TLR4 Mediated The Nuclear Translocation Of NF-κB And IRF-3 In HTNV Infected Vascular Endothelial Cells

Hantaan viruses(HTNV),which belong to the Hantavirus(HV) genus of the Bunyaviridae family, are enveloped single-stranded negative-sense RNA viruses. Hantaviruses are carried by numerous rodents throughout the world and can not cause diseases when they are existed in their natural hosts. In humans, they mainly cause two diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The total number of HFRS patients is about 150,000 annually all over the world. More than 90% of these cases occur in Asia. China is the country, in which the situation of HFRS is the most serious. HTNV is the most popular type of HV, which causes severe HFRS cases in China. The prevalence of HTNV brings great burdens on HFRS patients and the all society.Since HV was first isolated from its rodent hosts in 1978, researches have done much work on HV and the related apartments, and many achievements have been reached such as its` etiology, epidemic, pathogenesis, diagnosis, and many other apartments. But we still do not know about all of molecular pathogenesis of HFRS. There also do not have effective drug for HFRS treatment. So the research of effective anti-viral medicine and safe vaccine for HV are still hot point in long time.Many evidences supported that the immune response may be the main reason for HV related diseases. Innate immunity, as the first barrier to defense the invading infectious agents, plays important role during the infection of HV and the pathogenesis of HFRS. The immune response of HFRS may be induced by HTNV infected vascular endothelial cells, which are the target cells of HTNV. Toll-like receptor 4 (TLR4), which is an important kind of cellular membrane molecular of vascular endothelial cells, take part in the innate immune response of endothelial cells. Lipopolysaccharide (LPS) is the main ligand of TLR4, but many evidences imply that TLR4 also plays important role in the anti-viral immune response. HTNV can infect vascular endothelial cell, then induces the bodies` immune response. During the earlier researches, we found that the infection of HTNV 76-118 strain can induce the high expression of TLR4 in human umbilical vein endothelial cells (EVC304 cells), and then Dr. jiang et al. established an stable cell line, in which the gene of TLR4 was silenced by siRNA (EVC304 TLR4- cells). Their results showed TLR4 can mediate the high secretion of cytokines (IFN-β, IL-6 and TNF-α) after EVC304 cells are infected by HTNV, meanwhile before and after the infection of HTNV, the expression of TRIF, an important adaptor molecular in TLR4 signal pathway, is increased, but the expression of MyD88, which is another adaptor molecular in TLR4 signal pathway, have not obvious difference. Based on the above results, Indirect immunofluorescence assay (IFA) was used to detect the nuclear translocation of transcription factors NF-κB and IRF-3, and found the difference in some time-course after the infection of HTNV. Then intercellular adhesion molecule-1 (ICAM-1) was detected by RT-PCR and IFA in HTNV infected EVC304 TLR4- cells. But we did not find some important information. The major object of the researches is to clearly demonstrate the signal pathway of TLR4 in HTNV infected EVC304 cells, and to learn more about the pathogenesis of HFRS. We also hope our work can provide some useful data to the clinical therapy and the vaccinal designation of HFRS.1. The culture of HTNV and the infection of EVC304 cells by HTNVHTNV 76-118 was used to infect VERO cells. After 3 generations, the cells were freezed/thawed 3 times, centrifuged and collected the culture supernatant, then the HTNV supernatant was used to infect EVC304 TLR4+ cells and EVC304 TLR4- cells,respectively. The results show that the supernatant contain HTNV can infect the two kinds of cells effectively.2. Detection of TLR4 in EVC304 TLR4- cellsThe total RNA were extracted from equal number of EVC304 TLR4+ cells and EVC304 TLR4- cells, respectively. Then RT-PCR was used to detect the expression of TLR4 in mRNA level. The result shows that the mRNA expression of TLR4 has been decreased significantly in EVC304 TLR4- cells, and they can be used to test the functions of TLR4.3. The nuclear translocation of transcription factors NF-κB and IRF-3EVC304 TLR4- cells and EVC304 TLR4+ cells were infected by HTNV 76-118, respectively. The cells stimulated by LPS were selected as the positive control groups, and the cells without any stimulation were selected as the negative control groups. After 0.5h/1h/3h/6h/12h, IFA was used to detect the nuclear translocation of transcription factors NF-κB and IRF-3. After the cells are stimulated 0.5h, we do not find the nuclear translocation in all groups. In the 1h,3h,6h, we can detect the nuclear translocation of NF-κB and IRF-3 in the EVC304 TLR4+ cells, which are stimulated by LPS or HTNV, and we do not find the same results in the other groups. The nuclear translocation, which are detected in 6 hours, was the most obvious ones. But in 12 hours, we do not detect the obvious trace again.4. The expression of ICAM-1 before and after HTNV infectionEVC304 TLR4- cells and EVC304 TLR4+ cells were infected by HTNV 76-118, respectively. After 6 hours, RT-PCR and IFA were used to detect the expression of ICAM-1 in mRNA and protein levels. There are no significant difference between all the groups.Conclusion: TLR4 can mediate the nuclear translocation of transcription factors NF-κB and IRF-3 in HTNV infected EVC304 cells, and there was a positive relation between the nuclear translocation and infection time-course in some extent. The results indicate that TLR4 plays an important role in the nuclear translocation of NF-κB and IRF-3 caused by HTNV. The expression of ICAM-1 do not have significant difference before and after the infection of HTNV in all the groups.