Influence Of C-type Lectin CLEC4M On The Level Of Immune Molecule Loaded With HCV

Hepatitis C virus(HCV) infection is a one of the major worldwide public health problem, as well as a common and frequently-occurring disease that has been seriously endangering the human health. According to the epidemiological investigation and the World Health Organization(WHO) surveys, more than 170 million people are infected with HCV all over the world. After the initial infection, approximately 70%-80% of the patients can progress to chronic infection and some can result in liver fibrosis, cirrhosis and hepatocellular carcinoma. In China, it is estimated that about 30 million people are infected with HCV, accounting for 1/5 of the world’s population, seriously endangering their quality of life. According to the actual conditions and genotypes of HCV patients in China, combination of Pegylated interferon-α(PEG-IFN-α) with ribavirin(RBV) is still the main treatment. Since 2012, chronic hepatitis C was listed as one of the 12 priority diseases in China.Liver is the main target organ of the HCV infection. The process of HCV invading liver cells is a complex progress that requires multiple cell surface receptor molecules and the specific mechanism is still a controversial topic. The pathogenesis of HCV infection has been not clearly clarified due to lack of an ideal animal model in study. Recently, thanks to the establishment of HCV in vitro culture system and continuously update in the area of transgenic mice animal model, the research of HCV receptors is more deeply than before. HCV can escape from immune surveillance after entering the infected liver cells through its viral envelope proteins E1 and E2 binding the cell surface receptor CD81, scavenger receptor class B type 1, and tight binding protein family such as CLDN-1 and Occludin. The latest study found that the C-type lectin CLEC4 M may be an important receptor molecule which can mediate the endocytosis of HCV. CLEC4 M is an exogenous C-type lectin type II transmembrane protein located at 19p13.3 and about 46 k Da. CLEC4 M largely, specifically and selectively expressed on mature and immature liver sinusoidal endothelial cells, sinus lymphatic endothelial cells and placental capillaries. Its natural ligand for ICAM-3, mainly expressed on T cells, and the exogenous antigen after being processed and presented can be identified by numerous T-cell receptors(TCRs).Dendritic cells(DC) are known as the most powerful antigen-presenting cells by far, and and the representative feature of DC is to stimulate activation and proliferation of naive T-cell, which plays the pivotal role in the immune response. The relationship between DC and viral infection has become the research topic in recent years. DC are involved in anti-infection though the recognition of molecular structure of pathogen, subsequently promoting intracellular signal transduction pathway and activating the specific immune response, which will be characterized by the change of secreted level of cytokines or chemokines. DC is considered as a double-edged sword, however, because it can become communicators by acting as a "Trojan horse" at the same time of presenting the antigen of the virus. In our preliminary experiments, we found that CLEC4 M molecules can expressed abundantly on the surface of DC, so we speculate that CLEC4 M may mediate the molecular mechanisms of HCV invading the cell, escaping host immune attack, and thus affecting the immune function of regulatory T cells, which mainly leads to chronic HCV infection. So we plan to further study the impact of CLEC4 M after HCVcc-loaded on the body’s immune response. The experiment is funded by the National Natural Science Foundation of China(No: 81170390):1.The clinical significance of the expression of CLEC4 M on the Peripheral Blood Mononuclears(PBMCs) in CHC patients.Peripheral blood mononuclear cells(PBMCs) obtained from peripheral blood of and chronic HCV patients and healthy adults donorsuse were deteced expressive rate of expression of CLEC4M/CD14 on the cell surface among the HCV-na?ve patients, early virological response group, sustained virological response group and the healthy control group by flow cytometry, then analyzed the differences between the group. assessed the correlation between the expression of CLEC4M/CD14 and clinical parameters by using the Spearman correlation test in patients with chronic HCV.2.The Construction of a stable CLEC4 M overexpression of Huh7.5 cell lines.Cloning c CLEC4 M gene from CD14 + cells, we successfully constructed the GV166-CLEC4 M lentiviral expression vector followed by transfecting the vector into 293 T cells with lipofectamine and got the lentivirus. The Huh7.5 cells was transfected with the lentiviral and established a stable CLEC4 M overexpression of Huh7.5 cell lines. RT-PCR and Western blot were used to detect the expression of CLEC4 M in Huh7.5 cell.3.Construction of HCV culture in vitro..We used the p FL-J6/JFH Plasmid containing the full length sequence of HCV, was digested by restriction enzyme, then we carried out the DNA purification and T7 transcription in vitro to get the full-length HCV transcript. Next, Huh-7.5 cells were transfected with DMRIE-C transfection reagent. Collecting the HCV in cell culture solution then testing its susceptibility to HCVcc.4.Influence of CLEC4 M on the susceptibility to HCVcc.CLEC4M overexpression Huh7.5 cell lines were infected by HCVcc virus liquid with high infection efficiency. The monoclonal antibody interference group and mannan interference group, and tested the HCVcc infection of the change of CLEC4 M. Immunofluorescence and RT-PCR were used to detect the effect of CLEC4 M to HCVcc.5.Culture and identification of dendritic cells in vitro and the identification of CLEC4 M on DC.PBMCs were obtained from the peripheral blood of healthy adults donors. CD14+ monocytes were isolated by CD14 isolation beads and cultured in the presence of recombinant granulocyte macrophage colony stimulating factor(rh GM-CSF) and interleukin-4(IL-4). At the 5th days,tumor necrosis factor-α(TNF-α) were added to simulate the maturation of DC. The CD14+ separation efficiency and the expression of CLEC4 M on CD14+ were evaluated by flow cytometry. It was detected by flow cytometry, and CLEC4 Min CD14+ cells. The expression of surface markers CD83 and CD86 cells were also evaluated by flow cytometry to identify the DC maturation as well as the expression of CLEC4 M in mature DC.6.Influence of CLEC4 M on the level of immune response after HCV-loaded.CLEC4M blocking antibodies were also sued to block the expression of DC cell surface molecules CLEC4 M in our experiments. Then DC was infected by HCVcc, and setting the monoclonal antibody interference group and blank group. Supernatants were collected of each group and the expression of IL-12p70 and IL-10 were detected by ELISA. Conclusion:1.Detected the expression of CLEC4 M / CD14 in PBMCs of chronic HCV patients. The expression of the CLEC4M/CD14 in HCV-na?ve patients was significantly higher than that of the control group, EVR groupand SVR group, while there was no significant difference between the control group, EVR group, and SVR group. CLEC4 M was involved in the HCV infection and induced the immune response,which damaged the liver and accelerated the progression of the disease.2.Constructed a stable overexpressing human CLEC4 M of Huh7.5 cells. The overexpression of CLEC4 M of the Huh7.5 was infected by HCVcc, and got the susceptibility of HCVcc. All inlustrated CLEC4 M may influence the processing of HCV infection.3.Induced the culture of mature DC, detected the expression of CLEC4 M on mature DC cell surface. DC were infected by HCVcc, which set the monoclonal antibody interference group and mannan interference group were used as control..The expression of IL-12p70 and IL-10 were detected. The results showed that the expression of IL-12p70, IL-10 levels of the positive control groups were significantly higher than monoclonal antibody interference group and mannan interference group. The results indicated CLEC4 M mediated HCV infecting DC, which would affect the level of immune response.