The establishment of hepadnaviral infection and the success of ongoing viral replication are critically dependent on the state of the host hepatocyte. In addition, exogenous soluble agents such as cytokines (TNF alpha, IFN gamma) or hormones (glucagon) have been shown to influence hapadnaviral gene expression and replication. In vivo, liver cells have permanent contact to agents present in the portal blood. Endotoxin (lipopolysaccharid, LPS), a component of Garm-negative bacteria cell-walls, which is absorbed from the large intestine and enters the portal circulation, is one of the major biologically active agents. LPS mainly acts in an indirect fashion and stimulates host cells to produce and release endogenous mediators. We studied whether infection of primary duck hepatocytes (PDH) with duck hepatitis B virus (DHBV) in vitro was influenced by LPS.
Primary hepatocytes were isolated by collagenase perfusion of the liver from 2 to 4 week old ducklings. When added in low, physiological concentrations, LPS from E. coli O55:B5 (1 pg/ml to 10 ng/ml) reduced in a dose-dependent fashion expression of viral proteins as well as virus production by hepatocytes infected with DHBV positive duck serum at an MOI 10 to 100 and by hepatocytes from DHBV infected ducks. LPS reduced virus production 3- to 20-fold when added before the establishment of infection with a maximal effect within the first 24 hours after infection. When added thereafter or added to PDH from DHBV-infected ducks, a maximally 2-fold reduction was observed. This shows that LPS inhibits hepadnaviral replication in vitro in a dose-dependent fashion with a maximal efficiency if added before the establishment of infection.
To address the question wether LPS acts via stimulation of nonparenchymal liver cells (NPC), we prepared a cell fraction enriched for NPC by differential centrifugation (50×g for hepatocytes, 250×g for smaller NPC). NPC were characterized by phagozytosis to identify macrophages (i.e. Kupffer cells) and by receptor-mediated uptake of acetylated LDL to identify sinusoidal endothelial cells. NPC were treated with LPS (100 ng/ml) for 3 hours. After extensive washing and further incubation for 12 hours to allow production of e.g. cytokines, culture medium was collected and used instead of LPS to pretreat primary hepatocytes before DHBV infection. DHBV infection could be completely blocked by soluble mediators secreted into the culture medium by LPS-stimulated NPC. This indicates that LPS is able to block productive hepadnaviral infection in an indirect fashion.