STAT6 induces expression of Gas6 in macrophages to clear apoptotic neutrophils and resolve inflammation

Abstract

Efferocytosis of apoptotic neutrophils (PMNs) by alveolar macrophages (AMФs) is vital for resolution of inflammation and tissue injury. Here, we investigated the role of AMФ polarization and expression of the efferocytic ligand Gas6 in restoring homeostasis. In the murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), we observed augmented temporal generation of cytokines IL-4 and TSG6 in bronchoalveolar fluid (BALF). Interestingly, we also observed increased expression of antiinflammatory markers consistent with a phenotype shift in AMФs. In particular, AMФs expressed the efferocytic ligand Gas6. In vitro priming of bone marrow-derived macrophages (BMMФs) with IL-4 or TSG6 also induced MФ transition and expression of Gas6. TSG6- or IL-4-primed BMMФs induced efferocytosis of apoptotic PMNs compared with control BMMФs. Adoptive transfer of TSG6- or IL-4-primed BMMФs i.t. into LPS-challenged mice more rapidly and effectively cleared PMNs in lungs compared with control BMMФs. We demonstrated that expression of Gas6 during AMФ transition was due to activation of the transcription factor signal transducer and activator of transcription-6 (STAT6) downstream of IL-4 or TSG6 signaling. Adoptive transfer of Gas6-depleted BMMФs failed to clear PMNs in lungs following LPS challenge and mice showed severely defective resolution of lung injury. Thus, activation of STAT6-mediated Gas6 expression during macrophage phenotype transition resulting in efferocytosis of PMNs plays a crucial role in the resolution of inflammatory lung injury.

Keywords: Gas6; STAT6; macrophage.

Fig. 1.

MФ phenotype transition promotes efferocytosis. (A) BALF collected from mice challenged with LPS for the indicated time points was used for the measurement of TSG6 and IL-4 by ELISA. n = 6 mice per time point. *P < 0.05 vs. basal. (B) Immunoblots showing arginase-1, CD206, and Gas6 expression in AMФs isolated from BALF from mice challenged with LPS for the indicated time points. Results shown are representative blots with β-actin as a loading control. n = 6 mice per time point. *P < 0.05 vs. basal. (C) Gas6 secreted in BMMФ culture media was measured by ELISA. Results shown are mean ± SD of three experiments. *P < 0.001 vs. control. (D) Scheme of experimental protocol for PMN isolation and apoptosis induction. Apoptosis induction was analyzed by flow cytometry after staining with FITC Annexin V and Propidium Iodide. More than 90% of PMNs were apoptotic (Lower- and Upper-right quadrants) in irradiated group. (E and F) Assessment of efferocytosis by BMMФs in vitro. (E) BMMФs treated with TSG6 (400 ng/mL) or IL-4 (100 ng/mL) for the indicated time points were overlaid for 2 h with apoptotic PMNs labeled with CellTracker CMTPX red dye in the presence or absence of cytochalasin D (2 µM, 2 h). Then the cells were fixed in 1% PFA and analyzed by flow cytometry. BMMФ populations positive for both F4/80 and CMTPX PMNs were considered to be the BMMФs engulfing apoptotic PMNs (Upper-right quadrant). Results shown are from separate experiments. *P < 0.05 vs. control. (F) same as E with confocal analysis to assess efferocytosis by BMMФs. F4/80 antibody BMMФs in green; apoptotic PMNs in red. Quantified data from three different experiments are shown in bar graph (Lower right); *P < 0.05 vs. control. (G) In vivo imaging of PMNs in alveolar space and lung microvessels using high-resolution 2-photon excitation microscopy. BMMФs were stimulated with TSG6 (400 ng/mL) or IL-4 (100 ng/mL) for 6 h and then i.t.-instilled into mice (2 × 10⁶ cells/mouse). Animals simultaneously received LPS (10 mg/kg i.p.) and BMMФs i.t. At 24 h after LPS challenge and BMMФs i.t., 2-photon images were collected as described in Materials and Methods. (Scale bar, 50 μm.) Quantitative analysis of PMN density in lung (n = 30–33 for each bar of wild type) and number of MФs in alveolar space (n = 5, Right). Alveoli were outlined and fluorescent intensities of PMNs were quantified; value of the basal condition in wild-type mice with unstimulated BMMФs was normalized as 1 (Right). *P < 0.001 vs. basal level with unstimulated BMMФs.

Fig. 2.

Transcription factor STAT6 activation promotes Gas6 expression in BMMФs. (A) Schematics of STAT6 binding sites in the 5′-regulatory regions of mouse (m) and human (h) genes encoding Gas6. (B) BMMФs were pretreated with STAT6 inhibitor AS1517499 (1 µM) for 1 h, followed by TSG6 (400 ng/mL; Left) or IL-4 (100 ng/mL; Right) stimulation for the indicated time points. Cell lysates were used for immunoblot analysis to determine STAT6 phosphorylation at Y641 and total STAT6 expression. Representative blots from three independent experiments are shown. *P < 0.05 vs. control. (C) BMMФs pretreated with AS1517499 (1 µM, 1 h) followed by either TSG6 (400 ng/mL) or IL-4 (100 ng/mL) stimulation for 1 h were used for ChIP. TSG6 or IL-4 induced STAT6 binding to three sites (SB-1, SB-3, and SB-4) in the mGas6 promoter. Results are mean of three experiments normalized to those of input DNA and presented relative to control IgG values. *P < 0.05 vs. control. (D) BMMФs pretreated with AS1517499 were stimulated with either TSG6 (400 ng/mL) or IL-4 (100 ng/mL) for 1 h, and Gas6 mRNA expression was determined by RT-qPCR. *P < 0.05 vs. control; #P < 0.05 vs. TSG6- or IL-4–treated BMMФs without AS1517499 pretreatment. (E) Immunoblot analysis of Gas6 protein expression in BMMФs stimulated with TSG6 or IL-4 for the indicated time points with or without AS1517499 pretreatment. Results are representative of three independent experiments. *P < 0.05 vs. control; #P < 0.05 vs. TSG6- or IL-4–treated BMMФs without AS1517499 pretreatment.

Fig. 3.

Gas6 expression in MФs is required for efferocytosis. (A) BMMФs transfected with control or Gas6 siRNA (sc-35451) were used to determine Gas6 protein expression by immunoblot. *P < 0.05 vs. control siRNA group. (B) BMMФs transfected with control or Gas6 siRNA were primed with TSG6 and then incubated with CMTP-labeled PMNs for 2 h. In vitro efferocytosis was analyzed by flow cytometry as in Fig. 1E. *P < 0.05 vs. basal; ^#P < 0.05 vs. TSG6-treated control siRNA group. (C) BMMФs transfected with control or Gas6 siRNA were primed with TSG6 and adoptively transferred i.t. into mice challenged with LPS, followed by in vivo imaging of mouse lungs using high-resolution 2-photon excitation microscopy as in Fig. 1G. *P < 0.05 compared with control siRNA group without TSG6 stimulation. ^#P < 0.05 vs. control siRNA BMMФs primed with TSG6. (D) Proposed signaling mechanisms of inflammation resolution induced by TSG6 or IL-4 reprogramming of MФs. TSG6- or IL-4–induced STAT6 activation promotes Gas6 expression in MФs. Once secreted, Gas6 links phosphatidylserine (PtdSr) on apoptotic PMNs to TAM receptor kinases expressed on MФs. This results in PMN clearance and suppression of proinflammatory cytokine production, thereby resolving lung injury.

Fig. 4.

Adoptive transfer of TSG6-primed MФs via i.t. instillation suppresses LPS-induced lung injury. (A) Age- and weight-matched mice were challenged with a lethal dose of LPS (20 mg/kg i.p.) and i.t.-instilled with indicated number of TSG6- or IL-4–stimulated or unstimulated BMMФs. Mortality was monitored for 72 h. The mice instilled with TSG6- or IL-4–stimulated BMMФs (TSG6 MФs and IL-4 MФs) showed markedly increased survival compared with the unstimulated BMMФs (UT-MФs) group. *P < 0.05 vs. UT MФs group; n = 10 per group. (B) Lung vascular permeability (uptake of EBA) was assessed in LPS-challenged (10 mg/kg i.p.) mice concurrently instilled with UT or TSG6- or IL-4–stimulated MФs. The mice instilled with TSG6- or IL-4–treated MФs showed significantly reduced lung vascular permeability; n = 6 mice per group. *P < 0.05 vs. UT MФs group. (C) Ly6G population was measured by flow cytometric analysis in BALF of mice i.t.-instilled with either UT- or TSG6-treated BMMФs, and challenged with i.p. LPS (10 mg/kg) for 24 h. (D) BALF concentrations of proinflammatory (TNF-α, IL-6, IL-12, and IFN-γ) and antiinflammatory (IL-10 and IL-4) cytokines measured with ELISA. *P < 0.05 vs. UT MФs group.