STAT6 Signaling Mediates PPARγ Activation and Resolution of Acute Sterile Inflammation in Mice

Abstract

The signal transducer and activator of transcription 6 (STAT6) transcription factor promotes activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway in macrophages. Little is known about the effect of proximal signal transduction leading to PPARγ activation for the resolution of acute inflammation. Here, we studied the role of STAT6 signaling in PPARγ activation and the resolution of acute sterile inflammation in a murine model of zymosan-induced peritonitis. First, we showed that STAT6 is aberrantly activated in peritoneal macrophages after zymosan injection. Utilizing STAT6^-/- and wild-type (WT) mice, we found that STAT6 deficiency further enhanced zymosan-induced proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and macrophage inflammatory protein-2 in peritoneal lavage fluid (PLF) and serum, neutrophil numbers and total protein amount in PLF, but reduced proresolving molecules, such as IL-10 and hepatocyte growth factor, in PLF. The peritoneal macrophages and spleens of STAT6^-/- mice exhibited lower mRNA and protein levels of PPARγ and its target molecules over the course of inflammation than those of WT mice. The deficiency of STAT6 was shown to impair efferocytosis by peritoneal macrophages. Taken together, these results suggest that enhanced STAT6 signaling results in PPARγ-mediated macrophage programming, contributing to increased efferocytosis and inflammation resolution.

Keywords: PPARγ; STAT6; efferocytosis; macrophages; resolution of inflammation.

Figure 1

Enhanced STAT6 activation in peritoneal macrophages from WT mice and STAT6 deficiency in peritoneal macrophages, spleen, and lungs from STAT^−/− mice during zymosan-induced peritonitis. Wild-type (WT) and STAT6^−/− mice were injected i.p. with 1 mg zymosan (Zym) in 500 μL saline, and peritoneal lavage supernatant, spleens and lungs were collected at 6, 24, or 72 h after Zym injection. (A) Left: Immunofluorescence staining for phospho-STAT6 (green) and macrophage-specific marker (Mac3, Red) in peritoneal macrophages (PM) from WT mice. Images were captured at 400× magnification. Right: Quantification of phospho-STAT6 staining in Mac3-positive macrophages. The imaging medium was Vectashield fluorescence mounting medium containing DAPI. Scale bars = 50 μm. Representative results from three mice per group are shown. (B,C) The levels of IL-4 and IL-13 mRNA over time in PM and spleens analyzed by real-time PCR and normalized to that of hypoxanthine guanine phosphoribosyl transferase (Hprt) mRNA. (D) The abundance of IL-4 and IL-13 in peritoneal lavage fluid (PLF) as assessed by ELISA. (E) Left: Immunofluorescence staining for STAT6 (green) in peritoneal macrophages; 400× magnification. Right: Quantification of STAT6 staining using Vectashield fluorescence mounting medium containing DAPI. Scale bars = 50 μm. Representative results from three mice per group are shown. (F) Left: Western blot analysis of total and phosphorylated STAT6 in spleen and lung homogenates. Right: Densitometric analysis of the relative abundance of total and phosphorylated STAT6 normalized to that of β-actin at the indicated times. (G) Left: Western blot analysis of total and phosphorylated JAK3 in spleen. Right: Densitometric analysis of the relative abundance of phosphorylated JAK3 normalized to that of total JAK3 at the indicated times. Values represent the means ± S.E.M. of three (A,E–G) or five mice (B–D) per group. * p < 0.05 compared with saline control; + p < 0.05 for STAT^−/− mice vs. WT mice at a given time point.

Figure 2

The inflammatory response was exacerbated in STAT6^−/− mice. Peritonitis was induced as in Figure 1. The abundance of TNF-α (A), IL-6 (B), MIP-2 (C), IL-10 (D), and HGF (E) in peritoneal lavage fluid (PLF) as assessed by ELISA. Neutrophil count (F) and total protein abundance in PLF (G). The abundance of TNF-α (H), IL-6 (I), and MIP-2 (J) in serum as assessed by ELISA. Values represent the means ± S.E.M. of five mice per group. * p < 0.05 compared with saline control; + p < 0.05 for STAT^−/− mice vs. WT mice at a given time point.

Figure 3

PPARγ expression and activation are decreased in peritoneal macrophages from STAT6^-/- mice. Peritonitis was induced as in Figure 1. (A) Left: The mean fluorescence intensity (MFI) ratio of immunofluorescence staining for PPARγ (green) and DAPI (blue) for the nuclei in peritoneal macrophages (PM) from mice injected with zymosan at the indicated times; 400× magnification. Scale bars = 50 μm. Results are representative of three mice at each time point after zymosan treatment. Middle: Quantification of PPARγ staining. Right: The mean fluorescence intensity (MFI) ratio of basal PPARγ protein expression in PM from STAT6^−/− mice compared with WT controls. (B) Left: Changes in the levels of PPARγ, CD36, MMR, and Arg1 mRNAs over time in PM were analyzed by real-time PCR and normalized to that of Hprt mRNA. Right: The ratio of basal mRNA expression in PM from STAT6^−/− mice compared with WT controls. (C) Left: Changes in the levels of PPARγ, CD36, MMR, and Arg1 mRNAs over time in spleens were analyzed by real-time PCR and normalized to that of Hprt mRNA. Right: The ratio of basal mRNA expression in spleens from STAT6^−/− mice compared with WT controls. (D) Left: Western blot analysis of PPARγ, CD36, MMR, and Arg1 in spleen homogenates. Middle: Densitometric analysis of the relative abundance of the indicated proteins normalized to that of β-actin. Right: The ratio of basal protein expression in spleen homogenates from STAT6^−/− mice compared with WT controls. The values represent the means ± S.E.M. of at least three (A,D) or five mice in each group (B,C). * p < 0.05 compared with saline control; + p < 0.05 for STAT6^−/− mice vs. WT mice at a given time point.

Figure 4

STAT6 reduces efferocytic ability of peritoneal macrophages during zymosan-induced acute inflammation. Peritonitis was induced as in Figure 1. (A) Phagocytic indices in peritoneal lavaged macrophages. (B) Representative photomicrographs (400× magnification) showing cytospun-stained peritoneal lavaged cells at 72 h after zymosan injection. Arrowheads indicate peritoneal macrophages with engulfed apoptotic cells or fragments. Scale bars = 200 μm. Values represent the means ± S.E.M. of five mice per group. * p < 0.05 compared with saline control; + p < 0.05 for STAT6^−/− mice vs. WT mice at a given time point.