Investigating the Molecular Mechanism of TNF-? Production in Dengue Virus Infection

Abstract

Infection of dengue virus (DENV) threatens global public health due to its high prevalence and the lack of effective treatments Although the pathogenesis of severe dengue is still largely unknown an increase in a pro-inflammatory cytokine tumor necrosis factor-? (TNF-?) is reported to be involved in development of severe disorders However the molecular mechanisms underlying this inflammatory activation remain undefined This thesis is aimed at examining the underlying mechanisms of TNF-? production in DENV infection Results obtained may have implications on molecular pathogenesis and therapeutic targets in dengue diseases Although the activation of the transcription factor NF-κB is generally involved in pro-inflammatory responses such as TNF-? expression and also nitric oxide (NO) generation it has never been clarified in DENV infection Hence in the first part of thesis the role of NF-κB in TNF-? production and inducible nitric oxide synthase/nitric oxide (iNOS/NO) biosynthesis during DENV infection were investigated Results showed that in addition to TNF-? production in DENV-infected murine macrophage RAW264 7 cells iNOS was transcriptionally and post-translationally elevated and accompanied by NO generation Pharmacologically inhibiting NF-κB activation abolished iNOS/NO biosynthesis and TNF-? production With inhibition the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit long-termed (>24 hour) TNF-? production In contrast to TLR3 viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-? production These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-? expression The other critical regulator of TNF-? in DENV infection is the signaling receptor C-type lectin domain family 5 member A (CLEC5A) Based on a high-throughput study CLEC5A is one of target genes of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) In the second part of thesis the molecular regulation and the novel role of activated Nrf2 for CLEC5A-regulated TNF-? expression in DENV infection were clarified Results showed that DENV infection selectively activated Nrf2 Following endoplasmic reticular (ER) stress protein kinase R-like ER kinase (PERK) facilitated Nrf2-mediated transcriptional activation of CLEC5A to increase CLEC5A expression Signaling downstream of the Nrf2-CLEC5A interaction enhances Toll-like receptor 3 (TLR3)-independent TNF-? production following DENV infection Forced expression of the NS2B3 viral protein induced Nrf2 nuclear translocation/activation and CLEC5A expression which increases DENV-induced TNF-? production Animal studies confirmed Nrf2-induced CLEC5A and TNF-? in brains of DENV-infected mice These results demonstrate that DENV infection caused Nrf2-regulated TNF-? production by increasing levels of CLEC5A Taken together these data provide the detailed mechanism of TNF-? production during DENV infection The molecules involved in these pathways may be potential targets for anti-DENV diseases

Date of Award	2016 Sept 22
Original language	English
Supervisor	Yee-Shin Lin (Supervisor)