Deficiency of Stat1 in CD11c+ Cells Alters Adipose Tissue Inflammation and Improves Metabolic Dysfunctions in Mice Fed a High-Fat Diet

Abstract

CD11c⁺ macrophages/dendritic cells (MDCs) are increased and display the classically activated M1-like phenotype in obese adipose tissue (AT) and may contribute to AT inflammation and insulin resistance. Stat1 is a key transcription factor for MDC polarization into the M1-like phenotype. Here, we examined the role of Stat1 in obesity-induced AT MDC polarization and inflammation and insulin resistance using mice with specific knockout of Stat1 in MDCs (cKO). Stat1 was upregulated and phosphorylated, indicating activation, early and persistently in AT and AT MDCs of wild-type mice fed a high-fat diet (HFD). Compared with littermate controls, cKO mice fed an HFD (16 weeks) had reductions in MDC (mainly CD11c⁺ macrophage) M1-like polarization and interferon-γ-expressing T-helper type 1 (Th1) cells but increases in interleukin 5-expressing Th2 cells and eosinophils in perigonadal and inguinal AT, and enhanced inguinal AT browning, with increased energy expenditure. cKO mice compared with controls also had significant reductions in triglyceride content in the liver and skeletal muscle and exhibited improved insulin sensitivity and glucose tolerance. Taken together, our results demonstrate that Stat1 in MDCs plays an important role in obesity-induced MDC M1-like polarization and AT inflammation and contributes to insulin resistance and metabolic dysfunctions in obese mice.

Figure 1

HFD in WT mice increases Stat1 levels and phosphorylation in pAT and CD11c⁺ MDCs and adipocytes from pAT. tStat1 and pStat1 levels examined by Western blotting in pAT (A) and MDCs (B) and adipocytes (C) from pAT of male WT C57BL/6J mice fed ND or HFD for 1 week or 16 weeks. Correlation of mRNA levels of STAT1 and CD11c in visceral AT from obese humans (D). Data are mean ± SD. *P < 0.05, **P < 0.01.

Figure 2

cKO mice do not have altered HFD-induced weight gain and fat mass but have reduced liver weight. Stat1 mRNA in pAT and iAT (A) and Stat1 protein levels in adipocytes, CD11c⁺ MDCs, and CD11c⁻ SVCs isolated from pAT (B) of male cKO mice and littermate controls fed HFD (16 weeks). Body weight changes of male cKO mice and littermate controls fed HFD or ND (C). Total fat mass (D) and lean mass (E) determined with a PIXImus small animal densitometer and weight and relative ratio to body weight of the pAT pad (F) and liver (G) of male cKO mice and littermate controls fed ND or HFD (16 weeks). *P < 0.05. Con, control.

Figure 3

cKO mice on HFD exhibit an improved macrophage phenotype in pAT and iAT. Male cKO mice and littermate controls were fed HFD for 16 weeks. Flow cytometric analysis of F4/80⁺ macrophages and DCs in SVCs isolated from pAT (A and C) and iAT (B and D) of cKO and control mice showing total F4/80⁺ cells, percentages of CD11c⁺ (M1-like MDC) and CD206⁺ (M2-like macrophage) cells in F4/80⁺ cells, ratio of CD11c⁺ to CD206⁺ cells (A and B), and intracellular levels of IL-12 and TNF-α in CD11c⁺ (F4/80⁺) MDCs (C and D). Flow cytometric analysis of CD11c⁺CD64⁺ macrophages and CD11c⁺CD64⁻ DCs with CD11c⁺ MDCs of SVCs from pAT (E and G) and iAT (F and H) of cKO and control mice showing proportions of macrophages and DCs within MDCs (E and F) and intracellular levels of IL-12 and TNF-α in CD11c⁺ macrophages and DCs (G and H). RT-qPCR gene expression analysis of pAT (I) and iAT (J) of cKO and control mice showing mRNA levels of M1- and M2-like markers and cytokines. *P < 0.05, **P < 0.01. Con, control; MFI, mean fluorescence intensity.

Figure 4

T-cell–mediated inflammation and eosinophils are altered in pAT and iAT of cKO mice fed HFD. Male cKO mice and littermate controls were fed HFD for 16 weeks. Flow cytometric analysis of T cells in SVCs isolated from pAT (A and C) and iAT (B and D) of cKO and control mice showing CD3⁺ total T cell numbers, percentages of CD8⁺ and CD4⁺ T cells in total T cells (A and B), and intracellular expression of IFN-γ (for Th1), IL-5 (for Th2), and Tregs in CD4⁺ T cells (C and D). RT-qPCR gene expression analysis of pAT (E) and iAT (F) of cKO and control mice showing mRNA levels of T-cell markers and cytokines. RT-qPCR and flow cytometry analyses of pAT (G) and iAT (H) of cKO and control mice showing eosinophils (CD170⁺) and the related marker (CD170). *P < 0.05, **P < 0.01. Con, control.

Figure 5

cKO mice fed HFD have enhanced AT browning and improved energy metabolism. Male cKO mice and littermate controls were fed HFD for 16 weeks. Representative hematoxylin and eosin (H & E) staining of pAT and iAT sections and quantitation of adipocyte size in pAT and iAT of cKO and control mice (A). mRNA levels of browning/beige adipogenesis markers examined by RT-qPCR in iAT of cKO and control mice (B). Representative immunohistochemistry staining and quantification of UCP1 expression in pAT and iAT of cKO and control mice treated with β3-agonist (β3-AG, CL316,243) or vehicle control (C). Comprehensive Lab Animal Monitoring System analysis of cKO mice and littermate controls showing VO₂ (D), respiratory exchange ratio (E), food intake (F), and activity (G). n = 5 mice/group. TG content quantified in the liver and skeletal muscle of cKO and control mice (H). *P < 0.05, ***P < 0.001. Con, control.

Figure 5

cKO mice fed HFD have enhanced AT browning and improved energy metabolism. Male cKO mice and littermate controls were fed HFD for 16 weeks. Representative hematoxylin and eosin (H & E) staining of pAT and iAT sections and quantitation of adipocyte size in pAT and iAT of cKO and control mice (A). mRNA levels of browning/beige adipogenesis markers examined by RT-qPCR in iAT of cKO and control mice (B). Representative immunohistochemistry staining and quantification of UCP1 expression in pAT and iAT of cKO and control mice treated with β3-agonist (β3-AG, CL316,243) or vehicle control (C). Comprehensive Lab Animal Monitoring System analysis of cKO mice and littermate controls showing VO₂ (D), respiratory exchange ratio (E), food intake (F), and activity (G). n = 5 mice/group. TG content quantified in the liver and skeletal muscle of cKO and control mice (H). *P < 0.05, ***P < 0.001. Con, control.

Figure 6

cKO mice on HFD have improved insulin resistance. Male cKO mice and littermate controls were fed HFD for 16 weeks. Blood glucose levels after fasting for 6 h (A). ITT (B). n = 6–8 mice/group. GTT (C). n = 5 mice/group. Insulin-induced Akt phosphorylation determined by Western immunoblot analysis in pAT (D), skeletal muscle (E), and liver (F). *P < 0.05, **P < 0.01. Con, control.

Figure 7

Reducing eosinophils does not affect insulin sensitivity in HFD-fed cKO mice. Male cKO mice that had been fed HFD for 12 weeks were injected intraperitoneally with 40 μg of anti-mouse IL-5 antibody (Ab) or isotype control once every 72 h for a total of 11 injections. Eosinophil content in pAT and iAT analyzed using flow cytometry at 2 days after the 11th injection (A). ITT performed at 2 days after the ninth injection (B). n = 4 mice/group. GTT performed at 2 days after the 10th injection (C). n = 4 mice/group. Macrophages and DCs (D) and T cells (E) analyzed using flow cytometry and mRNA levels of UCP1 and Prdm16 examined by RT-qPCR (F) in AT at 2 days after the 11th injection. *P < 0.05, ***P < 0.001.

Figure 8

Potential mechanisms for alterations in AT inflammation and improvements in insulin sensitivity in mice with STAT1 ablation in MDCs.