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

Saroj Nepal,Chinnaswamy Tiruppathi,Yoshikazu Tsukasaki,Joseph Farahany,Manish Mittal,Jalees Rehman,Darwin J Prockop,Asrar B Malik

doi:10.1073/pnas.1821601116

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.

Highlights

Efferocytosis of apoptotic neutrophils (PMNs) by alveolar macrophages (AMVs) is vital for resolution of inflammation and tissue injury
We showed that MV secretes the protein TNF-α-stimulated gene-6 (TSG6), which is essential for MV phenotype transition; further, it promotes the resolution of sepsis-induced acute lung injury [14]
To elucidate mechanisms of efferocytosis activated during MV phenotype transition, we focused on the role of IL-4 and TSG6 signaling

Summary

Results

MV Phenotype Transition Promotes Efferocytosis in Alveoli. To determine whether AMV phenotype shift occurs during resolution of endotoxin-induced inflammatory lung injury, we injected LPS (10 mg/kg i.p.) into wild-type (WT) mice. We determined the effects of Gas knockdown on MV efferocytosis in vitro and observed significantly reduced engulfment of PMNs compared with control TSG6-primed BMMVs (Fig. 3B). We suppressed Gas expression using siRNA in MVs, and observed decreased MV efferocytosis in vitro (SI Appendix, Fig. S1 A and B), further supporting the requisite role of Gas in the mechanism of efferocytosis in phenotype-shifted MVs. In addition, we determined the effects of Gas depletion in BMMVs in clearing lung airspace PMNs in vivo. In contrast to the active efferocytosis of airspace PMNs induced by instillation of TSG6-primed BMMVs (Fig. 1G), we found that instillation of Gas6-depleted BMMVs failed to clear PMNs (Fig. 3C) Based on these results, we propose a model for the mechanism of resolution of lung injury by MVs through activation of PMN efferocytosis following phenotype transition of MVs (Fig. 3D). We observed that i.t. instillation of TSG6-primed BMMVs significantly reduced lung vascular permeability (Fig. 4B), PMN accumulation in BALF (Fig. 4C), and generation of inflammatory cytokines in BALF (Fig. 4D)

Discussion

C Con-siRNA

Materials and Methods