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Perforin and granzymes work in synergy to mediate cholangiocyte injury in experimental biliary atresia
Pranavkumar Shivakumar1, Reena Mourya1, Jorge A Bezerra1
1Division of Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Pediatric Liver Care Center, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Insights
Perforin and granzymes from cytotoxic cells cause bile duct injury in biliary atresia. Inhibiting both perforin and granzymes is necessary to prevent this condition in infants.
Area of Science:
- Immunology
- Hepatology
- Cellular Biology
Background:
- Biliary atresia is a severe obstructive cholangiopathy in infants leading to cirrhosis.
- Activated natural killer (NK) cells expressing Nkg2d are implicated in bile duct injury.
- The precise mechanisms of cytotoxic cell involvement require further elucidation.
Purpose of the Study:
- To investigate the roles of perforin and granzymes in rotavirus (RRV)-induced experimental biliary atresia in neonatal mice.
- To define the mechanisms of bile duct epithelial injury mediated by cytotoxic cells.
Main Methods:
- Cell lysis assays, flow cytometry, and quantitative PCR were employed.
- In vivo studies utilized a neonatal mouse model of RRV-induced biliary atresia.
- Pharmacological inhibition of granzymes using nafamostat mesilate (FUT-175) was assessed.
Main Results:
- Perforin and granzymes from NK and CD8 T cells contribute to cholangiocyte lysis.
- Perforin deficiency delayed but did not prevent bile duct obstruction in vivo.
- Inhibition of granzymes with FUT-175 in perforin-deficient mice reduced jaundice and improved survival.
- Granzyme A and B mRNA increased in infants with biliary atresia, while perforin did not.
Conclusions:
- Perforin and granzymes play complementary roles in cytotoxic cell-mediated epithelial injury.
- Effective prevention of experimental biliary atresia requires simultaneous inhibition of both perforin and granzymes.
Background & Aims:
Biliary atresia represents obstructive cholangiopathy in infants progressing rapidly to cirrhosis and end-stage liver disease. Activated NK cells expressing Nkg2d have been linked to bile duct injury and obstruction by establishing contact with cholangiocytes. To define the mechanisms used by cytotoxic cells, we investigated the role of perforin and granzymes in a neonatal mouse model of rotavirus (RRV)-induced biliary atresia.
Methods:
We used complementary cell lysis assays, flow cytometric analyses, quantitative PCRs and in vivo systems to determine the mechanisms of bile duct epithelial injury and the control of the tissue phenotype in experimental biliary atresia.
Results:
RRV-infected hepatic NK and CD8 T cells increased the expression of perforin and injured cholangiocytes in short-term culture in a perforin-dependent fashion. However, the loss of perforin in vivo delayed but did not prevent the obstruction of bile ducts. Based on the increased expression of granzymes by perforin-deficient cytotoxic cells in long-term cytolytic assays, we found that the inhibition of granzymes by nafamostat mesilate (FUT-175) blocked cholangiocyte lysis. Administration of FUT-175 to perforin-deficient mice after RRV infection decreased the development of jaundice, minimized epithelial injury, and improved long-term survival. However, the inhibition of granzymes alone in wild-type mice was not sufficient to prevent the atresia phenotype in newborn mice. In infants with biliary atresia, hepatic Granzymes A and B mRNA, but not Perforin, increased at the time of portoenterostomy.
Conclusions:
Perforin and granzymes have complementary roles mediating epithelial injury by NK and CD8 T cells. The prevention of experimental biliary atresia can only be achieved by inhibiting both granules.
