Activation of necroptosis in human and experimental cholestasis

Abstract

Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3−/−) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro. We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3−/− mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.

The in vivo evidence for regulated necrosis

“…The latter group also found increased RIPK3 and pMLKL in samples of patients with primary biliary cholangitis, which leads to cholestasis within the liver. 227 An interesting study specifically looked at the involvement of necroptosis in childhood IBD comparing samples of patients with ulcerative colitis, Crohn's disease, and allergic colitis, and reported reduced caspase-8 but increased RIPK3 and MLKL levels in inflamed tissue samples from patients suffering from Crohn's disease or allergic colitis. 228 These results are consistent with data showing elevated RIPK3 in Crohn's disease, especially in Paneth cells.…”

O-GlcNAc transferase suppresses necroptosis and liver fibrosis

“…Therefore, necroptosis is considered as a highly inflammatory form of cell death. In light of the data collected from patients, a myriad of studies in recent years have investigated the contribution of necroptosis in different mouse liver injury models and demonstrated the importance and 6 complexity of necroptosis in these pathological conditions (11)(12)(13)(14)(15)(16). However, the mechanism that governs necroptotic hepatocyte death is largely unknown.…”

MicroRNA‐506 promotes primary biliary cholangitis–like features in cholangiocytes and immune activation

“…In agreement with these PBC features, our data indicate that miR‐506 induces cell senescence by increasing p21 expression and stimulates cellular stress by increasing ROS levels, ER stress, and DNA damage; this altered phenotype did not result in baseline cholangiocyte cell death but sensitized cells against the caspase‐3‐dependent apoptosis induced by toxic bile acids. In contrast, the necrotic cell death pathway “necroptosis,” which was reported to be active in hepatocytes of an acute cholestatic animal model and PBC patients, did not have any role in bile acid–induced cholangiocyte death. The proapoptotic effect of miR‐506 may be related, to a certain degree, to its direct targeting of AE2 mRNA as experimental down‐regulation of AE2 in cholangiocytes has been shown to favor bile salt–induced apoptosis .…”