Cell death was analysed by WST-1 based assay and data are expressed as a percentage of transmission obtained in basal survival conditions without Fas-agonist. knockout mice (mice challenged with ConA developed severe acute hepatitis that sometimes led to death (4 out of the 12 enrolled animals), a co-treatment with Etanercept (ETA), a TNF- decoy receptor, significantly reduced liver injury and prevented any death. However, ETA did not fully protect mice from hepatitis (Fig.?1a). This suggested that other factors must take part in the hepatocyte death process, potentially such as FasL and/or TRAIL which have been shown to be involved during ConA-induced liver injury in WT mice2, 8C12. Thus, the severity of ConA-induced hepatitis could be significantly reduced in presence of FasL antagonists33. In agreement with this hypothesis, FasL, Fas and DR5 transcripts were significantly up-regulated in mice after ConA treatment, as in their WT littermates (mice (Fig.?1b). As Fas activation with the Fas-agonist mAb-Jo2 has been shown to induce acute liver injury14, we used this hepatitis model to explore the role of RIPK1 under Fas signaling. While injection of a low dose (0,15?mg/kg) of mAb-Jo2 provoked a moderate hepatitis in WT mice, the same treatment elicited more important liver damages in mice, as shown by higher levels of serum transaminases (Fig.?2a) and by the increased quantity of necrotic areas and of TUNEL positive cells, 6?h after the injection (Fig.?2b). Nevertheless, no individual died from this treatment. In the LY573636 (Tasisulam) Fas agonist-induced liver injury model, hepatocytes are believed to pass away by apoptosis since hepatocyte-specific caspase-8 deficient mice are guarded from liver injury34. Activation of c-Jun N-terminal kinases (JNKs) is also believed to take part in the apoptotic death process of LY573636 (Tasisulam) hepatocytes in different murine hepatitis models35, including those induced by Fas36. Therefore, we decided to investigate the apoptotic response by assessing the amount of cleaved caspase-3 and by analyzing JNK1/2 activation in the livers of and mice. As expected, the cleaved caspase-3 labelling revealed positive hepatocytes round the portal veins of WT mice (Fig.?2c). Moreover, activation of JNK1/2, revealed by enhanced phosphorylation, was detected 6?h LY573636 (Tasisulam) after mAb-Jo2 administration. Amazingly, both markers were greatly enhanced in the liver of the mice (Fig.?2d and see Supplementary Fig.?S1). Altogether, these data revealed that RIPK1 deficiency in liver parenchymal cells sensitized mice to Fas agonist-induced liver injury due to increased hepatocyte apoptosis. The enhanced hepatocyte death observed in mice was also associated with an exacerbated liver inflammation, as shown by the increased upregulation of TNF-, IL-1 and IL-6 transcripts detected in livers after mAb-Jo2 administration, when compared to similarly treated mice from ConA-induced liver injuries. (a) Levels of serum ALT, 11?h after PBS or ConA injection in mice, potentially pre-treated with ETA. Quantity of mice for each group: PBS (n?=?6), ConA (n?=?10) and ETA?+?ConA (n?=?5). (b) Levels of hepatic FasL, Fas, IFN-, TRAIL and DR5 transcripts in or mice 7?h after treatments with PBS or ConA (n?=?3C5), (ns: non-significant). For all those graphs, each circle represents an individual. Open in a separate window Physique 2 RIPK1 deficiency sensitizes mice to Fas-mediated liver injuries. (a) Levels of serum ALT and AST, 3 and 6?h after mAb-Jo2 injection in and mice (n?=?6C7). (b) Pictures of liver tissue sections, stained by H&E (upper panels) or analysed by TUNEL (in reddish) and DAPI (in blue) immunofluorescence (lower panels) issued from and mice, 6?h after mAb-Jo2 injection. Yellow arrows show necrotic areas, PV: portal vein. (c) Immunostaining of cleaved caspase-3 in the livers of and mice, 6?h after mAb-Jo2 injection. (d) Mean levels of cleaved caspase-3 (left panel) and of JNK phosphorylation status (right panel) in the livers of (n?=?5) and (n?=?5) mice, collected 6?h after mAb-Jo2 injection (see corresponding Western blots in Supplementary Fig.?S1). (e) LY573636 (Tasisulam) Levels of hepatic IL-1, IL-6 and TNF- transcripts in or mice, 6?h after PBS (n?=?3 mice) or mAb-Jo2 injection (n?=?6C7 LY573636 (Tasisulam) mice). For all those graphs, each circle represents an individual. Since we Rabbit Polyclonal to Granzyme B previously reported that, unlike their WT counterparts, RIPK1-deficient hepatocytes succumb by apoptosis upon TNF- sensing, we next investigated the potential contribution of TNF- in the Fas-agonist induced hepatitis model. Especially since the.