29.03 Murine Model of Viral-Induced Hepatic Fibrosis

Posted on January 18, 2016

I. N. Lobeck1, P. Dupree1, B. Donnelly1, S. Mohanty1, A. Walther1, M. McNeal2, G. Tiao1 2Cincinnati Children’s Hospital Medical Center,Division Of Infectious Disease,Cincinnati, OH, USA 1Cincinnati Children’s Hospital Medical Center,Division Of Pediatric General And Thoracic Surgery,Cincinnati, OH, USA

Introduction: Biliary atresia (BA) is a devastating neonatal obstructive cholangiopathy that progresses to fibrosis and end-stage liver disease by two years of age. Despite re-establishment of biliary drainage by portoenterostomy, many infants develop fibrosis requiring liver transplant. The mechanisms by which fibrosis occurs remain unknown. In the murine model of BA, Rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling human BA and is used to study mechanistic aspects of the disease. Infected mice however, experience a high mortality by DOL14. The aim of this study was to develop a model of a virus-induced biliary obstruction that allows survival beyond two weeks to determine if fibrosis occurs.

Methods: Rotavirus is a double-stranded RNA virus composed of eleven genes encoding 6 structural proteins (VP1-VP4, VP6 and VP7) and 6 nonstructural proteins (NSP1-NSP6). When two rotavirus strains simultaneously infect a host cell, the resulting progeny contain different combinations of the parental strain’s genome and are called reassortants. To better understand the RRV genes involved in the pathogenesis of BA, single and double gene reassortants were created by co-culture of RRV and TUCH (T), a simian rotavirus strain that does not cause the model. In the progeny, TR2 and TR4 are composed of ten genes from the TUCH parent with the VP2 or VP4 gene respectively, from RRV. TR2,4 contains both VP2 and VP4 RRV genes with the remaining nine genes from the TUCH parent. The VP4 gene was utilized due to its vital role in cholangiocyte tropism. TR2,4 was used due to its ability to replicate at a lower titer than RRV.

We have previously shown 80% mortality by DOL14 with RRV injected on DOL0-3. Thus, in our new model, newborn pups underwent intraperitoneal inoculation of the viruses on DOL4 in an effort to improve survival. Mice were monitored for biliary obstructive symptomatology. At DOL28, bile ducts and livers were harvested and evaluated by an independent pathologist using the Ishak fibrosis scoring system. Analysis was performed using the Chi-square test.

Results: Mice were inoculated with RRV (n=100), TR2 (n=50), TR4 (n=65) or TR2, 4 (n=100). TR2 resulted in minimal symptomatology (14% on DOL11) and no mortality. Ninety-four percent of TR4 mice exhibited obstructive symptoms on DOL11, with minimal mortality (6% by DOL28). RRV and TR2,4 displayed similar symptom presentation (96% and 99%, respectively, on DOL11) and no significant difference in morbidity (42% versus 38% by DOL28). Upon histologic analysis, no TR2 mice were found to have fibrosis. Twenty-two percent of TR4 and 40% of RRV mice exhibited Ishak grade 3-5 fibrosis. A significantly greater amount of fibrosis was seen in TR2,4 mice (63%).

Conclusion: The novel virus strain, TR2,4, can induce fibrosis by DOL28 to a significantly higher level than RRV without increase in mortality. This is the first murine viral-induced fibrosis model in a neonate.

29.04 Activation of pDCs by Rhesus Rotavirus VP4: Evidence of Innate Immune Response in Biliary Atresia

Posted on January 18, 2016

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.