P. D. Dupree1, I. Lobeck1, B. Donnelly1, A. Walther1, M. McNeal2, E. Janssen3, S. Mohanty1, G. Tiao1 2Cincinnati Children’s Hospital Medical Center,Infectious Disease,Cincinnati, OH, USA 3Cincinnati Children’s Hospital Medical Center,Immunobiology,Cincinnati, OH, USA 1Cincinnati Children’s Hospital Medical Center,Pediatric Surgery,Cincinnati, OH, USA
Introduction: Biliary atresia (BA) is a progressive obstructive cholangiopathy of infancy which leads to cirrhosis, end stage liver disease, and often times require liver transplantation. The etiology of BA still remains unclear but a current hypothesis suggests a perinatal viral infection triggers the host’s inflammatory immune response. Previously we identified the VP4 gene on Rhesus Rotavirus (RRV) as the major determinant for the induction of the murine model of BA. Using a non-pathogenic strain, Ro1845, we generated a reassortant (Ro1845 R(VP4)) with 10 genes from the parent strain and one gene, VP4, from RRV. We demonstrated that Ro1845 R(VP4) is able to infect and replicate in cholangiocytes similar to that of RRV, as well as induce the BA model. Using Ro1845 R(VP4) we also showed that RRV’s VP4 plays a role in NK cell activation. A previous study found that plasmacytoid dendritic cells (pDCs) activate NK cells in the murine model of BA. We hypothesized that RRV’s VP4 plays an important role in activation of pDCs which subsequently activate the NK cells.
Methods: BALB/c mice were injected within the first 24 hours of life with saline, RRV, Ro1845, or Ro1845 R(VP4). Livers and bile ducts were harvested on post-infection (PI) day 7 or 10. Miltenyi column purification kits were used to isolate pDCs, CD8+ cells, and NK cells from the harvested livers. Viral titers and flow cytometry were performed on the immune cell populations. In order to isolate a larger quantity of pDCs, adult BALB/c mice were injected with B16 Flt-3 ligand, which causes expansion of the dendritic cell population. Naive pDCs were isolated from B16 Flt-3 ligand injected mice and used for RRV infection.
Results: The purified NK cells and CD8+ cells harvested from the livers on PI day 7 were found to have all three viral strains present. However, virus was only detected in the pDCs isolated from RRV and Ro1845 R(VP4) infected mice (1.6±0.1 x 103, 2.4±1.0 x 103 respectively) but not in Ro1845 infected mice. At day 10 PI, virus persisted in both the NK cells, and pDCs, of RRV and Ro1845 R(VP4) infected mice but was not detectable in Ro1845 infected immune cells. Flow cytometry demonstrated a significant increase in the activation of pDCs isolated from RRV (39.9%) and Ro1845 R(VP4) ( 42.4%) infected mice compared to Ro1845 infected mice (27.8%, p<0.05). Viral titers measured in naïve pDCs infected with RRV in-vitro demonstrated the evidence of virus replication in pDCs (9.3±1.8 x 105).
Conclusion: RRV VP4 plays an important role in the activation of pDCs and NK cells. Measurement of viral yield demonstrated that non-disease inducing strains (Ro1845) were capable of infecting CD8+ and NK cells but only those strains expressing the RRV VP4 have the potential to infect the pDCs. These findings further define the pathogenesis of biliary atresia and may have implications for future therapies to block the progression of the disease.