Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.The etiologies of acute liver injury are diverse and its overall public health burden is considerable. Liver injury from acetaminophen (APAP) overdose is the most common cause of death from over-the-counter drugs and is the leading cause of acute liver failure in the developed world.^{1, 2, 3} Hepatic dysfunction from autoimmune hepatitis has a prevalence of 10–20/100 000.^{4, 5} Other etiologies of acute liver failure include idiosyncratic reaction to medications such as tetracycline, severe viral or alcoholic hepatitis, acute fatty liver of pregnancy, and idiopathic causes. Clinical complications resulting from liver failure include hepatic encephalopathy, impaired protein synthesis, and coagulopathies. Moreover, there are no effective means to reverse liver failure once advanced disease sets in – regardless of etiology – and transplantation frequently remains the only option for survival.⁶Concanavalin-A (ConA) is a lectin derived from the jack-bean plant with a unique ability to induce hepatitis in a well-described murine model of acute hepatic injury. ConA stimulates mouse CD4⁺ T-cell subsets to mediate insult to hepatocytes. The resulting cytokine release can further lead to recruitment and activation of innate inflammatory mediators, which perpetuate an insidious cycle of inflammation and hepatocellular injury.^{7, 8, 9}APAP is a widely used analgesic and antipyretic. Although usually considered safe at therapeutic doses, at higher doses APAP causes acute liver failure characterized by centrilobular hepatic necrosis.^{1, 10} At therapeutic doses, >90% of APAP is metabolized by glucuronidation and sulphation and its metabolites are excreted via the renal system. Of the remaining APAP, roughly 2% is excreted intact in the urine, and approximately 8% is metabolized by the cytochrome P450 system to N-acetyl-p-benzo-quinone imine (NAPQI), which is highly reactive.^{11, 12} Hepatic glutathione (GSH) then induces the formation of a safely excretable APAP-protein adduct. However, at toxic doses of APAP, GSH becomes depleted and NAPQI is able to exert harmful effects by forming covalent bonds with mitochondrial proteins, inhibiting the Ca²⁺-Mg²⁺-ATPase and inducing mitochondrial dysfunction.^{1, 2} This disturbance leads to a decrease in ATP synthesis, disruption of cellular membrane, and eventually hepatocyte death.¹³Although GSH depletion and the resulting toxic metabolites are prerequisites for APAP hepatotoxicity, there is evidence that the severity of liver injury may depend on subsequent participation of innate immunity.^{10, 14, 15, 16} In particular, APAP-induced injury has been reported to be contingent on activation of the NLRP3 inflammasome via DAMPs released from injured hepatocytes. Inflammasome activation cleaves Caspase 1 inducing IL-1β release and galvanizing intrahepatic neutrophils and inflammatory monocytes, which exacerbate injury.¹⁷ However, alternate studies using transgenic mice suggest that NLRP3 inflammasome is largely dispensable for APAP toxicity.¹⁸ Thus the role of inflammasome activation in APAP toxicity is controversial and may be dependent on the precise experimental conditions or strain of mice employed.Apoptosis and necrosis are classically understood processes of cell death that denote either organized Caspase 8-dependent programmed cell death or non-programmed disorganized death, respectively. In contrast to necrosis, which leads to the release of DAMPs and sustains inflammation, apoptosis produces cell fragments called apoptotic bodies, which phagocytic cells are able to engulf before the contents of the cell can spill out onto the surrounding space and activate innate immunity. ‘Necroptosis'' is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. Necroptosis requires the co-activation of RIP1 and RIP3 kinases. Both in vitro and in vivo investigations have suggested that APAP can induce cellular demise via necrosis or Caspase 8-dependent apoptosis, which is determined, in part, by ATP availability from glycolysis.¹⁹ Zhang et al.²⁰ recently confirmed that RIP1 is necessary in APAP-induced necroptosis. Similarly, Takemoto et al.²¹ showed that RIP1 inhibition protects against reactive oxygen species (ROS)-induced hepatotoxicity in APAP-induced acute liver injury. Further, a recent report suggested that selective inhibition of RIP3 using the anticancer drug Dabrafenib alleviates APAP injury.²²In the ConA model of acute liver injury, experiments using apoptosis-resistant mice expressing mutant FADD revealed that ConA alone induced primarily necrotic cell death, whereas ConA combined with d-galactosamine induced apoptosis and necrotic cell death.²³ Zhou et al.²⁴ reported that Necrostatin-1 (Nec-1) prevents autoimmune hepatitis in mice via RIP1- and autophagy-related pathways. Another recent report investigated the role of RIP1, RIP3, and PARP-1 in murine autoimmune hepatitis. This study found that in cases where death of mouse hepatocytes is dependent on TRAIL and NKT cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by RIP1 or PARP-1 inhibitors.²⁵ Our goal in the current study was to investigate, in parallel, the effects of RIP1 and RIP3 blockade in diverse models of acute liver injury. Our work suggests that modulating necroptosis may have divergent effects, depending on the etiology of hepatic injury and the specific component of the necrosome being targeted.