Zenke Kosuke

Inducible nitric oxidase (iNOS) encoded by Nos2 is a representative IFNγ-inducible effector molecule that plays an important role in both innate and adaptive immunity. In the present study, we demonstrated that full-length NF-κB p105 (p105), which is a precursor of NF-κB p50 (p50), is required for full activation of IFNγ-induced iNOS expression in the RAW264.7 mouse macrophage cell line. In comparison to wild-type (WT) RAW264.7 cells, p105 KO RAW264.7 (p105 KO) cells completely lost IFNγ-induced iNOS expression. Despite the limited effect of exogenous expression of p50 in p105 KO cells on IFNγ-induced Nos2 promoter activity, p105 expression fully restored IFNγ-induced Nos2 promoter activity to a level comparable to that of WT cells, suggesting an important role for full-length p105 in IFNγ-induced iNOS expression. While the expression and phosphorylation of JAK1 and STAT1 were rather enhanced in p105 KO cells, the phosphorylation of c-Jun downstream of MAPK signaling was decreased. IFNγ-induced phosphorylation of ERK, a kinase for IFNγ-induced c-Jun phosphorylation, was not significantly reduced in p105 KO cells, although the nuclear activity of ERK was significantly decreased due to its reduced translocation to the nucleus. Expression of iNOS, nuclear translocation of ERK, and phosphorylation of c-Jun were restored by stable supplementation of p105 in p105 KO cells. These results suggest that p105 is required for the nuclear translocation of ERK and the subsequent phosphorylation of c-Jun, which are necessary for full activation of IFNγ-induced iNOS expression. Reduced nuclear translocation of ERK in p105 KO cells was also observed in the activation of ERK following serum starvation, further suggesting that the involvement of p105 in ERK nuclear translocation is not limited to IFNγ-stimulated cells but is a more general function of p105.

Abstract LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow–derived macrophages from TLR2−/− mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β–induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Heat shock proteins (HSPs) are molecular chaperones that have recently been shown to function as host factors (HFs) for virus multiplication in fish as well as in mammals, plants, and insects. HSPs are classified into families, and each family has multiple isoforms. However, no comprehensive studies have been performed to clarify the biological importance of these multiple isoforms for fish virus multiplication. Betanodaviruses are the causative agents of viral nervous necrosis in cultured marine fish and cause very high mortality. Although the viral genome and encoded proteins have been characterized extensively, information on HFs for these viruses is limited. In this study, therefore, we focused on the HSP70 and HSP90 families to examine the importance of their isoforms for betanodavirus multiplication. We found that HSP inhibitors (17-AAG, radicicol, and quercetin) suppressed viral RNA replication and production of progeny virus in infected medaka (Oryzias latipes) cells. Thermal stress or virus infection resulted in increased expression of some isoform genes and facilitated virus multiplication. Furthermore, overexpression and knockdown of some isoform genes revealed that the isoforms HSP70-1, HSP70-2, HSP70-5, HSP90-α1, HSP90-α2, and HSP90-β play positive roles in virus multiplication in medaka. Collectively, these results suggest that multiple isoforms of fish HPSs serve as HFs for betanodavirus multiplication.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a major threat to marine cage culture in tropical and subtropical waters; however, controlling the disease remains challenging. In this study, we constructed DNA vaccines encoding a cysteine protease of C. irritans (pcDNA3.1-cp2-full-myc and pcDNA3.1-cp2-partial-myc) and examined the protective efficacy of the vaccines. The results of the challenge experiment showed that the number of parasites recovered from fish immunized with the DNA vaccines was lower than that recovered from control fish (phosphate-buffered saline-injected and mock vector-injected groups); this difference was statistically significant when pcDNA3.1-cp2-full-myc was used for vaccination (p < 0.05). In addition, the cysteine protease was found to be relatively conserved among different isolates of the parasite. Thus, the protease may be a potential antigen candidate for the development of a vaccine against multiple strains of C. irritans.