A Stat6/Pten Axis Links Regulatory T Cells with Adipose Tissue Function

Abstract

Obesity and type 2 diabetes are associated with metabolic defects and adipose tissue inflammation. Foxp3⁺ regulatory T cells (Tregs) control tissue homeostasis by counteracting local inflammation. However, if and how T cells interlink environmental influences with adipocyte function remains unknown. Here, we report that enhancing sympathetic tone by cold exposure, beta3-adrenergic receptor (ADRB3) stimulation or a short-term high-calorie diet enhances Treg induction in vitro and in vivo. CD4⁺ T cell proteomes revealed higher expression of Foxp3 regulatory networks in response to cold or ADRB3 stimulation in vivo reflecting Treg induction. Specifically, Ragulator-interacting protein C17orf59, which limits mTORC1 activity, was upregulated in CD4⁺ T cells by either ADRB3 stimulation or cold exposure, suggesting contribution to Treg induction. By loss- and gain-of-function studies, including Treg depletion and transfers in vivo, we demonstrated that a T cell-specific Stat6/Pten axis links cold exposure or ADRB3 stimulation with Foxp3⁺ Treg induction and adipose tissue function. Our findings offer a new mechanistic model in which tissue-specific Tregs maintain adipose tissue function.

Keywords: Borcs6; C17orf59; Foxp3; PTEN; STAT6; T cells; Tregs; adipose tissue function; cold exposure; metabolic function; metabolism; regulatory T cells.

Figure 1. Frequencies and induction of regulatory T (Treg) cells in fat depots

(A) Representative FACS plots for the identification of fat-residing CD4⁺T cells purified from BAT, scWAT or visWAT of young lean female Balbc Foxp3 GFP reporter mice. CD4⁺T cells were gated on live CD14⁻, F4/80⁻, CD8a⁻, CD11b⁻, CD11c⁻, B220, sytox and CD4⁺. (B) Representative FACS plots for the identification of fat-residing CD4⁺CD25⁺Foxp3 GFP⁺Tregs in BAT, scWAT or visWAT. (C) Box-and-whisker plots for frequencies of Foxp3 GFP⁺ Tregs residing in fat-depots as indicated in B. n=22 per group from 5 independent experiments. (D) Representative FACS plots for in vitro Treg induction assays using limited TCR stimulation and naïve CD4⁺T cells purified from different fat depots. (E) Box-and-whisker plots for in vitro Treg induction assays of fat-residing CD4⁺T cells. n=6 per group. (F) Box-and-whisker plots of absolute Treg numbers obtained from Treg induction experiments starting with identical numbers of naïve CD4⁺T cells from respective fat-tissues. n=6 per group. (G) Representative confocal microscopy images of CD4⁺T cells purified from mice upon in vivo cold exposure (4 days at 8°C). (H) Foxp3⁺CD3⁺T cells per high power field in samples from (G). n=8 per group. Data are shown as means±SEM from 2 independent experiments. (I) Ex vivo Treg frequencies purified from fat-depots of young Balbc mice upon in vivo cold exposure (24 h at 4°C). n=9 per group. (J) In vitro Treg induction assays of fat-residing naïve CD4⁺T cells after in vivo cold-exposure (24 h at 4°C). n=6 per group. Data are presented as box-and-whisker plots with min and max values for data distribution, ** = P<0.01, *** = P<0.001.

Figure 2. Beta-adrenergic stimulation promotes T cell tolerance

(A) Representative confocal microscopy images of CD4⁺T cells purified from Balbc Foxp3 GFP reporter mice after in vivo treatment with CL (2 d, 1 mg/kg i.p.). (B) Foxp3⁺CD3⁺T cells per high power field in samples from (A). n=5 per group, P=0.0003. (C) Representative FACS plots for the identification of ex vivo CD4⁺CD25⁺Foxp3 GFP⁺Tregs from BAT upon in vivo treatment with CL (3 d, 1 mg/kg i.p.). (D) Summary graph for ex vivo Treg frequencies purified from fat-depots of young Balbc mice as in (C). n=6 per group. (E) Summary graph for in vitro Treg induction assays with naïve CD4⁺T cells from adipose tissues after in vivo treatment with CL (3 d, 1 mg/kg i.p.). n=6 per group. (F) Representative FACS plots for the identification of ex vivo CD4⁺CD25⁺Foxp3⁺Tregs from fat depots of WT or mice lacking all three beta adrenergic receptors (betaless mice). (G) Summary graph for ex vivo Treg frequencies purified from inguinal lymph nodes of WT or betaless mice. n=11 per group. (H) Summary graph for ex vivo Treg frequencies purified from fat depots of WT mice or betaless mice. n>12 per group. (I) Summary graph for in vitro Treg induction assays of naïve CD4⁺T cells purified from fat depots or inguinal lymph nodes of WT or betaless mice. n=6 per group. Data are presented as box-and-whisker plots with min and max values for data distribution or as means±SEM. * = P<0.05, ** = P<0.01, *** = P<0.001.

Figure 3. Role of Tregs induced by ADRB3 stimulation or cold exposure for adipose tissue function

(A) Representative FACS plots demonstrating Treg depletion efficacy in inguinal lymph nodes after 3 d of mCD25 antibody treatment. (B) Representative FACS plots demonstrating Treg depletion efficacy in inguinal lymph nodes 48 h after administration of diphtheria toxin. (C) mRNA expression of genes involved in BAT function upon treatment with CL in vivo in the presence or absence of Tregs. Tregs were depleted using anti-CD25 depleting antibodies. n=6 per group. (D–F) mRNA expression of genes involved in BAT (D), scWAT (E) and visWAT (F) function after cold exposure (4°C, 24h) in the presence or absence of Tregs. Tregs were depleted in Foxp3 DTR mice by administration of diphtheria toxin. n=6 per group. (G) In gain-of-function experiments, CD4⁺CD25⁺Foxp3GFP⁺Tregs were adoptively transferred into congenic recipients. Analyses of BAT function by qPCR was performed 1 wk after transfer. n=6 per group. Data are presented as box-and-whisker plots with min and max values for data distribution. * = P<0.05, ** = P<0.01, *** = P<0.001.

Figure 4. Role of Stat6 in T cell tolerance of fat-residing T cells

(A) Depicted are the top 5 fold-changes for upregulated genes in CD4⁺T cells purified from brown vs. white fat by mRNA expression profiling. The cut-off for reading counts was set to 30 and pseudogenes have been manually removed. (B) Representative confocal microscopy images of CD4⁺T cells purified from Balbc control mice or Balbc mice after in vivo treatment with CL (3 d, 1 mg/kg i.p.). (C) Quantitative RT-qPCR analyses of Stat6 mRNA abundance in CD4⁺T cells purified from Balbc mice upon cold exposure (1 wk, 8°C). n=4 mice per group from 2 independent experiments. (D) Stat6 mRNA expression in CD4⁺T cells purified from BAT, scWAT or visWAT of young lean Balbc mice. n=5 mice per group. (E) Representative confocal microscopy images for p-Stat6 induction in CD4⁺T cells from inguinal lymph nodes after CL treatment (3 d at 1 mg/kg i.p.) and in vivo cold exposure (4°C, 24 h). (F) Quantification of p-Stat6⁺CD4⁺T cells per high power field in samples from (E) after cold exposure. n=4 per group. (G) Quantification of p-Stat6⁺CD4⁺T cells per high power field in samples from (E) after CL treatment. n=4 per group. (H) Histogram of p-Stat6 detection in pre-activated CD4⁺T cells after 10 nM CL stimulation for 15 min in vitro. (I) Representative FACS plots for the identification of ex vivo CD4⁺CD25⁺Foxp3⁺Tregs from BAT of WT or Stat6ko mice. (J) Summary graph for ex vivo Treg frequencies purified from fat depots of WT mice or Stat6ko mice. n=10 per group. (K) Summary graph for in vitro Treg induction assays of naïve CD4⁺T cells purified from fat depots of WT or Stat6ko mice. n=6 per group. (L) Summary graph for in vitro Treg induction assays of naïve CD4⁺T cells purified from BAT, scWAT and visWAT Stat6ko mice that were treated with vehicle or CL (3 d, 1 mg/kg) in vivo. (M) Representative FACS blots for ex vivo Treg frequencies purified from inguinal lymph nodes of WT mice or mice with constitutively active Stat6 (Stat6VT mice). n=10 per group. (N) Summary graph for ex vivo CD4⁺CD25⁺Foxp3⁺ Treg frequencies purified from fat depots of Stat6VT⁺ or Stat6VT⁻ mice. n=4 per group. Data are presented as box-and-whisker plots with min and max values for data distribution. * = P<0.05, ** = P<0.01, *** = P<0.001.

Figure 5. Role of Pten in T cell tolerance of fat-residing CD4⁺T cells

Quantitative RT-qPCR analyses of Pten mRNA abundance in (A) CD4⁺T cells purified from BAT, scWAT or visWAT of young lean Balbc animals. n=5 per group, (B) CD4⁺T cells purified from inguinal lymph nodes after in vivo cold exposure of Balbc animals. n=5 per group and in (C) CD4⁺T cells purified from inguinal lymph nodes of Stat6ko animals, n=4 per group. (D) Representative FACS plots identifying ex vivo CD4⁺CD25⁺Foxp3⁺Tregs from inguinal lymph nodes of WT or mice transgenetically-overexpressing Pten (PtenTg animals). (E) Summary graph for the identification of ex vivo CD4⁺CD25⁺Foxp3⁺Tregs purified from BAT, scWAT or visWAT of WT or PtenTg mice. n=5 per group. (F) Summary graph for in vitro Treg induction assays using limited TCR stimulation of naïve CD4⁺T cells purified from fat depots of WT or PtenTg animals. n=6 per group. (G) Summary graph for in vitro Treg induction assays using limited TCR stimulation of naïve CD4⁺T cells of PtenTg animals in the presence or absence of ADRB3 stimulation [0.001 nM CL]. n=5 per group. (H) Ex vivo Treg frequencies purified from fat-depots of PtenTg mice upon in vivo ADRB3 stimulation (3 d, 1 mg/kg CL). n=8 for PtenTg control group and n=4 for PtenTg + CL group. (I) Representative FACS plots for in vitro Treg induction assays with or without Pten inhibitor (500 nM) using naïve CD4⁺T cells purified from inguinal lymph nodes of WT mice. (J) Summary graph of Pten inhibition for in vitro Treg induction assays of naïve CD4⁺T cells purified from inguinal lymph nodes of WT animals. n=4 per group from 2 independent experiments. Data are presented as box-and-whisker plots with min and max values for data distribution or as means±SEM. * = P<0.05, ** = P<0.01, *** = P<0.001.

Figure 6. Cold exposure or ADRB3 stimulation induces a tolerogenic proteome signature in CD4⁺T cells

(A+B) Proteins associated with selected GeneOntology terms Biological Function (GOBP) were grouped using unsupervised hierarchical clustering of the z-scored MaxLFQ-intensities across the indicated experimental groups; CL vs. NaCl (A) and cold vs. RT (B). GOBP annotations are depicted in purple. (C+D) Proteins associated with the Foxp3 regulatory network were grouped using unsupervised hierarchical clustering of the z-scored MaxLFQ-intensities across the indicated experimental groups; CL vs. NaCl (C) and cold vs. RT (D). (E–G) Combined volcano plot of the pairwise comparison between CD4⁺T cell proteomes purified from cold/CL- vs. RT/NaCl-treated mice (E), NaCl- vs. CL-treated mice (F) and NaCl- vs. CL-treated Stat6ko mice (G). Expression fold changes (t-test difference, log₂) were calculated and plotted against the t-test p-value (–log₁₀). Proteins associated with Foxp3 regulatory networks (red) and the C17orf59 homologue (blue) are highlighted. Their position on the right side of the plot indicates a higher abundance upon cold/CL- (E) or CL-treatment (F+G). (H+I) Quantitative RT-qPCR analyses of Borcs6 mRNA abundance in CD4⁺T cells purified from mice after in vivo ADRB3 stimulation (H) or cold exposure (I). n=5 mice per group. Data are presented as box-and-whisker plots with min and max values for data distribution. * = P<0.05, ** = P<0.01.

Figure 7. ADRB3 stimulation or cold-exposure increases C17orf59 protein expression in CD4⁺T cells

(A) Representative confocal microscopy images for C17orf59 expression of CD4⁺T cells purified from inguinal lymph nodes of mice exposed to cold in vivo (24 h, 4°C). (B) C17orf59⁺CD4⁺T cells per high power field in samples from CD4⁺T cells from (A). n=9 for control group at room temperature and n=6 for group at 4°C. (C) Representative high magnification confocal images for C17orf59 expression of CD4⁺T cells purified from inguinal lymph nodes of mice subjected to ADRB3 stimulation (3 d, 1 mg/kg CL). (D) C17orf59⁺CD4⁺T cells per high power field in samples from (C). n=5 per group. (E) Stimulated emission depletion (STED) microscopy of cytoplasmatic C17orf59 expression in CD4⁺T cells purified from inguinal lymph nodes of mice upon cold exposure. (F) Representative confocal microscopy images for Lamp2 and mTOR expression in CD4⁺T cells purified from inguinal lymph nodes of mice subjected to ADRB3 stimulation or cold exposure in vivo. (G+H) Single cell magnifications and depiction of single stainings of samples from (F) after cold exposure (G) or ADRB3 stimulation (H). (I) mTOR⁺Lamp2⁺T cells per high power field in samples from CD4⁺T cells purified from inguinal lymph nodes of mice subjected to CL (3 d, 1 mg/kg CL). n=6 per group. (J) mTOR⁺Lamp2⁺T cells per high power field in samples from CD4⁺T cells purified from inguinal lymph nodes of mice subjected to cold exposure (24 h, 4°C) in vivo. n=6 per group. (K) Representative confocal microscopy images for C17orf59 expression in CD4⁺T cells purified from inguinal lymph nodes of WT or Stat6ko mice. (L) Negative staining control of C17orf59. Shown are stainings with secondary antibody in the absence of primary antibodies using CD4⁺T cells from WT mice. Data are presented as box-and-whisker plots with min and max values for data distribution. * = P<0.05, ** = P<0.01, *** = P<0.001.