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

Abstract

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

Keywords: RRV; VP4; cholangiocytes; natural killer cells; rotavirus.

Fig. 1.

PhyML analysis of rotavirus VP4 and bile duct viral titers 1 and 7 days after infection. A: PhyML analysis was performed to determine the homology of Rhesus rotavirus (RRV) VP4 with other strains. B: a focus-forming assay (FFA) was performed on bile ducts harvested on postinoculation (PI) days 1 and 7 from newborn pups inoculated with 1.5 × 10⁶ focus-forming units (FFU)/pup of each strain of rotavirus. Six strains were able to infect the bile ducts and replicate to similar titers at 1 day PI with higher viral yields at 7 days PI: RRV, GRV, PA260, Ro1845, Cu-1, and TUCH. *P < 0.05 vs. RRV on PI day 1, #P < 0.05 vs. RRV on PI day 7; n = 6–10 mice.

Fig. 2.

Immunofluorescence detection of rotavirus in the liver parenchyma. Livers harvested 7 days PI, stained for CK-19 (green), rotavirus (red), and nucleus (blue) and overlaid to demonstrate colocalization (yellow-orange), indicated by arrows, confirmed the presence of rotavirus in the portal triad of pups inoculated with RRV, GRV, CU-1, PA260. and Ro1845. Magnification ×20.

Fig. 3.

Histological appearance of liver parenchyma following rotavirus infection. Representative hematoxylin and eosin (H&E)-stained livers from mice 7 days PI with various rotavirus strains show periportal inflammation (black arrows) only in strains that are capable of inducing biliary atresia (BA) [RRV and capra rotavirus strain (GRV)]. Inflammation was seen to a lesser extent with CU-1 but was absent in Ro1845, PA260, and saline controls. There were 15–20 mice in each group. Magnification ×20.

Fig. 4.

Histological appearance of the extrahepatic bile ducts following infection with various rotavirus strains. Representative H&E stained bile duct sections 7 days PI with rotavirus strains shows bile duct obstruction only in those strains that cause symptoms and mortality (RRV and GRV) but a patent bile duct was seen in the other strains that do not induce the model, including CU-1, PA260, Ro1845 and saline controls. Magnification ×10; n = 15–20 mice.

Fig. 5.

Cholangiograms demonstrating biliary obstruction. Methylene blue dye was injected into the gall bladders of mice 12–14 days postinoculation. Bile duct obstruction was seen in strains that cause symptoms and mortality (RRV and GRV). Bile ducts remained patent after infection with CU-1 and Ro1845. There were 5–10 mice in each group.

Fig. 6.

In vivo experiments with the Ro1845^R(VP4) reassortant. A: dsRNAs extracted from the single-gene reassortant were separated by SDS-PAGE and silver stained to reveal the migration rates of the gene segments. Arrow indicates gene substitution. B: pups inoculated with the Ro1845^R(VP4) reassortant induced symptoms of BA and mortality similar to RRV (n = 23 to 36 pups). *P < 0.05 vs. RRV, #P < 0.05 vs. Ro1845R(VP4). C: FFA revealed Ro1845^R(VP4) was able to replicate in bile ducts to the same high titer 7 days PI as RRV and Ro1845 (P = 0.249; n = 6–10). D: representative cholangiograms demonstrated patency of the bile duct in pups infected with Ro1845 but obstruction of the bile duct in pups infected with Ro1845^R(VP4) 12 days PI (n = 5–10).

Fig. 7.

NK cell activation and IFN-γ production is increased in the presence of the RRV VP4. A and B: flow cytometry of lymphocyte population harvested from the livers of 7 days PI pups illustrated a significant increase in the number of activated NK cells present in the model-inducing strains RRV and Ro1845^R(VP4) over saline controls, but not in Ro1845. Red quadrants show the percentage of CD69+ cells in the CD49b+ population (2 livers per sample and 3 samples per strain). C: EliSpot data obtained from purified NK cells isolated from RRV and Ro1845^R(VP4) shows increased production of IFN-γ. *P < 0.05; n = 4.

Fig. 8.

Cytotoxicity of NK cells obtained from RRV and Ro1845^R(VP4) livers. NK cells harvested from the model-inducing strains RRV and Ro1845^R(VP4) showed a significantly higher killing of uninfected cholangiocytes compared with those harvested from Ro1845 or saline controls. All strains where significantly different vs. saline. *P < 0.05; n = 4.