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

Abstract

Background and 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.

Live cell evaluation of granzyme delivery and death receptor signaling in tumor cells targeted by human natural killer cells

“…Based on reports of GrzA inhibition, we reasoned that the pan-tryptase inhibitor, nafamostat mesylate (NM), would inhibit both GrzA-and GrzK-induced luciferase activity. [33][34][35][36] After preincubation with recombinant GrzA and GrzK, NM inhibited biosensor activation induced by both enzymes, although the magnitude of GrzA inhibition was greater than that of GrzK ( Figure 1F). Although subtle, these findings represent the first report of GrzK inhibition by nafamostat mesylate.…”

Pathogenesis of biliary atresia: defining biology to understand clinical phenotypes

“…100 The expression of perforin and granzymes by cytotoxic T cells and natural killer cells seem to be key effector molecules. 126 The similarities between the phenotypes produced by the loss of CD8 + T cells or Ifnγ expression suggest that both factors work in parallel to promote duct obstruction (Figure 2). 84,100 Although these data pointed to key roles of the T H 1 cell response in pathogenesis of experimental biliary atresia, the administration of RRV into newborn mice engineered to lack a T H 1 cell response by the inactivation of Stat1 gene unexpectedly resulted in the full obstructive phenotype, in addition to focal cystic dilatations of extrahepatic bile ducts.…”

Innate Immunity and Pathogenesis of Biliary Atresia

“…IL-8, mostly produced by macrophages but also by cholangiocytes (90), recruits and modulates the action of neutrophils (85), basophils, monocytes, and T cells (64,67,90); while IL-15, secreted primarily by DCs, attracts and regulates the activity of natural killer (NK), natural killer T (NKT), and gamma-delta cells (89). The recruited inflammatory effector cells are engaged to target specifically the biliary epithelium in a contact dependent manner (91), through IFN-γ-related cytokines (48) and/or cytotoxic agents (perforins, granzymes) (92). Recruited neutrophils produce reactive oxygen species (ROS), leukotrienes, and neutrophil defensins (90).…”

“…Recruited neutrophils produce reactive oxygen species (ROS), leukotrienes, and neutrophil defensins (90). NK cells, activated by DCs via IL-15 (89), induce cholangiocyte death in a contact-dependent manner through Natural killer group 2d (Nkg2d) ligand that interacts with ribonucleic acid export 1 (RAE1) receptors, expressed in infected cells (91) and via the secretion of IFN-γ, perforins, and granzymes (92). In a similar fashion, the cytotoxic power of neonatal CD8 + T cells is exerted through cytotoxic agents (perforin, granzymes, IFN-γ) (92) and in a contact-dependent manner by invading the epithelium (27).…”