*p<0.01, compared with corresponding poly I:C-treated groups #p<0.01, compared with corresponding poly I:C-treated WT groups.Ĭultured peritoneal macrophages from wild-type (WT) and CD11b −/− mice were incubated with C圓-labeled poly I:C (10 μg/ml) at 37☌ for 15 or 30min or C圓-labeled poly C (10 μg/ml) for 30min. Data are shown as means ± SEM from three independent experiments in triplicate. CD11b immunostaining showed that Mac-1 co-localized with surface-bound C圓-poly I:C (E). The confocal experiments revealed that more C圓-labeled poly I:C bound to the surface of WT macrophages than bound to the surface of CD11b −/− macrophages, and the binding of C圓-labeled poly I:C was inhibited by fibrinogen only in WT macrophages (D). Flow cytometry showed that wild-type (WT) macrophages had higher surface binding of FITC-labeled poly I:C than CD11b −/− macrophages, and the poly I:C binding was significantly inhibited by fibrinogen (1 μM) in WT macrophages and RAW264.7 cells but not CD11b −/− macrophages (B, C). Surface binding assay further delineated the interaction between poly I:C and Mac-1 on cell surface (B–E). Only poly I:C-coated beads can pull down the CD11b subunit of Mac-1 in either primary macrophages or Raw 264.7 macrophage cells (A). This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.ĭsRNA-binding assay was performed by using poly I:C-coated agarose beads, unconjugated beads, or pol圜-coated beads. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. ![]() dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. The present study demonstrated that CD11b/CD18 (Mac-1 ), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. ![]() Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages.
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