Cytoplasm-translocated Ku70/80 Complex Sensing HBV DNA Induces Inflammation-Associated Chemokine Secretion

Hepatitis B,caused by HBV infection,is a worldwide health concern with around 35 million HBV carriers develop chronic infections.As research develops these decades,now we are clear that when the liver of chronic HBV patient become inflamed,it means that the immune system proceed reactive to the hepatocytes suffering rapid HBV replication,and the HBV-specific CD8+ T cells act as the dominant effector cells,besides NK,NKT and other subtypes.However,precisely how these cells are recruited remains unclear.That is also what are interested in.In the present study,combined experiments in vivo and in vitro,we got some answers about this question.1.We analyzed some HBV-associated gene-expression chip data on the Gene Expression Omnibus(GEO)website,and found that the mRNA expression of inflammation-associated chemokine CCL5 is quite high in the liver samples from patients suffering HBV-associated hepatitis,compared to samples of normal livers.Also in the serum of HBV patients receiving antiviral treatment for 48 weeks,CCL5 concentration is much lower than their serum before treatment.These data seem to implicate an important role of chemokine CCL5 in hepatitis B.2.Agree with data in HBV patients,Ad-HBV infected mice also appeared to express much CCL3 and CCL5 in the livers,suffering lymphocyte infiltration and high serum ALT meanwhile.Given that CCR5,main receptor of CCL3 and CCL5,is reported to be important in hepatitis B,these inflammation-associated chemokines may be critical in lymphocyte recruitment of hepatitis B.3.Furthermore,HBV plasmid transfection also showed chemokine upregulation in liver-derived endothelial cells,but not hepatocyte-derived cells.As HBV virus replication is reported to suppress innate sensing pathways in hepatocytes,chemokine upregulation might also be suppressed in these transfected cells.Which means the endothelial cells could be the main source of chemokine CCL3 and CCL5 in the liver.4.Cytoplasm-translocated Ku70/80 complex in liver-derived cells sensed cytosolic HBV DNA and promoted inflammation-associated chemokine secretion.In HepG2.2.15 cells,Ku70/80 were also observed to colocalize with HBcAg,which suggested them to sense HBV DNA.Knockdown of Ku70 expression would impact the upregulation of chemokine CCL3,CCL5.5.We carried out Co-EP experiment against Ku70 protein in HBV DNA transfected cells and found PARP1 to bind Ku70/80 complex?There is a colocalization of PARP1 and Ku70/80 complex in cytoplasm upon HBV DNA sensing.RNAi of PARP1 downregulated chemokines,too.IRF1 served as the key transcription factor downstream.IRF1 assembled to Ku70/80 sensing complex and transported into nuclear Soon after HBV DNA stimulation.With the data above,we found a new kind of pathway upregulating chemokine CCL3 and CCL5,mediated by Ku70/80 sensing HBV DNA and passed through PARP1 and IRF1.Since chemokine CCL5 is a crucial factor in hepatitis B as the microarray chip data tells and chemokines have essential roles in lymphocyte recruitment,this pathway may be critical in the initiation of hepatitis B.