The Interplay Of Toll-like Receptor Signaling And Gas6/ProS-TAM System In Regulating Inflammatory Responses Of Mouse Macrophages

Background and objectives:Inflammation is an important immune event, which includes local and systemic immune responses initiated by pathogen infection or tissue injury. Acute inflammation is a protective mechanism to remove invading pathogens and repair tissue injury. However, it must be properly regulated because unrestrained chronic inflammation can lead to a variety of diseases. Macrophages regulate inflammation via phagocytosis and inflammatory cytokine production. Immune heameostasis is maintained by the interplay of inflammation and its negative regulatory system. Most studies focus on the mechanisms that negative regulatory system inhibits Toll-like receptor (TLR)-triggered inflammatory responses. Here, we investigate effect of TLR activation on a negative regulatory system——Gas6/ProS-TAM, and phagocytosis in mouse peritoneal macrophages.Materials and methods:Mouse peritoneal macrophages were isolated and identified by immune staining with F4/80. Gene expression was examined using qRT-PCR, Western blot and ELISA. TAM (Tyro3, Axl and Mer) receptor tyrosine kinases knock-out mice were used. Mouse neutrophils were isolated and cultured at37℃in serum-free medium for24h to induce spontaneous apoptosis. The apoptotic rate of neutrophils was assessed by flow cytometry. Phagocytosis was assessed by fluorescence microscopy and flow cytometry analysis.Results:Macrophages constitutively express TAM receptors and their ligands——Gas6and ProS. Gas6and ProS inhibit the inflammatory cytokine expression by macrophages in an autocrine manner.TLR ligands (poly(I:C) for TLR3, LPS for TLR4and CpG for TLR9) suppress Gas6and ProS expression in macrophages. In turn, down-regulation of Gas6and ProS facilitates the TLR-mediated inflammatory cytokine production.Poly(I:C), LPS and CpG activate respective receptors and inhibit phagocytosis of apoptotic cells through NF-κB pathway, whereas promote bacteria uptake via IRF3activation. Gas6and ProS promote uptake of apoptotic cells. Type I interferons (including IFN-α and IFN-β) enhance phagocytosis of bacteria, but have no obvious effect on engulfment of apoptotic cells. Further, TLR activation on macrophage regulates expression of various phagocytosis-related genes. Conclusions:TLR activation induces inflammatory cytokine production by macrophages and triggers inflammation. Gas6/ProS-TAM system inhibits inflammation by suppressing pro-inflammatory cytokine production and promoting phagocytosis of apoptotic cells. On the other hand, TLR activation inhibits Gas6/ProS expression in macrophages, which facilitates inflammation. The results describe a self-regulatory mechanism of TLR signaling and Gas6/ProS-TAM system in inflammation.