The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-ß synthesis in macrophages.

Double-stranded RNA (dsRNA) induces phosphorylation of Toll-like receptor 3 (TLR3) at tyrosine 759 and subsequently triggers signaling pathways to promote interferon-ß (IFN-ß) production. In this study, we found that dsRNA stimulation induces biphasic TLR3 Tyr-759 phosphorylation in macrophages. In addition to the immediate TLR3 Tyr-759 phosphorylation, we identified a second wave of Tyr-759 phosphorylation accompanied by an increase of both Src and ifn-ß transcription in the later phase of dsRNA stimulation. Interestingly, Src phosphorylated TLR3 Tyr-759 in vitro and in vivo. However, knockdown of Src abolished the late phase of TLR3 Tyr-759 phosphorylation and decreased the nuclear accumulation of interferon regulatory factors 3 and 7 (IRF3 and -7) and IFN-ß production. Reintroduction of Src restored all of these molecular changes. Notably, via down-regulation of Src, dsRNA-elicited TLR3 Tyr-759 phosphorylation, the nuclear accumulation of IRF3/IRF7, and IFN-ß generation were inhibited in inducible nitric-oxide synthase (iNOS)-null macrophages. TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-ß production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-ß expression). Ectopic expression of wild type TLR3, but not its 759-phenylalanine mutant, restored Src activity and ifn-ß transcription. Taken together, these results suggested an essential role of the iNOS/Src/TLR3 axis in IFN-ß production in macrophages.

Butyrate reduced lipopolysaccharide-mediated macrophage migration by suppression of Src enhancement and focal adhesion kinase activity.

Macrophage motility is vital in innate immunity. Lipopolysaccharide (LPS)-mediated macrophage migration requires the enhancement of Src expression and enzymatic activity, which can be regulated by inducible nitric oxide synthase (iNOS). As a major short-chain fatty acid with histone deacetylase (HDAC) inhibitor activity, butyrate exerts anti-inflammatory effect by regulating the expression of cytokines. However, the influence of butyrate on macrophage movement was vague. In this study, we observed that butyrate inhibited migration of both RAW264.7 and rat peritoneal macrophages elicited by LPS. Unlike its myeloid relatives (i.e. Lyn, Fgr and Hck) whose expression was almost unaltered in the presence or absence of butyrate in LPS-treated macrophages, LPS-mediated Src induction was greatly suppressed by butyrate and that could be attributable to reduced level of the src transcript. Similar phenomenon was also detected in LPS-treated macrophages exposed to another HDAC inhibitor, trichostatin A (TSA). Consistent with the indispensability of iNOS in promoting macrophage mobilization via Src up-regulation and the activation of both Src and FAK, we did observe concomitant decrement of iNOS, Src and the suppressed activity of Src and FAK in butyrate- or TSA-pretreated macrophages following LPS exposure. These results imply that by virtue of reduction of Src, butyrate could effectively hamper LPS-triggered macrophage locomotion.