We can observe the same protein in several spots with decreasing molecular mass

latory role of AXL in epithelial tissue is less well understood, and is of particular relevance in the HCV infected liver due to its regulation of SOCS3. Following stimulation of mouse dendritic cells with IFN, AXL was shown to highjack IFN signalling by binding to the IFN receptor IFNAR1 to prevent its signal transduction. Simultaneously, AXL mediated the formation of STAT1 homodimers, to induce the expression of SOCS1 and SOCS3. We have previously shown that AXL is induced by HCV infection in vitro and have subsequently chosen to examine the functional relevance of AXL up-regulation by HCV. Here we confirm that AXL is up-regulated during HCV infection in vitro and in vivo and that AXL expression in the liver is driven primarily by type I/III IFN signalling, as well as inflammatory signalling pathways. Moreover, AXL reduces activation of the innate immune response by IFN in hepatocytes, limiting the antiviral response to HCV. Lastly, ATL-962 web patients possessing the theIFNL3 rs12979860 responder SNP demonstrated reduced baseline AXL expression in the liver and a stronger peripheral blood mononuclear cell AXL up-regulation after the first injection of IFN. Material and Methods Patient samples Liver biopsies were collected from untreated patients chronically infected with HBV, patients with HCV genotype 1/3 infection and low fibrosis and HCV genotype 1 infection with high fibrosis. All `low fibrosis’ samples were confirmed histologically to have Metavir fibrosis score 1 and steatosis Grade 1, unless otherwise stated. Peripheral blood mononuclear cell RNA was obtained from 15 healthy controls, as well as 18 genotype 1 HCV patients at baseline and 12 h after the first interferon injection using PAXgene blood RNA tubes. All genotype 1 patients were genotyped for the rs12979860 IFNL3 SNP by Taqman genotyping as described in. Ethics approval and patient consent for the research use of blood and biopsies was provided for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19730426 all samples. Cell culture and virus infection Huh-7 and HepG2 cell lines were grown in Dulbecco’s minimal essential medium with 10% fetal bovine serum. HCV RNA was 2 / 16 HCV Induced AXL Suppresses the Hepatic Type I Interferon Response transcribed, electroporated, and baculovirus transfection of HepG2 cells was performed as in. Fugene HD was used to transfect full length HCV and subgenomic replicon RNA into Huh-7 cells to examine short term viral RNA replication. Cytokines IFN was obtained from Roche, IFN from Biogen Idec, IFN from Jomar Bioscience and recombinant IFN3 andIL6 were obtained from R&D Systems. Chemical inhibition, gene knockdown and overexpression Huh-7 cells were treated for 24 h with 50 M SP600125 or 25 M BAY11-7082, to inhibit JNK or NFB signalling respectively. To inhibit STAT mediated signalling, Huh-7 cells were transfected with 10 nM siRNA against STAT1 or STAT3 for 24 h, with RNAiMax or AllStars scrambled siRNA as a control. AXL expression was knocked down using 25 M Mission siRNA for 24 h, prior to 24 h of IFN treatment. A stable polyclonal Huh7 cell line overexpressing AXL was established by transfecting the pCMV6-AXL ORF PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19728371 plasmid with Fugene HD according to the manufacturers protocol, and selecting with G418. The empty vector pCMV6-Entry was maintained under G418 selection as well, and was used as a control. GAS, ISRE, AP-1 and NF-B activity reporter assays Firefly luciferase reporter plasmids containing the gamma-activated sequence, interferon-stimulated response element, activator protein 1 and nuclear factor