Cholangiocyte expression of α₂β₁-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

Abstract

lation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as ␣2␤1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express ␣2␤1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether ␣2␤1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the ␣2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the ␣2␤1-integrin. Newborn mice were pretreated with a monoclonal antibody against the ␣2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed ␣2␤1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-␣2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the ␣2␤1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection. rhesus rotavirus; bile ducts; cholangiopathy BILIARY ATRESIA (BA) is a unique disease of infancy in which affected children develop fibroinflammatory obliteration of the biliary tract. It is the most common indication for pediatric liver transplantation (24). Because pathogenic viruses have been found in the livers of children afflicted with biliary atresia (10,12,18,26,29), a proposed etiology for biliary atresia is perinatal infection by a virus triggering inflammatory destruction of the biliary epithelium (3,23,32). A murine model of biliary atresia (30) supports a viral pathogenesis where newborn mice infected with rhesus rotavirus (RRV) develop inflammation within the portal tract (31) and extrahepatic bile duct obstruction (9, 27).Recently, it has been shown that in the murine model of biliary atresia, RRV targeted the biliary epithelial cell (cholangiocyte) for infection (1, 31). To determine the basis for cholangiocyte susceptibility to RRV, an in vitro model of RRV infection of the two dominant cell types found within the liver (cholangiocytes and hepatocytes) was established. Consistent with the in vivo findings, RRV was better able to replicate in cholangiocytes than hepatocytes. Because the ability of rotavirus to infect cells appears to be regulated by cell-surface expression of the integrins ␣ 2 ␤ 1 , ␣ 4 ␤ 1 , ␣ v ␤ 3 , and ␣ x ␤ 2 , which serve as viral receptors (8,13,14,16,21), the cholangiocyte was surveyed for integrin expression and found to uniquely express the ␣ 2 ␤ 1 -integrin when compared with hepatocytes. Based on this informat...

Cooperation between IL-7 Receptor and Integrin α2β1 (CD49b) Drives Th17-Mediated Bone Loss

“…Thus, we tested the effect of Ha1/29 mAb directed against a2 integrin chain on IL-7-induced bone loss. The Ha1/29 mAb has shown potent blocking activity in vivo (29,30). Three-dimensional reconstructions of trabecular BV indicated that the anti-a2b1 mAb reduced trabecular bone damage associated with IL-7 treatment compared with mice treated with a control IgG mAb (Fig.…”

A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

“…BHK21-T7 cells (baby hamster kidney cell line expressing T7 polymerase; obtained from Ursula Buchholz, NIH, Bethesda, MD) were maintained in DMEM supplemented with nonessential amino acids, L-glutamine, penicillin-streptomycin, amphotericin B, and 10% fetal calf serum. The mouse cholangiocyte cell line (mCL), generously provided by the laboratory of James Boyer (Yale Liver Care Center, Hartford, CT), was cultured as previously described (51). All media were purchased from Gibco/BRL, and cells were grown at 37°C with 5% CO 2 .…”

Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia

“…MA104 cells (BioWhittaker, Walkersville, MD) were grown in Dulbecco's modified Eagle's medium (DMEM) (Cellgro) supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL, Gaithersburg, MD), 0.01% penicillin-streptomycin (GIBCO BRL), 0.01% L-glutamine (GIBCO BRL), and 0.005% amphotericin B (Cellgro). The mouse cholangiocyte cell line (mCL) generously provided by the laboratory of James Boyer (Yale Liver Care Center, Hartford, CT) was cultured as described previously (12). Sixteen culture-adapted rotavirus strains were grown in MA104 cells and used in this study: NCDV, WC-3, ST-3, VA-70, DS-1, WI-61, EMcN, EB, OSU, and RRV (Monica McNeal, Cincinnati Children's Hospital Medical Center); CU-1 (ATCC, Manassas, VA): TUCH (Dr. Karol Sestak, Tulane National Primate Research Center and Tulane University School of Medicine); PA260 (Dr. Vito Matella, University of Bari Aldo Moro); GRV (Dr. Osamu Nakagomi, Nagasaki University); KU (Dr. Koki Taniguchi, Fujita Health University); and Ro1845 (Yasutaka Hoshino, National Institute of Allergy and Infectious Diseases).…”

“…Intraperitoneal injection of neonatal BALB/c mice with Rhesus rotavirus (RRV) leads to symptoms (jaundice, acholic stools, bilirubinuria) and bile duct obstruction that mimic human disease (1,18). Previous studies demonstrated the unique tropism of RRV to the cholangiocyte (biliary epithelial cells) (12,23) and that development of the murine model is virus strain dependent (1). In a previous study, it was shown that a simian rotavirus strain, SA11-FM (having a fast moving VP4 gene) induced symptoms, obstruction, and mortality whereas another similar simian strain, SA11-SM (having a different VP4 gene that was slow moving when run on a polyacrylamide gel), showed some clinical signs of hepatobiliary injury without significant mortality (1).…”