Tomoaki Yoshida

To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(-)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-β, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-β Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(-) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(-) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

The effect of TGF-β1 on CpG DNA-induced type I IFN production was examined by reconstituting a series of signaling molecules in TLR 3 signaling. TGF-β1 inhibited CpG DNA-induced IFN-α4 productivity in HeLa cells. Transfection of IFN regulatory factor (IRF)7 but not TNF receptor-associated factor (TRAF)6 and TRAF3 into cells triggered IFN-α4 productivity, and TGF-β1 inhibited IRF7-mediated type I IFN production in the presence of TRAF6. TGF-β1 induced ubiquitination of TRAF6, although CpG DNA did not induce it. Moreover, TGF-β1 accelerated the ubiquitination of TRAF6 in the presence of CpG DNA. TGF-β1 ubiquitinated TRAF6 at K63 but not K48. TGF-β1 also induced ubiquitination of IRF7. Further, TGF-β1 did not impair the interaction of IRF7 and TRAF6. CpG DNA induced the phosphorylation of IRF7 in the presence of TRAF6, whereas TGF-β1 inhibited the IRF7 phosphorylation. Blocking of TRAF6 ubiquitination abolished the inhibition of CpG DNA-induced type I IFN production by TGF-β. Taken together, TGF-β was suggested to inhibit CpG DNA-induced type I IFN production transcriptionally via ubiquitination of TRAF6.

Here we report that LPS induces osteoclast (OC) formation in murine RAW 264.7 macrophage cells in RPMI-1640 medium but not in α-minimum essential medium (α-MEM) as the original culture medium. LPS-induced OC formation in both media was examined to clarify the differential response. Receptor activator of NF-κB ligand induced OC formation in either α-MEM or RPMI-1640 medium. However, LPS-induced OC formation in RAW 264.7 cells maintained in RPMI-1640 medium, but not α-MEM, which was also supported by mouse bone marrow-derived macrophages, although they were less sensitive to LPS than RAW 264.7 cells. LPS augmented the expression of nuclear factor of activated T-cells (NFATc1) as a key transcription factor of osteoclastogenesis in cells maintained in RPMI-1640 medium, but reduced it in cells maintained in α-MEM. A high concentration of LPS was cytotoxic against cells maintained in α-MEM. Glutathione exclusively present in RPMI-1640 medium prevented LPS-induced cell death in α-MEM and augmented LPS-induced NFATc1 expression, followed by enhanced LPS-induced OC formation. LPS induced higher generation of reactive oxygen species in α-MEM than RPMI-1640 medium. An antioxidant enhanced LPS-induced OC formation, whereas a pro-oxidant reduced it. Taken together, redox balance in the culture condition was suggested to regulate in vitro LPS-induced OC formation.

The effect of LPS on the production of vascular endothelial growth factor (VEGF) was examined using RAW 264.7 macrophage cells. LPS induced VEGF production in RAW 264.7 cells and mouse peritoneal cells. LPS induced VEGF production via the expression of hypoxia inducible factor-1α and LPS-induced VEGF production was dependent on the activation of p38 MAPK and NF-κB activation· Transforming growth factor (TGF)-β1 augmented LPS-induced VEGF production, although TGF-β1 alone did not induce VEGF production. The augmentation of LPS-induced VEGF production by TGF-β1 was inhibited by a p38 MAPK inhibitor and was correlated with the phosphorylation of Smad3. The enhancing effect of TGF-β1 on LPS-induced VEGF production was observed in vivo in the skin lesions of mice receiving a subcutaneous injection of LPS. Taken together, it is suggested that LPS induced the VEGF production in macrophages and that it was augmented by TGF-β1 in vitro and in vivo.

The inhibitory effect of valproic acid (VPA) on lipopolysaccharide (LPS)-induced inflammatory response was studied by using mouse RAW 264.7 macrophage-like cells. VPA pretreatment attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen-activated protein kinases. VPA reduced phosphorylation of MDM2, an ubiquitin ligase and then prevented LPS-induced p53 degradation, followed by enhanced p53 expression. Moreover, p53 small interfering RNA (siRNA) abolished the inhibitory action of VPA on LPS-induced NF-κB p65 transcriptional activation and further LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. VPA prevented LPS-induced degradation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and up-regulated the PTEN expression. Taken together, VPA was suggested to down-regulate LPS-induced NF-κB-dependent transcriptional activity via impaired PI3K/Akt/MDM2 activation and enhanced p53 expression. A detailed mechanism for inhibition of LPS-induced inflammatory response by VPA is discussed.