Perforin and granzymes work in synergy to mediate cholangiocyte injury in experimental biliary atresia

Abstract

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.

Keywords: Children; Cholangiocyte; Cholestasis; Immunity; Jaundice; Liver; Neonates; PKO; RRV; Rhesus rotavirus type A; WT; ffu; fluorescence-forming units; perforin knockout; wild type.

Fig. 1. Perforin expression and cholangiocyte lysis

(A) mRNA expression for Perforin in extrahepatic bile ducts from WT mice at 3, 5 and 7 days after saline or RRV challenge as a ratio to mouse Gapdh; N=3 per time-point; *=P<0.001. (B) Median Fluorescence Intensity (MFI) histograms showing perforin expression by NK and CD8 T cells after saline or RRV challenge. N=3 per time-point; Mean ± SD MFI values are shown. Lower panels show percent cholangiocyte lysis after incubation with NK (C) or CD8 T cells (D) from RRV- WT (grey bars), PKO (black bars) and saline-injected mice (open bars). Hepatic NK and CD8 T cells were obtained from a pool of 6–8 livers; *=P<0.01, **= P<0.003, ns: not significant.

Fig. 2. Impact of perforin deficiency on outcome and cholangiocyte lysis after RRV infection

(A) RRV infection resulted in poor growth and onset of jaundice, acholic stools and bilirubinuria in WT and PKO mice, but mortality was delayed in PKO mice (Kaplan-Meier survival analysis, N=9-33 per group in each experiment). In panel (B), sections of extrahepatic bile ducts from RRV-challenged WT (top panel) and PKO (middle panel) mice show severe cholangitis and lumenal obstruction at 5-7 days. Lower panel shows normal anatomy in age-matched control PKO mice. Arrows: subepithelial inflammation; arrowheads: intact duct epithelium; asterisks: lumen. Percent cholangiocyte lysis after incubation with NK (C) and CD8 T cells (D) from RRV-WT (grey bars), PKO (black bars) and saline-injected mice (open bars). Hepatic NK and CD8 T cells were obtained from a pool of 6–8 livers; *=P<0.001.

Fig. 3. Contribution of NK cell granzymes to cholangiocyte lysis

(A) Granzyme-B levels in supernatants following co-culture of PKO NK cells and cholangiocytes (mCL). Arrows indicate non-detectable levels in saline group (blue) or in the RRV group (green) with a transwell between NK and mCL. N=6-8 mice per group; *=P<0.0001. (B) Percent lysis after co-culture of PKO NK cells with mCL in the presence or absence of 10μg/mL granzyme-B antibodies or 15mg/mL FUT-175. Hepatic NK cells were obtained from a pool of 6–8 livers; *=P<0.006. (C) Scatter plots showing granzyme-B expression by NK and CD8 T cells from PKO mice 7 days after RRV infection. Numbers represent percent NK or CD8 T cells expressing granzyme-B; N=3 mice per time-point. (D) mRNA expression of Granzyme A and B in PKO mice expressed as a ratio to mouse Gapdh. N=3 replicates per time-point; *=P<0.003.

Fig. 4. Prevention of experimental biliary atresia by FUT-175 treatment of perforin-deficient mice

(A) Daily injections of FUT-175 improved long-term weight, jaundice, acholic stools, bilirubinuria and long-term survival of Perforin knockout (PKO) mice challenged with RRV. N=9-29 mice per group; *=P<0.001; Kaplan-Meier survival analysis shows increased survival after FUT-175 treatment (P=0.013). (B) Longitudinal sections of extrahepatic bile ducts from PKO mice showing normal histology after saline (top panel), typical duct inflammation and obstruction after RRV (middle panel), and prevention of duct obstruction in mice receiving FUT-175 (lower panel). Arrows point to inflammation, arrowheads to the duct epithelium, and asterisks to the lumen.