乙型肝炎病毒（HBV）感染会演变为急性或慢性肝脏疾病，从脂肪变性、肝炎、纤维化到肝硬化和肝细胞癌（HCC）。在慢性肝脏疾病中，纤维化伴随着增加的间质硬度，被视为疾病进展的强力预测指标。本研究旨在确定肝纤维化组织中增加的肝脏硬度调节HBV感染性的机制。 为了进行体外研究，HBV转染的HepG2.2.15细胞在具有2 kPa（柔软）或24 kPa（硬性）刚度的聚硅氧烷凝胶上培养，模拟健康或纤维化肝脏的硬度。为了进行体内研究，通过每周两次注射CCl4而在C57Bl/6亲本和HBV+转基因（HBVTg）小鼠中诱导肝纤维化，持续6周。 我们发现，与软凝胶连接的肝细胞中HBV标志物水平更高，同时细胞上清液中的OPN含量升高，并且抗病毒干扰素刺激基因（ISGs）被抑制。这表明，预期的“纤维化”硬度会增加磷酸积石藻（OPN）含量并释放出并抑制抗病毒天然免疫，从而导致肝细胞中HBV标志物表达的上升。体外实验结果通过HBVTg小鼠CCl4给药（HBVTg CCl4）的数据得到证实。这些小鼠在诱导肝纤维化后显示出更高的HBV RNA、DNA、HBV核心抗原（HBcAg）和HBV表面抗原（HBsAg）水平，伴随着Col1a1、SMA、MMPs和TIMPs mRNA的升高以及肝脏硬度的增加。值得注意的是，CCl4诱导肝细胞的增强纤维化活化以及肝脏硬度在HBVTg小鼠中高于对照小鼠。HBV标志物和OPN水平的升高对应着HBVTg CCl4小鼠与HBVTg对照小鼠中ISG活化的降低。 根据我们的数据，我们得出结论：肝脏硬度提高OPN水平以限制肝细胞中的抗病毒ISG激活，促进HBV感染性的增加，从而促进末期肝脏疾病的进展。版权所有©2023 Bybee, Moeun, Wang, Kharbanda, Poluektova, Kidambi, Osna和Ganesan。

Hepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue.For in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks.We found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite "fibrotic" stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice.Based on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression.Copyright © 2023 Bybee, Moeun, Wang, Kharbanda, Poluektova, Kidambi, Osna and Ganesan.