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Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

Previously, we have demonstrated the induction of Src in lipopolysaccharide (LPS)-stimulated macrophages. In this study, we observed that pharmacological blockade or knockout of inducible nitric-oxide synthase (iNOS) reduced LPS-mediated Src induction and macrophage migration. Either SNAP (a NO donor) or 8-Br-cGMP (a cGMP analogue) could rescue these defects in iNOS-null macrophages, which indicated the participation of NO/cGMP in LPS-elicited Src expression and mobilization. In addition, Src family kinase (SFK)-specific inhibitor, PP2, inhibited SNAP- and 8-Br-cGMP-evoked motility implicating the involvement of SFKs downstream of NO/cGMP. Analysis of the expression of SFKs indicated LPS dramatically induced Src, which could be attributable to the increased level of the src transcript. Attenuation of Src by src-specific siRNA reduced LPS- and SNAP-evoked mobilization in Raw264.7 macrophages, and reintroduction of avian Src could rescue their motility. Furthermore, LPS-mediated Src induction led to increased FAK Pi-Tyr-397 and Pi-Tyr-861, which was also iNOS-dependent. With these findings, we concluded that iNOS was important for LPS-mediated macrophage locomotion and Src was a critical player in this process.

Lithium chloride (LiCl) has long been used as a mood stabilizer for bipolar mood depression patients. However, its biological effects on immune cells are unclear yet. In this study, we observed that upon LiCl stimulation, the motility and the content of total protein tyrosine phosphorylation in RAW264.7 macrophages and murine peritoneal macrophages (PEMs) were significantly increased. The inhibition of LiCl-induced macrophage migration by PP2 (an inhibitor for Src family kinases (SFKs)) suggested the involvement of SFKs in this process. Interestingly, LiCl induced NF-kB activation, and while Src was greatly induced, the expression of its myeloid relatives (i.e. Lyn, Fgr, Hck) was almost unaltered in RAW264.7 cells. Knockdown of Src suppressed LiCl-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by siRNA-resistant Src. Consistent with Src and migration increment was iNOS-dependent in macrophages, markedly reduced Src expression, activity and cell migration were observed in iNOS-null PEMs treated with LiCl. Moreover, FAK knockdown suppressed LiCl-stimulated macrophage motility, suggesting the involvement of FAK in this process. Remarkably, similar increment of iNOS, Src, FAK Pi-Tyr861 and migration ability could also be detected in RAW264.7 treated with other GSK3β inhibitors (i.e. SB216763 and Kenpaullone). These results corroborate that through inhibition of GSK3β, the iNOS/Src/FAK axis occupies a critical role in macrophage locomotion in response to LiCl.

Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97(Eps8)) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.