Genotypical comparison of T_reg and T_conv cells isolated from brown adipose tissue (BAT) and spleen tissue (SPL) in cold- and warm-conditioned animals generated with an Illumina Mouse Expression Array.

<p>(A) Gene expression profiles comparing T_reg (top) or T_conv (bottom) cell populations between spleen and adipose tissue samples isolated from warm-conditioned animals (left) or between cells isolated from cold vs warm-conditioned animals (right). Numbers indicate genes either up- or downregulated more than 2-fold (cut-off: dotted line), with the number of significantly different (p<0.05) genes shown in brackets with an asterisk. (B) Volcano plot comparing gene expression and significance values between T_reg and T_conv genes isolated from BAT in warm-conditioned animals. Key up- or downregulated genes in T_reg cells are annotated (Foxp3, Il10, Cxcl1/2, Tcf7, Ifng) and serve as quality control to the published consensus T_reg-cell signature. (C) Hierarchical clustering of the top-30 upregulated genes and the top-10 downregulated genes in warm-conditioned brown adipose tissue T_reg cells versus spleen T_reg cells. (D) Comparison of BAT-T_reg-specific genes with visceral adipose tissue (VAT)-specific genes. We first determined 430 genes to upregulated in BAT warm-conditioned T_reg cells, with 222 genes being significantly altered (p<0.05). We then overlaid BAT T_reg-upregulated genes with VAT T_reg tissue specific expression gene data. 181 genes were matched between both microarary chips, with 169 genes also upregulated in VAT, and only 12 genes specific for BAT (left). The corresponding analysis of the 516 genes upregulated in cold BAT T_reg cells versus warm spleen T_reg cells revealed 194 genes to be significantly upregulated. 158 could be matched to VAT T_reg-specific genes, of which 148 were VAT-specific, whereas only 10 were specific for BAT. P-values indicate the significance of overrepresentation of BAT T_reg-specific genes in the VAT T_reg signature. (E) Comparison of VAT-T_reg specific genes on BAT warm (left) or BAT cold (right) gene signatures. Of 1839 genes specifically overexpressed in VAT T_reg cells, 1059 were statistically significantly (p<0.05) upregulated. Of these 1059 genes, 829 were also detectable in the BAT T_reg microarray. When comparing the VAT T_reg signature to warm BAT T_reg cells, 660 genes were overrepresented in VAT, whereas cold BAT T_reg cells show 685 genes to be overrepresented in VAT.</p

Physiological parameters.

<p>(A) Body weight (BW) and (B) adipose tissues weights of T_reg cell-proficient (PBS) and T_reg cell-deficient (DT) mice after cold exposure. BAT, brown adipose tissue; scWAT, subcutaneous white adipose tissue, aWAT, abdominal white adipose tissue. (C) Blood glucose, (D) serum non-estherified fatty acids (NEFA) and (E) serum triglycerides in PBS and DT mice. Values are mean ± SD (n = 9–10); *P<0.05 (Student’s t-test).</p

Inflammatory status of adipose tissue.

<p>Real-time RT-PCR analysis of (A) brown adipose tissue (BAT) and (B) subcutaneous white adipose tissue of T_reg cell-proficient (PBS) and T_reg cell-deficient (DT) mice after cold exposure. Ucp1, uncoupling protein 1; Cidea,cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; Dio2,deiodinase, iodothyronine, type II; Pparg, peroxisome proliferator-activated receptor gamma; Prdm16, PR domain containing 16; Cd68, Cd68 antigen; Ccl2,chemokine (C-C motif) ligand 2; Tnfa, tumor necrosis factor alpha; Ifng, interferon, gamma; Mrc1, mannose receptor, C type 1; Mgl1, macrophage galactose-type C-type lectin 1; Arg1,arginase 1; Il-10, interleukin 10; Il-4, interleukin 4. Data are mean ± SD (n = 9–10); *p<0.05 (Student’s t-test). (C) Representative hematoxylin and eosin (H&E) staining (left) and immunohistochemical anti-MAC-2 staining (right; brown color) in BAT from PBS and DT mice. Scale bar 100 μm. Quantification of MAC-2 positive area (panel below MAC-2 staining) as a percentage of total area.</p

Principal biological processes affected by T_reg cell depletion in BAT.

<p>Principal biological processes affected by T_reg cell depletion in BAT.</p