Yuan Liu1, Wenbo Wang2, Ziying Zou3, Zonghai Hu3, Quanshui Fan2, Jie Xiong4. 1. Clinical Laboratory, The General Hospital of Western Theater Command, Chengdu, China. liuyuan198231@163.com. 2. Center for Disease Control and Prevention of Western Theater Command, Chengdu, China. 3. Clinical Laboratory, The General Hospital of Western Theater Command, Chengdu, China. 4. Clinical Laboratory, The General Hospital of Western Theater Command, Chengdu, China. xiongjie1969@126.com.
Abstract
BACKGROUND: Hepatitis C virus (HCV) has been classified as a strictly hepatotropic pathogen for a long time, and hepatocytes are target cells for HCV infection. More and more studies showed non-liver cells supported HCV entry and replication, such as macrophages. The mechanisms of HCV entry into macrophages are still not clear. AIMS: This study aims to determine the way of HCV entry into macrophages. METHODS: Cell culture-derived infectious HCV particles (HCVcc) were prepared using Huh7 cells transfected with HCV RNA. CD81-knockdown cells were obtained through siRNA transfection. HCV RNA levels were determined by RT-qPCR. Flow cytometry analyses were used to determine cell surface levels of CD11b, CD68, and CD81. ELISA and western blotting were performed to quantify the protein levels of IL-1β, IL-6, and TNF-α. Phagocytic ability was determined by neutral red uptake assay. RESULTS: CD81 knockdown could not inhibit HCVcc entry into macrophages. The entry of HCV into macrophages could not be blocked by pooled IgG from chronic hepatitis C patient's sera. Macrophages derived from THP-1 cells displayed stronger phagocytic capacity, which also swallowed more HCV RNA. Treatment of macrophages with endocytic inhibitor, methyl-β-cyclodextrin, decreased the internalization of HCV. HCV uptake by macrophages was related to the reorganization of F-actin cytoskeleton and PI3Ks activation. HCV infection significantly increased the expression of IL1β and IL6 in macrophages and promoted apoptosis of macrophages. CONCLUSIONS: HCV entry into macrophages mainly depends on phagocytosis of macrophages.
BACKGROUND:Hepatitis C virus (HCV) has been classified as a strictly hepatotropic pathogen for a long time, and hepatocytes are target cells for HCV infection. More and more studies showed non-liver cells supported HCV entry and replication, such as macrophages. The mechanisms of HCV entry into macrophages are still not clear. AIMS: This study aims to determine the way of HCV entry into macrophages. METHODS: Cell culture-derived infectious HCV particles (HCVcc) were prepared using Huh7 cells transfected with HCV RNA. CD81-knockdown cells were obtained through siRNA transfection. HCV RNA levels were determined by RT-qPCR. Flow cytometry analyses were used to determine cell surface levels of CD11b, CD68, and CD81. ELISA and western blotting were performed to quantify the protein levels of IL-1β, IL-6, and TNF-α. Phagocytic ability was determined by neutral red uptake assay. RESULTS:CD81 knockdown could not inhibit HCVcc entry into macrophages. The entry of HCV into macrophages could not be blocked by pooled IgG from chronic hepatitis Cpatient's sera. Macrophages derived from THP-1 cells displayed stronger phagocytic capacity, which also swallowed more HCV RNA. Treatment of macrophages with endocytic inhibitor, methyl-β-cyclodextrin, decreased the internalization of HCV. HCV uptake by macrophages was related to the reorganization of F-actin cytoskeleton and PI3Ks activation. HCV infection significantly increased the expression of IL1β and IL6 in macrophages and promoted apoptosis of macrophages. CONCLUSIONS:HCV entry into macrophages mainly depends on phagocytosis of macrophages.
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