善家 孝介

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.

In spite of the growing attention given to medaka (Oryzias latipes) as an excellent vertebrate model, an effective gene knockdown system has not yet been established using cultured cells of this fish species. In this study, a gene knockdown system using short interfering RNA (siRNA) in medaka cell lines was established through the optimization of transfection conditions. By extensive screening of several medaka cell lines and transfection reagents, OLHNI-2 cells and X-tremeGENE siRNA Transfection Reagent were selected as the best combination to achieve high transfection efficiency of siRNA without cytotoxic effect. Knockdown conditions were then refined using the endogenous heat shock protein 90 (Hsp90) genes as the siRNA targets. Among the parameters tested, cell density, serum concentration in the culture medium, and duration of transfection improved knockdown efficiency, where the target mRNA in cells transfected with each of the siRNAs was reduced from 12.0% to 26.7% of the control level. Our results indicate that the established knockdown system using siRNA is a promising tool for functional analysis of medaka genes in vitro.