Blocking TGFβ sensitizes mice to acute cold exposure.

a, Decreased lipid accumulation in BAT from AAV-DIO-TGFβ1-treated mice as determined by H&E histology. Data are representative of two independent experiments. b, Wild-type mice treated with TGFβ-neutralizing antibody (Ab)) for 3 weeks are sensitive to cold exposure(n = 6 mice). c, Wild-type mice treated with TGFβ inhibitor (sb431542) for 3 weeks are sensitive to cold exposure (n = 8). d, e, Forced expression of TGFβ1-AAV increases the TH (d) and TUBB3 (e) immunostaining in the salivary glands from Il17rc−/− mice (n = 6 mice). f, g, Forced expression of AAV-TGFβ1 increases TH (f) and TUBB3 (g) immunostaining in the salivary glands from Rag2−/− mice (n = 6 mice). Scale bars, 50 μm. Data are mean ± s.e.m. and representative of two or three independent experiments. P values were determined by two-way ANOVA (b, c) or unpaired Student’s two-sided t-test (dg).

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Hu, B., Jin, C., Zeng, X. et al. γδ T cells and adipocyte IL-17RC control fat innervation and thermogenesis. Nature 578, 610–614 (2020). https://doi.org/10.1038/s41586-020-2028-z

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