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 innervation^1,2. 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.

Extended Data Fig. 1

γδT cells promote adaptive thermogenesis. a, Reduced oxygen consumption (VO2) of Tcrδ^−/− mice during cold exposure measured by indirect calorimetry (n=9 mice). Data are mean ± SEM and analyzed by Two-way ANOVA. b, Increased lipid accumulation in the BAT of Tcrδ^−/− mice (H&E histology), scale bar=100um. Data represent at least 2-3 independent experiments with similar results. c, No significant difference of food intake and physical movement in WT and Tcrδ KO mice by indirect calorimetry (n=9 mice). d, No significant change of EMG RMS muscle activity in WT and Tcrδ KO mice during cold exposure (n=4 mice). e, A significant population of γδT cells in BAT are Vγ6Vδ1+ (n=6 mice). f, Flow cytometry analysis of γδ Τ cells in BAT. g, cold exposure (6hr) upregulate IL-17F mRNA in BAT (n=5 mice). Data represent at least 2-3 independent experiments with similar results. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA

Extended Data Fig. 2

IL-17RC deficiency predispose mice to cold sensitivity and obesity. a, b, Il17rc mRNA expression level shown by translating ribosomal affinity purification (TRAP) from (a) ADIPONECTIN positive cells (ADIPONECTIN-TRAP) (n=3 mice)and (b) UCP1-positive cells (UCP1-TRAP, BAT) (BAT, iWAT n=3 mice; eWAT n=2 mice); c, Il17ra^−/− mice are not sensitive to cold exposure (WT n=13, Il17ra^−/−n=10 mice). d, Increased lipid accumulation in the BAT of AdIl17RCKO mice (H&E histology), scale bar =100um. Data represent at least 2-3 independent experiments with similar results. e, UCP1-Cre;Il17rcfl/fl KO mice are sensitive to acute cold exposure (WT n=7, UCP1-Cre;Il17rcfl/fl KO n=6 mice). f, AdIl17RCKO;Vγ6Vδ1 transgenic mice are sensitive to acute cold exposure (WT n=8; AdIl17RCKO;Vγ6Vδ1, AdIl17RCKO n=5 mice). g, Decreased high fat diet-induced oxygen consumption (VO2) by indirect calorimetry (2wks after the start of high fat diet feeding) (n=8 mice). h, i, Physical movement (X-total counts) and food intake are not different in WT and AdIl17RCKO mice (n=8). j, k, iWAT (j) and eWAT (k)H&E histology of HFD treated WT and AdIl17RCKO mice (n=8 mice). Scale bar =50um. Data represent at least 2-3 independent experiments with similar results. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA.

Extended Data Fig. 3

IL-17RC signaling deficiency impairs sympathetic innervation in adipose and multiple tissue a, Reduced sympathetic innervation of BAT in Rag2 KO mice by TH (green) and tubb3 (yellow) staining (n=6 mice). b, Reduced sympathetic innervation of BAT in Il17f^−/− but not Il17a^−/− mice by TH (green) and tubb3 (yellow) staining (WT n=6, Il17f^−/− n=5, Il17a^−/− n=3 mice). Data are mean ± SEM and analyzed by One-way ANOVA and Bonferroni’s multiple comparisons test. Scale bar=50um. c, Reduced sympathetic innervation of iWAT in AdIl17RCKO mice by TH staining (n=6 mice). Scale bar=100um. d, FOS immunoreactivity and thermogenic response to CNO administration. Scale bars, 100 μm. Data represent 2-3 independent experiments with similar results. e, Forced expression of S100b by s100b-AAV rescues the cold sensitivity in AdIl17RCKO mice (n=5 mice). f, Decreased lipid accumulation in the s100b-AAV treated BAT (H&E histology). Data represent 2 independent experiments with similar results. g, Forced expression of S100b by s100b-AAV increases adipose innervation in AdIl17RCKO mice by TH (green) and TUBB3 (yellow) staining (n=6 mice). h, Forced expression of Clstn3β by clstn3β-DIO-AAV rescues the cold sensitivity in AdIl17RCKO mice (n=5 mice). i, Decreased lipid accumulation in the clstn3β-AAV treated BAT (H&E histology), scale bar =100um. j, Forced expression of clstn3β by clstn3β-DIO-AAV increases adipose innervation in AdIl17RCKO mice by TH (green) and TUBB3 (yellow) staining (n=6 mice). scale bar=50um. Data represent 2 independent experiments with similar results. k, Reduced sympathetic innervation of salivary glands (SG) in Il17rc KO mice by TH staining (green) and TUBB3 immunostaining (yellow) of WT and Il17rc KO SG (n=6 mice). l, Reduced sympathetic innervation of SG in Tcrδ KO, Il17f KO but not Il17a KO mice by TH (green) and TUBB3 (yellow) staining (WT n=6, Tcrδ KO n=6, Il17f KO n=6, Il17a KO n=3 mice). Data are mean ± SEM and analyzed by One-way ANOVA and Bonferroni’s multiple comparisons test. m, Reduced neuronal innervation of SG in Rag2 KO mice by TH (green) and TUBB3 (yellow) staining. (n=4 mice). n, No significant difference of sympathetic innervation of SG in AdIl17rcKO mice by TH (green) and TUBB3 (yellow) staining (n=3 mice). Scale bar=50um. Data represent at least 2-3 independent experiments. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA. o, Reduced sympathetic innervation of bronchi in Rag2^−/− and Tcrδ^−/− mice by TH and TUBB3 staining (WT n=8, Rag2 KO n=3, Tcrδ KO n=4, Il17rc KO n=3 mice). Data represent at least 2 independent experiments with similar results. Scale bar=50um. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA.

Extended Data Fig. 4

Reduced Tgfb1 and collagen genes in AdIl17rcKO mice a, Pathway enrichment analysis of genes downregulated both at the RNA and the protein level in the AdIl17RCKO BAT compared with littermate controls. b, Reduced TGFβ1, Col1a1, Col3a1, Col5a1 and Ncor2 mRNA expression in BAT of AdIl17rcKO mice (n=3 mice). Data represent at least 2-3 independent experiments. c, d, e, Reduced TGFβ1 mRNA expression in BAT, SG and Lung of Il17rc KO, Tcrδ KO, Il17f KO and Rag2 KO mice (BAT (WT n=7, Il17rc KO n=6; WT n=5, Tcrδ KO n=6, Il17f KO n=6; WT, Rag2 KO n=4 mice); SG (WT n=4, Il17rc KO n=4; WT, Tcrδ KO, Il17f KO n=3; WT, Rag2 KO n=3 mice); Lung (WT n=5, Il17rc KO n=4; WT n=6, Tcrδ KO n=4, Il17f KO n=6; WT, Rag2 KO n=6 mice)). Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test, One-way ANOVA and Bonferroni’s multiple comparisons test.

Extended Data Fig. 5

TGFβ blocking sensitize mice to acute cold exposure a, Decreased lipid accumulation in the TGFβ1-AAV treated BAT (H&E histology). Data represent 2 independent experiments with similar results. b, WT mice treated with TGFβ neutralizing antibody for 3 weeks are sensitive to cold exposure(n=6 mice). c, WT mice treated with TGFβ inhibitor for 3 weeks are sensitive to cold exposure(n=8). d, e, Forced expression of TGFβ1-AAV increases the SG TH (d) and TUBB3 (e) immuno-staining in Il17rc KO mice (n=6 mice). f, g, Forced expression of TGFβ1-AAV increases SG TH and TUBB3 immuno-staining in Rag2 KO mice by TH(f) and TUBB3 (g) staining (n=6 mice). scale bar=50um. Data represent 2-3 independent experiments. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA.

Figure 1.. γδ T cells and adipocyte Il17rc control adaptive thermogenesis.

a, b, c, d, Rag2^−/−, Tcrδ^−/−, Tcrα^−/− but not muMT^−/− mice are sensitive to acute cold exposure (WT littermate n=13, Rag2^−/− n=10; WT littermate n=15, muMT^−/− n=10; WT littermate n=13, Tcrα^−/− n=9; WT littermate n=10, Tcrδ^−/− n=9 mice). Data are mean ± SEM and analyzed by Two-way ANOVA. e, Vγ6Vδ1 transgenic mice are protected from cold exposure (Vγ6Vδ1- n=8, Vγ6Vδ1+ n=7 mice); Il17f^−/− mice (f) but not Il17a^−/− mice (g) are sensitive to acute cold exposure (WT littermate n=10, Il17f^−/− n=10, WT littermate n=11, Il17a^−/− n=13 mice). Data represent at least 2-3 independent experiments. Data are mean ± SEM and analyzed by Two-way ANOVA. h, Il17rc^−/− mice are sensitive to cold exposure (n=9 mice); i, AdIl17RCKO mice are sensitive to cold exposure (WT littermate n=11, AdIl17RCKO n=9 mice). j, AdIl17RCKO mice are more obese on HFD; k, AdIl17RCKO mice have impaired glucose tolerance on HFD; l, m, AdIl17RCKO mic have increased lipid droplet deposition in BAT and liver on HFD (n=8 mice). Data represent at least 2-3 independent experiments with similar results. Data are mean ± SEM and analyzed by Two-way ANOVA.

Figure 2.. IL-17RC signaling deficiency impairs innervation in brown fat.

a, AdIl17RCKO mice have reduced oxygen consumption (VO2) after acute cold exposure by indirect calorimetry (n=8 mice). b, No significant change of oxygen consumption (VO2) after CL 316,243 injection indirect calorimetry (n=6 mice). Data are mean ± SEM and analyzed by Two-way ANOVA. c, d, Reduced sympathetic innervation of BAT in AdIl17RCKO mice by TH (c) and TUBB3 immunostaining; (d) of WT and AdIl17RCKO BAT (n=6 mice). Data represent at least 3 independent experiments with similar results e, f, Reduced sympathetic innervation of BAT in Tcrδ KO mice by TH (e) and TUBB3 (f) staining. Data represent at least 3 independent experiments with similar results g, h Increased sympathetic innervation of BAT in Vγ6Vδ1 Tg mice by TH (g) and TUBB3 (h) staining (n=6 mice). Data represent at least 3 independent experiments with similar results. i, j, Thermogenic response to CNO administration in wild-type and Il17rc-knockout mice (WT littermate n= 5, Il17rc KO n= 8 mice) (i), and wild-type and Vγ6Vδ1 Tg mice (WT littermate n=7, Vγ6Vδ1 Tg n=6 mice). (j). Scale bar=50um. Data represent at least 2-3 independent experiments with similar results. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA.

Figure 3.. IL-17RC promotes sympathetic innervation in BAT through TGFβ1 signaling

a, b, Reduced TGFβ1 and Col1a1, Col1a2, Col3a1, Col5a1, Col5a3 and Ncor2 protein expression (a) (n=5 mice) and RNA (b) (n=3 mice) in AdIl17RCKO BAT. Data are analyzed by unpaired Student’s two-sided t-test and as described in the method section c, Reduced TGFβ1 mRNA in isolated mature adipocytes from AdIl17RCKO BAT (n=3 mice). Data are mean ± SEM and analyzed by One-way ANOVA and Bonferroni’s multiple comparisons test. d, Forced expression of TGFβ1 by TGFβ1-DIO-AAV rescues the cold sensitivity in AdIl17RCKO mice (n=6 mice). e, f, Forced expression of TGFβ1 by TGFβ1-DIO-AAV increases adipose innervation in AdIl17RCKO mice by TH(e) and TUBB3 (f) staining (n=6 mice). scale bar=50um. Data represent 2-3 independent experiments. Data are mean ± SEM and analyzed by unpaired Student’s two-sided t-test and Two-way ANOVA.