Volume 26, Issue 3, Pages 493-508.e4 (September 2017) The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity  Zhe Huang, Ling Zhong, Jimmy Tsz Hang Lee, Jialiang Zhang, Donghai Wu, Leiluo Geng, Yu Wang, Chi-Ming Wong, Aimin Xu  Cell Metabolism  Volume 26, Issue 3, Pages 493-508.e4 (September 2017) DOI: 10.1016/j.cmet.2017.08.003 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Cold Exposure Markedly Induces the Expression of FGF21, but Not Its Receptors in Thermogenic Adipose Tissues Twelve-week-old male C57BL/6J mice were housed at thermoneutral condition (30°C) for 2 weeks before cold exposure at 6°C for various time periods as indicated. (A–D) The mRNA expression of Fgf21 in inguinal subcutaneous WAT (scWAT) (A), interscapular BAT (B), epididymal WAT (eWAT) (C), and liver (D), as determined by real-time PCR analysis. (E–H) The protein concentration of FGF21 in scWAT (E), BAT (F), eWAT (G), and liver (H) as determined by ELISA. (I) The relative mRNA abundance of the Fgf21 gene in stromal vascular fraction (SVF) and mature adipocyte (MA) fraction isolated from scWAT of mice housed at 30°C or 6°C for 6 days. (J) Serum levels of FGF21 as determined by ELISA. (K and L) The relative mRNA abundance of the Klb (K) and Fgfr1 (L) in SVF and MA fractions isolated from scWAT of mice housed at 30°C or 6°C for 6 days. Data represent mean ± SEM; n = 4–6 per group; repeated with three independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 A-FGF21KO Mice Exhibit Impaired Adaptive Thermogenesis and Beiging of scWAT during Chronic Cold Exposure Twelve-week-old male A-FGF21KO mice and their FGF21lox/lox (control) littermates housed at 30°C for 2 weeks were subjected to cold exposure at 6°C or continued to be housed at 30°C for 6 days. (A) Rectal temperature of mice measured at different time points after cold exposure. (B) Representative infrared images showing A-FGF21KO and control mice housed at 30°C or 6°C. The right panels are quantification of the mean surface temperature in the inguinal and interscapular regions above scWAT (top) and BAT (bottom), respectively. (C) Whole-body oxygen consumption (VO2) in A-FGF21KO and control mice during 24-hr light/dark cycles by comprehensive laboratory animal monitoring system analysis. Data were normalized with body weight (BW). (D) Real-time PCR analysis for mRNA expression of several thermogenic genes, including uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor α (Cidea), elongation of very long chain fatty acids protein 3 (Elovl3), and carnitine palmitoyltransferase 1b (Cpt1b) in scWAT. (E) Western blot analysis for UCP1 protein expression in scWAT. The bottom panel is the densitometric analysis for the relative abundance of UCP1 normalized with β-Tubulin. (F and G) Representative images of scWAT stained with H&E (F) and with an anti-UCP1 antibody (G). Scale bar, 20 μm. (H) Quantification of mitochondrial DNA (mtDNA) normalized with genomic DNA (gDNA) in scWAT as determined by real-time PCR analysis. (I) Western blot analysis for proteins from four complexes in the electron transport chain (ETC) (CI, NDUFB8; CII, SDHB; CIII, UQCRC2; CV, ATP5A) in protein lysates from scWAT. (J) Basal and norepinephrine (NE)-stimulated oxygen consumption rate (OCR) in the explants of scWAT. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S1–S3. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Adipose-Derived FGF21 Is Required for Cold-Induced Recruitment of Eosinophils and M2 Macrophages, and Proliferation and Commitment of Adipocyte Precursor Cells in scWAT (A and B) SVF isolated from scWAT of 12-week-old male C57BL/6J mice was differentiated into primary adipocytes for 8 days and treated with recombinant FGF21 (rmFGF21, 1 μg/mL) or PBS (as a vehicle control) for 48 hr. The mRNA expression of Ucp1 (A) and the UCP1 protein expression (B) in the adipocytes were measured by real-time PCR and western blot analyses, respectively. Rosiglitazone (Rosi) (1 μM, dissolved in DMSO) was used as a positive control for induction of UCP1. (C–J) Inguinal scWAT was harvested from 12-week-old male A-FGF21KO mice and their control littermates housed at 30°C for 2 weeks before subjecting to exposure at 6°C or 30°C for 2 days. The mRNA expression of Adiponectin (Adn) (C), IL-4, IL-13, and IL-5 (D) in scWAT as determined by real-time PCR analysis. (E) Representative fluorescence-activated cell sorting (FACS) plots showing the frequency of eosinophils (F4/80+ CD11b+ Siglec F+ SSChi) in SVF. The right panel is the quantification of the percentage of eosinophils in total SVF cells from scWAT. (F) Representative FACS plots showing the abundance of type 2 innate lymphoid cells (ILC2s, Lin− CD5– CD45+ CD25+ CD127+) in SVF. The right panel is the quantification of the percentage of ILC2s in Lin− cells. (G) Representative FACS plots showing the proliferating adipocyte precursors (APs) (Lin− CD31– CD45– Sca-1+ and PDGFRα+) in SVF as determined by staining for Ki67 in APs. The right panel is the quantification of the percentage of proliferating cells in APs. (H) Expression of the beige-specific markers, TMEM26 and CD137, on APs. The upper panels are representative histograms of FACS analysis for TMEM26 and CD137. The lower panels are quantifications of mean fluorescence intensity (MFI) of TMEM26 and CD137 in APs. (I) The mRNA expression of the AP markers (Pdgfra and IL-4Ra) and beige lineage-specific markers (Tmem26, Tnfrsf9, and Ear2) in scWAT as determined by real-time PCR analysis. (J) Representative FACS plots of macrophages (M1, F4/80hi CD11b+ CD11c+; M2, F4/80hi CD11b+ CD206+) in SVF. Right panels are quantifications for the percentage of M1 and M2 macrophages in total macrophages (Mφ). Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S4. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 FGF21 Mediates Cold-Induced Expression of CCL11 via ERK1/2 in White Adipocytes (A) The mRNA expression of Ccl11, Ccl24, and Ccl26 in scWAT of 12-week-old male C57BL/6J mice subjected to 6°C exposure for various time periods was measured by real-time PCR analysis. (B and C) Twelve-week-old A-FGF21KO mice or their control littermates were housed at 30°C for 2 weeks before exposure at 6°C or 30°C for another 2 days. The mRNA expression of Ccl11 in scWAT (B) and serum level of CCL11 (C) determined by real-time PCR analysis and ELISA, respectively. (D) The mRNA expression of Ccl11 in mature adipocytes (MA) and SVF fractionated from scWAT of 12-week-old male C57BL/6J mice subjected to 6°C or 30°C for 2 days was determined by real-time PCR analysis. (E and F) SVF cells isolated from scWAT of C57BL/6J mice were differentiated to white adipocytes, followed by treatment with rmFGF21 (1 μg/mL) or PBS (as a vehicle control) for 24 hr. The mRNA expression of Ccl11 in differentiated adipocytes (E) and CCL11 protein secreted into conditioned medium (F) was measured by real-time PCR analysis and ELISA, respectively. (G) Primary adipocytes were pre-incubated with the ERK inhibitors PD98059 (30 μM) and U0126 (10 μM), phosphatidylinositide 3-kinase inhibitor LY294002 (50 μM), or DMSO as control for 1 hr before treatment with rmFGF21 (1 μg/mL) for 24 hr. The mRNA expression of Ccl11 was determined by real-time PCR analysis. Data represent mean ± SEM; n = 4–6 per group; repeated with three independent experiments; ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not statistically significant. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Ablation of β-Klotho in Mature Adipocytes Impairs Cold-Induced Recruitment of Eosinophils and Biogenesis of Beige Cells in scWAT Twelve-week-old male A-KLBKO mice and their KLBlox/lox (control) littermates housed at 30°C for 2 weeks were subjected to cold exposure at 6°C or continued to be housed at 30°C for the indicated time periods. (A) Rectal temperature of mice housed at 30°C or 6°C for 24 hr. (B–H) scWAT collected from mice housed at 6°C or 30°C for 2 days were subjected to analysis for the mRNA expression of Ccl11 in scWAT by real-time PCR (B), the percentage of eosinophils in SVF (C), the percentage of proliferating APs (D), the MFI of beige-specific adipocyte precursor cell markers TMEM26 (E) and CD137 (F), and the percentages of M1 (G) and M2 (H) macrophages in total macrophages from scWAT were determined by flow cytometric analysis as in Figure 3. (I and J) The mRNA expression of several thermogenic genes (I) and protein expression of UCP1 (J) in scWAT from mice housed at 6°C or 30°C for 6 days as determined by real-time PCR and western blot analyses, respectively. The right panel of (J) is the densitometric analysis for the relative abundance of UCP1 normalized with β-Tubulin. (K and L) Representative images of H&E staining (K) and immunohistochemical staining for UCP1 (L) in scWAT from mice exposed to 6°C or 30°C for 6 days. Scale bar, 20 μm. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S5. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 CCL11 Is Obligatory for Cold-Induced Recruitment of Eosinophils and Beiging in scWAT (A) Schematic diagram for the treatment regime. Twelve-week-old male C57BL/6J mice housed at 30°C for 2 weeks were treated with a neutralizing antibody against CCL11 (anti-CCL11, 100 μg/kg/day) or the same amount of isotype antibody by daily bilateral subcutaneous injection into inguinal scWAT and then housed at 6°C or 30°C. (B–D) scWAT was harvested at 2 days after exposure at 6°C or 30°C and analyzed for the percentage of eosinophils in SVF by flow cytometry (B), and mRNA expression of eosinophil markers (Siglec F and IL-4) (C) and beige-specific AP markers (D) by real-time PCR analysis. (E and F) scWAT collected at 3 days after exposure at 6°C or 30°C were analyzed for the mRNA expression of a panel of thermogenic genes by real-time PCR analysis (E), and protein expression of UCP1 by western blot analysis (F). The right panel of (F) is the densitometric analysis for the relative abundance of UCP1 normalized with β-Tubulin. (G and H) Representative images of H&E staining (G) and immunohistochemistry for UCP1 (H) in scWAT from mice exposed to 6°C or 30°C for 3 days. Scale bar, 20 μm. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Replenishment of Recombinant CCL11 Reverses Impairments in Cold-Induced Beiging of scWAT in A-FGF21KO Mice (A) Schematic diagram for the experimental procedures. Twelve-week-old male A-FGF21KO mice housed at 30°C for 2 weeks were treated with recombinant mouse CCL11 protein (20 μg/kg/day) or PBS by daily bilateral subcutaneous injection into inguinal scWAT and subjected to cold challenge (6°C). Wild-type control mice receiving PBS were used as a control. (B–F) scWAT harvested at 2 days after cold challenge or thermoneutral exposure was analyzed for the percentage of eosinophils in SVF by flow cytometry (B) and mRNA expression of eosinophil markers (Siglec F and IL-4) (C) and beige-specific adipocyte precursor markers (D) by real-time PCR analysis. The mRNA expression level of several thermogenic genes (E) and protein expression of UCP1 (F) in scWAT collected at 3 days post cold challenge or thermoneutral exposure were measured by real-time PCR and western blot analysis, respectively. The right panel of (F) is the densitometric analysis for the relative abundance of UCP1 normalized with β-Tubulin. (G and H) Representative images of H&E staining (G) and immunohistochemistry for UCP1 (H) in scWAT from mice at 3 days post cold challenge or thermoneutral exposure. Scale bar, 20 μm. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not statistically significant. See also Figure S6. Cell Metabolism 2017 26, 493-508.e4DOI: (10.1016/j.cmet.2017.08.003) Copyright © 2017 Elsevier Inc. Terms and Conditions