| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 103 === Dengue virus (DENV) infection is a global and aggressive mosquito-borne infectious disease which causes both dengue fever and dengue hemorrhagic fever. Unfortunately, there are no effective vaccines and therapeutic antiviral drugs for clinical use. Accumulated clinical evidence shows that DENV infection induces a high level of anti-inflammatory cytokine interleukin (IL)-10 in patients with severe dengue hemorrhagic fever and dengue shock syndrome as compared with those with mild dengue fever. It is important that the intrinsic antibody-dependent enhancement (ADE) of infection causes higher production of IL-10 which promotes viral replication; however, the underlying molecular mechanisms of IL-10 regulation are still unclear. This study is aimed at investigating the pathogenic role and regulatory mechanism of IL-10 production during ADE of DENV infection. First, I established an in vitro model of ADE infection and discovered that the presence of monoclonal anti-envelope (E) antibody increased the infectivity of DENV in human monocytic THP-1 cells. The effects of ADE were further studied by determining protein expression and transcriptional activation of IL-10. We previously demonstrated that DENV infection induces IL-10 production by deactivating glycogen synthase kinase (GSK)-3β in a sequential protein kinase A (PKA)- and phosphoinositide (PI) 3-kinase/PKB-regulated manner. Under ADE infection, DENV not only caused a significant increase in PI3K and PKA activities, but also induced phosphorylation of PKB at Ser473 and GSK-3β at Ser9. Silencing cAMP response element-binding protein (CREB) decreased IL-10 production. Pharmacological inhibition of spleen tyrosine kinase (Syk), PI3K, and PKA reduced IL-10 production has been confirmed following ADE of DENV infection. Moreover, inhibiting Syk also decreased ADE-induced phosphorylation of PKB at Ser473, GSK-3β at Ser9, and CREB at Ser133, indicating Syk may act upstream of PI3K/PKB/GSK-3β/CREB pathway for ADE-induced IL-10 production. The heat-inactivated DENV was unable to induce IL-10 production in THP-1 cells, whereas ultraviolet-deactivated DENV induced IL-10 production normally. The knockdown of C-type lectin superfamily member 5 (CLEC5A) expression in THP-1 cells showed a significant decrease in IL-10 production after DENV infection. The viral load which is not serotype affected the IL-10 response. Regarding ADE-enhanced IL-10/ suppressor of cytokine signaling 3 (SOCS3) expression may interfere with the antiviral response, results showed that genetically and pharmacologically inhibiting IL-10 signaling (including CREB, Syk, PI3K, PKA, and CLEC5A) significantly retarded DENV replication and NS4B expression no matter whether it is with or without ADE of DENV infection. These results show that IL-10 is beneficial for DENV replication and the target IL-10 may be a potential antiviral treatment.
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