a, b, Real time-PCR analysis of markers of alternative and classical activation in BAT (a) and WAT (b) of wild type (WT) mice chronically housed at 30 °C, 22 °C, or acutely subjected to a 4 °C from 22 °C (n=4 per temperature). Expression of all genes is normalized to their relative expression at 30 °C in WT mice. c-e, Expression of alternative activation markers Arg-1, CD206, and CD301 was monitored by flow cytometry in BAT (c, d) and WAT (e) macrophages of wild type (WT), IL4/IL13^−/−, and STAT6^−/− mice housed at 30 °C, 22 °C, and 4 °C (n=4-5 per genotype and temperature). f, Alternative activation of tissue macrophages was monitored at 22 °C and 4 °C by quantifying expression of CD301. BM (bone marrow). *P < 0.05, **P < 0.01, ***P < 0.001 comparison between WT at 30 °C and 22 °C, or between 22 °C and 4 °C. φP < 0.05, φφφP < 0.001 comparison between WT and various knockout mice at the same temperature.

a, Core body temperature of WT, IL4/IL13^−/−, and STAT6^−/− mice during a cold challenge at 4°C (n=8 per genotype and temperature). b, c, Real-time PCR analysis of thermogenic genes in BAT of WT, IL4/IL13^−/−, and STAT6^−/− mice housed at 30 °C, 22 °C or subjected to 4 °C cold challenge (n=4-5 per genotype and temperature). Expression of all genes is normalized to their relative expression at 30 °C in WT mice. d, Expression of alternatively activated mRNAs in BAT of IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice housed at various temperatures (n=5 per genotype and temperature). e, Core body temperature of IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice during exposure to 4 °C (n=5-6 per genotype and temperature). f, BAT of IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice was analyzed by real-time PCR for expression of thermogenic and β-oxidation genes (n=5 per genotype and temperature). Expression of all genes is normalized to their relative expression at 30 °C in IL4Rα^L/L mice. g, Serum free fatty acid (FFA) levels in WT, IL4/IL13^−/−, and STAT6^−/− mice housed at 30 °C, 22 °C, and 4 °C (n=5-8 per genotype). h, Serum FFAs in IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice housed at the three temperatures (n=5-11 per genotype). i, Representative gross and microscopic (haematoxylin and eosin staining) histology and of BAT from WT, IL4/IL13^−/−, and STAT6^−/− mice at 22 °C and after exposure to 4°C for 6 hours. j, Representative gross and microscopic (haematoxylin and eosin staining) histology of BAT from IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice at 22 °C and after 6 hour exposure to 4°C. k, Immunoblot analysis for serine phosphorylated perilipin, total perilipin, serine phosphorylated-HSL and total HSL in 3T3-L1 adipocytes treated with PIA, CL-316243, IL4 or macrophage conditioned medium (± IL4 and AMPT) for 15 min. PIA (N6-phenylisopropyl adenosine), AMPT (α-methyl-p-tyrosine). l, Glycerol release by 3T3-L1 adipocytes after 6h treatment with PIA, CL-316243 (CL), IL4 or macrophage conditioned medium (n=5-7). *P < 0.05, **P < 0.01, ***P < 0.001 compared to comparison between WT or IL4Rα^L/L at 30 °C and those at 22 °C, or at 22 °C and 4 °C. φP < 0.05, φφP < 0.01, φφφP < 0.001 comparison between knockouts and WT or IL4Rα^L/L at the same temperature.

a, Expression of tyrosine hydroxylase in WT and STAT6^−/− peritoneal macrophages treated with vehicle (veh) or IL4, n=5 per genotype and condition. b, Noradrenaline secretion by WT and STAT6^−/− bone-marrow-derived macrophages stimulated with IL4 or LPS (n=5 per genotype and condition). c, e Tyrosine hydroxylase expression in BAT (c) and WAT (e) macrophages of WT and STAT6^−/− mice at 30 °C, 22 °C and 4 °C (n=5 per genotype and temperature). d, f, Noradrenaline content of BAT (d) and WAT (f) at 22 °C and 4 °C of WT and STAT6^−/− mice (n=4-5 per genotype and temperature). *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT. φP < 0.05, φφP < 0.01, φφφP < 0.001 compared to WT with IL4 at 4 °C samples.

a,b, Expression of alternative activation marker CD301 (a) and TH (b) in adipose tissue macrophages from IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice treated with vehicle (Veh) or IL4 for 6 hours at 22 °C (n=4-5 per genotype and condition). c, Real-time PCR for thermogenic genes in BAT of IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice treated with Veh or IL4 for 6 hours at 22 °C (n=4-5 per genotype and condition). d, Serum free fatty acid (FFA) levels in IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice treated with Veh or IL4 for 6 hours at 22 °C (n=4-5 per genotype and condition). e, f, Quantification of energy expenditure in IL4Rα^L/L and IL4Rα^L/LLysM^Cre mice treated with vehicle (Veh) or IL4 (n=7-9 per genotype and condition). (e) Oxygen consumption (VO₂) and (f) respiratory exchange ratio (RER). g, h, Quantification of energy expenditure in WT mice after macrophage depletion (n=8 per condition). Mice were injected with empty liposomes (Lipo) or clodronate-containing liposomes (Clod) 24 hours prior to energy expenditure studies. All data were collected during the light cycle. *P < 0.05, **P < 0.01, ***P < 0.001 compared to IL4Rα^L/L with Veh. φP < 0.05, φφP < 0.01, φφφP < 0.001 compared to IL4Rα^L/L with IL4.