Infection of liver sinusoidal endothelial cells with Muromegalovirus muridbeta1 involves binding to neuropilin-1 and is dynamin-dependent.

Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles, including some viruses. Endocytosis in LSEC is mainly via the clathrin-coated pit mediated route, which is dynamin-dependent. LSEC can also be a site of infection and latency of betaherpesvirus, but mode of virus entry into these cells has not yet been described. In this study we have investigated the role of dynamin in the early stage of muromegalovirus muridbeta1 (MuHV-1, murid betaherpesvirus 1, murine cytomegalovirus) infection in mouse LSECs. LSEC cultures were freshly prepared from C57Bl/6JRj mouse liver. We first examined dose- and time-dependent effects of two dynamin-inhibitors, dynasore and MitMAB, on cell viability, morphology, and endocytosis of model ligands via different LSEC scavenger receptors to establish a protocol for dynamin-inhibition studies in these primary cells. LSECs were challenged with MuHV-1 (MOI 0.2) ± dynamin inhibitors for 1h, then without inhibitors and virus for 11h, and nuclear expression of MuHV-1 immediate early antigen (IE1) measured by immune fluorescence. MuHV-1 efficiently infected LSECs in vitro. Infection was significantly and independently inhibited by dynasore and MitMAB, which block dynamin function via different mechanisms, suggesting that initial steps of MuHV-1 infection is dynamin-dependent in LSECs. Infection was also reduced in the presence of monensin which inhibits acidification of endosomes. Furthermore, competitive binding studies with a neuropilin-1 antibody blocked LSEC infection. This suggests that MuHV-1 infection in mouse LSECs involves virus binding to neuropilin-1 and occurs via endocytosis.

Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endothelial Cells (LSECs) in Analyses of Hepatic Drug Distribution.

Development of the new generation of drugs (e.g., oligo- and polynucleotides administered intravascularly either as free compounds or as nano-formulations) frequently encounters major challenges such as lack of control of targeting and/or delivery. Uncontrolled or unwanted clearance by the liver is a well-known and particularly important hurdle in this respect. Hence, reliable techniques are needed to identify the type(s) of liver cells, receptors, and metabolic mechanisms that are responsible for unwanted clearance of these compounds.We describe here a method for the isolation and culture of the major cell types from mouse liver: hepatocytes (HCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs). The presently described protocol employs perfusion of the liver with a collagenase-based enzyme preparation to effectively transform the intact liver to a single cell suspension. From this initial cell suspension HCs are isolated by specified centrifugation schemes, yielding highly pure HC preparations, and KCs and LSECs are isolated by employing magnetic-activated cell sorting (MACS). The MACS protocol makes use of magnetic microbeads conjugated with specific antibodies that bind unique surface antigens on either KCs or LSECs. In this way the two cell types are specifically and separately pulled out of the initial liver cell suspension by applying a magnetic field, resulting in high purity, yield, and viability of the two cell types, allowing functional studies of the cells.If the drug compound in question is to be studied with respect to liver cell distribution of intravascularly administered drug compounds the isolated cells can be analyzed directly after isolation. Detailed studies of receptor-ligand interactions and/or dynamics of intracellular metabolism of the compound can be conducted in primary surface cultures of HCs, LSECs, and KCs established by seeding the isolated cells on specified growth substrates.