ABSTRACT
Double-stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll-like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA-TLR3-mediated signaling are largely unknown. Here we show that c-Src tyrosine kinase is activated by dsRNA in human monocyte-derived dendritic cells, and is recruited to TLR3 in a dsRNA-dependent manner. DsRNA-induced activation of interferon-regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase-deficient cells, and restored by adding back c-Src, suggesting a central role of c-Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA-containing endosomes in response to dsRNA, and colocalizes with c-Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3-mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.
Subject(s)
Endosomes/enzymology , Endosomes/virology , Protein-Tyrosine Kinases/physiology , RNA, Double-Stranded/antagonists & inhibitors , Rhinovirus/physiology , Signal Transduction/physiology , Toll-Like Receptor 3/physiology , Animals , CSK Tyrosine-Protein Kinase , Cells, Cultured , Dendritic Cells , Endosomes/physiology , Enzyme Activation/physiology , HeLa Cells , Humans , Interferon Regulatory Factor-3/metabolism , Mice , Mice, Knockout , Protein-Tyrosine Kinases/metabolism , RNA, Double-Stranded/physiology , Rhinovirus/genetics , STAT1 Transcription Factor/metabolism , Toll-Like Receptor 3/metabolism , src-Family KinasesABSTRACT
An extract of the edible mushroom Agaricus blazei Murill (AbM) has known antitumor and anti-infection properties, probably mainly by stimulating mononuclear phagocytes of the native immune system. The aim of this work was to study the effect of AbM on the production by human monocytes and human umbilical vein endothelial cells (EC) of pro-inflammatory cytokines (IL-1beta, IL-6, IL-8, TNFalpha), the anti-inflammatory/T regulatory cytokine IL-10 and the pro-Th1 cytokine IL-12. AbM, in concentrations from 1-15%, induced a considerable and dose-dependent increase in production of IL-8, IL-6, TNFalpha and IL-1beta in monocyte cultures. The biosynthesis reached a plateau at a concentration of 10% of AbM, and was most pronounced for the three former cytokines. AbM did also dose-dependently stimulate EC production of IL-8,I L-6 and TNFalpha, but at lower levels compared with the monocytes. AbM did neither induce synthesis of cytokines IL-10 nor IL-12 in monocytes or EC. Our results demonstrate the differential effect of AbM stimulation on the magnitude of pro-inflammatory cytokines produced by monocytes and EC.
