Hantaan Virus Induced Antiviral Immunity And Signal Transduction By TLR4 In Vascular Endothelial Cell

Hantaviruses are enveloped viruses which define a unique genus within the Bunyaviridae and contain a tripartite negative-stranded RNA genome. The viral RNA segments, L, M and S, encode an RNA dependent RNA polymerase, two envelope glycoprotein (G1 and G2), and nuclecapsidoprotein (NP), respectively. Pathogenic hantaviruses mainly cause two kinds of acute infectious diseases: one is hemorrhagic fever with renal syndrome (HFRS), which is charactered by fever, vascular hemorrhage, kidney injury or renal failure and immunologic dysfunction, another is hantavirus cardiopulmonary syndrome (HCPS). In China HTNV is the most prevalent type of Hantavirus, which causes HFRS with severe symptoms and high mortality. Hantavirus is also as an important biological weapon, and it affects the military affairs. Though the Hantavirus was recovered from rodent hosts in 1981 in China and much work have been done by scientists all over the world, there are no specific and effective therapeutic medicine or regimen for hantaviruses diseases, and the pathogenesis of hantaviruses infections have not yet been disclosed and elucidated.Vascular endothelial cells are the primary target of hantavirus. A subsequent decrease of blood platelet and damage of small vessels or blood capillary is the main pathological change. It is supported by many reports that the immune respone induced by hantavirus is the main reason of dysfunction of endothelial cells and pathologicity of hantavirus. But the interaction between immuno moleculars and cytokines and membrane moleculars is still not clear. Toll-like receptors are like the eyes of the innate immunity and essential for recognition of invading pathogens. Toll-like receptors activate signal transduction pathway in cell and lead to cytokines secretion. It is thought the first barrier of host to defense the invading pathogens. To understand the initial events that lead to disease, it would be helpful to examine viral interactions with host endothelial cells and to characterize the innate antiviral responses associated with Toll-like receptors. Therefore, it is helpful to understand the antiviral mechanism of host to suppress or activate the TLRs expression and to regulate the signal pathway of them. In this study, we select the TLR4 as the target molecular to research the anti-hantavirus immunity in human vascular endothelial cell. RNA interference was used to slience the TLR4 in EVC-304 cell line. The main object is to demonstrate the pathological mechanism of HFRS for TLRs in vascular endothelial cells. We also hope this is benefit for designing drugs designment and vaccines development.1. HTNV induces Toll-like receptors expression change in human vascular endothelial cellsThe EVC-304 cells were infected by HTNV 76-118. After 6 h, the semi-quantity RT-PCR was used to detect the mRNA expression of five TLRs respectively. It indicated that TLR4 was upregulated while TLR3 was downregulated. There is no difference of TLR2, TLR7 and TLR9 mRNA expression between infected and uninfected cells. The recognized ligands of the TLRs were used as positive control and the results is credible. In the past, it is deemed that TLR4 is the receptor for LPS in most reports but now it is found that TLR4 can still recognize structure components from viruses. This point was confirmed in this study.2. siRNA expression vectors construction and stable cell establishingRNA interference is a natural process in many organisms by which double-stranded RNA mediates sequence-specific post-transcriptional silencing of homologous genes. The specific gene function was shut off by RNA interference. The P-silencer 4.1-CMV vector which contains CMV Pol II promoter was selected as expression vector. Nine siRNAs were selected according to the sequence of TLR4(NM-138554). After the double strand DNA were synthesized and annealed, the expression vector P-silencer 4.1-CMV was constructed. Fifty five bp bands were got by PAGE electrophoresis after the recombinant plasmids were digested by Hind III and BamH I, the plasmids were sequenced by Invitrogen Corporation and confirmed right.The 9 recombinant plasmids were transfected into EVC-304 cells separately. Five high suppression rate siRNA named TS1, TS4, TS5, TS6 and TS8 were selected after the mRNA were analysed by RT-PCR. Among them the better silence effect were 89% and 90%, which is induced by TS4 and TS6. The 5 plasmids were transfected into EVC-304 cells and screened by G418. Finally, 3 strains of stable cell lines were established. The TS4 cell line was the most potent silence effect which surpresse more than 98% TLR4 expression in both mRNA and protein level, and the cell line was used in the next experiment.3. The relationship between cytokine secretion and TLR4 expresionBoth EVC-304 and EVC-304 TLR4- cells were infected by HTNV 76-118. After 48 h, the supernatant of cell cultrue was collected separately and detected by ELISA kit as the manuscripture's manunal. It indicates that there is not difference of IFN-αand IL-8 between EVC304 cell and EVC-304 TLR4- cell whether in infected or uninfected by HTNV 76-118. Furthermore, the secretion increase more significantly after LPS induced. HTNV 76-118 infection significantly increase IFN-β, IL-6 and TNF-αsecretion from EVC-304 cells, while there is no distinct difference in EVC-304 TLR4- cells in infected or uninfected cells.4. TLR4-mediated cytokines signaling pathwaysBoth EVC-304 and EVC-304 TLR4- cells were infected by HTNV 76-118. After 48 h, total RNA was prepared and FQ-PCR was used to detect the mRNA expression of MyD88 and TRIF, which are important adaptor molecular in TLR4 signaling pathway. The results indicate that TRIF mRNA expression in EVC-304 is about 3 times more than that of in EVC-304 TLR4- cells. In both cell lines, there is no obvious difference of the MyD88 mRNA expression. The same results were obtained by Western blot assay.Conclusion: The infection of the vascular endothelial cell line, EVC-304 by HTNV 76-118 can induce change of TLRs, and among them the expression of TLR4 is upregulated remarkably. TLR4 is a key molecular that mediated the augmentation of several cytokines, IL-6, IFN-βand TNF-α. secreted by EVC-304 cell. The main signal pathway is MyD88-independent after the TLR4 is activated by HTNV 76-118 infection. The results mentioned above demonstrate that TLR4 plays an important role in the antiviral immunity of host against the infection of HTNV. A further research of innate immunity will highlight the pathogenesis of HFRS.