The Chemical Inhibitors of Cellular Death, PJ34 and Necrostatin-1, Down-Regulate IL-33 Expression in Liver
Abstract
Interleukin-33 (IL-33), a cytokine belonging to the IL-1 family, is crucially involved in inflammatory pathologies including liver injury and is linked to various modes of cell death. However, a link between IL-33 and necroptosis or programmed necrosis in liver pathology remains elusive. This study aimed to investigate the regulation of IL-33 during necroptosis-associated liver injury. The possible regulation of IL-33 during liver injury by receptor-interacting protein kinase 1 (RIPK1) and poly(ADP-ribose) polymerase 1 (PARP-1) was investigated in mice in vivo and in hepatic stellate cells in vitro. Liver immunohistopathology, flow cytometry, serum transaminase measurement, ELISA, and qPCR-based cytokine measurement were carried out. Using a chemical approach, it was shown that pretreatment of mice with Necrostatin-1 (Nec-1), an inhibitor of RIPK1, and/or PJ34, an inhibitor of PARP-1, significantly protected mice against concanavalin A (ConA) liver injury (aspartate aminotransferase (AST)/alanine aminotransferase (ALT)) associated with down-regulated hepatocyte-specific IL-33 expression. In contrast, the expression level of most systemic cytokines (except for IL-6) or activation of liver immune cells was not altered by chemical inhibitors, although there was an increased infiltration of neutrophils in the liver. During polyinosine-polycytidylic acid (Poly(I:C))-induced acute hepatitis, liver injury and hepatocyte-specific IL-33 expression were also inhibited by PJ34 without any protective effect of PJ34 in CCl4-induced liver injury. Moreover, PJ34 down-regulated the protein expression of IL-33 in activated hepatic stellate cells by a cocktail of cytokines or staurosporine in vitro. In conclusion, the study evidenced that Nec-1/PJ34 is a potent inhibitor of liver injury and that Nec-1/PJ34 down-regulated hepatocyte-specific IL-33 expression in the liver in vivo or in hepatic stellate cells in vitro, suggesting IL-33 as a possible readout of necroptosis-involved liver pathologies.
Introduction
Liver injuries involve different mechanisms of cell death such as apoptosis, necrosis, or necroptosis (also called programmed necrosis) depending upon the death stimuli, cellular environment, and immune responses. These cellular death mechanisms determine the fate of the liver. Caspase-independent programmed necrosis is a novel form of cell death that is dependent on receptor-interacting protein (RIP) kinases, namely RIPK1 and RIPK3, upon engagement of several stimuli like the death receptors of tumor necrosis factor (TNF)-α superfamily. In the liver, hepatocytes undergo non-apoptotic cell death during partial hepatectomy and in steatohepatic livers with induction of RIPK3 expression in hepatocytes. The concanavalin A (ConA)-induced hepatic injury mimics immune cell-mediated fulminant hepatitis or autoimmune hepatitis in humans. In mice, ConA induces activation of liver natural killer T (NKT) cells and T cells that lead to hepatocyte death via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a TNF family member. Most studies reported that ConA-induced hepatic cell death was rather necrotic than apoptotic (independent of caspases). ConA-induced hepatic cell death was shown to be associated with RIPK1 and RIPK3, and conditional deficiency of hepatocyte caspase-8 sensitized mice to increased ConA liver injury associated with increased formation of necrosome. A protective effect of Necrostatin-1 (Nec-1), an inhibitor of RIPK1, has been found in ConA-induced acute hepatic injury in mice. Furthermore, poly(ADP-ribose) polymerase 1 (PARP-1) is now known to be involved in a RIPK1-RIPK3-dependent cascade of TRAIL-induced necroptosis, and it has been shown that ConA liver injury is a relevant TRAIL-induced necroptotic model. Polyinosine-polycytidylic acid (Poly(I:C)) is a synthetic analog of viral double-stranded RNA and mediates liver injury through Toll-like receptor 3 (TLR3). Liver injury induced by Poly(I:C) involves TNF receptor-associated factor 6 (TRAF6), receptor-interacting protein 1 (RIP1), and NF-κB activation. Carbon tetrachloride (CCl4)-induced acute liver toxicity in mice is used as a model of liver fibrosis with cytochrome-lipid-peroxidation-dependent free radical injury.
In the liver, interleukin-33 (IL-33), a member of the IL-1 family and also proposed as an alarmin cytokine, is expressed by hepatocytes in ConA hepatitis, in Poly(I:C)-induced liver injury, and in liver ischemia/reperfusion injury. Furthermore, NKT cells are responsible for the specific hepatocyte-IL-33 expression, and TRAIL is required to induce IL-33 in hepatocytes in this hepatic model. At a functional level, IL-33 interacts via its specific receptor ST2 and common chain IL-1RAcP to mediate its cytokine activity. In murine ConA hepatitis, administration of recombinant IL-33 induced protective effects by triggering anti-apoptotic effects, decreasing pro-inflammatory cytokines, and recruiting regulatory T cells (Tregs) in the liver.
IL-33 is proposed to be released in the functional/active form during necrotic cell death as an alarmin while it is inactivated during apoptotic cell death by caspases, and a link between IL-33 and necroptotic cell death remains elusive. In the present study, the expression of IL-33 and its chemical inhibition in ConA and Poly(I:C)-induced acute liver injury were investigated. A chemical inhibition approach was used by selective inhibition of RIPK1 activity by Nec-1 and PARP-1 inhibition by PJ34 during ConA-induced, Poly(I:C)-induced, and CCl4-induced acute hepatitis. The consequence in activation and recruitment of liver immune cells and cytokine levels during ConA hepatitis was investigated following Nec-1 and PJ34 treatment in mice. It was shown that RIPK1/PARP-1 inhibition by Nec-1+PJ34 protected mice against ConA liver injury. In conclusion, the pharmacological inhibition of liver injury by Nec-1/PJ34 down-regulated hepatocyte-specific IL-33 expression in the liver in vivo or in hepatic stellate cells in vitro.
Materials and Methods
Animals and Treatment Protocol
C57Bl/6 8–10-week-old wild-type mice were injected intravenously with ConA to induce acute hepatitis at a dose of 12 mg/kg body weight. Mice were sacrificed at 10 hours (for liver enzymes, mRNA, and protein expression of IL-33), 12, and 24 hours (for flow cytometry experiments) following ConA administration. Poly(I:C) was intravenously injected at a dose of 30 μg/mouse, and mice were sacrificed at 8 and 16 hours post-injection. Control mice received a similar volume of vehicle in each treatment group. For Necrostatin-1 (Nec-1) and PJ34 experiments, C57Bl/6 wild-type mice were pretreated with intraperitoneal administration of 10 mg/kg of PJ34 (1 hour before) or intravenous administration of 125 μg/mouse of Nec-1 (15 minutes before), or with both inhibitors prior to ConA or Poly(I:C) injection. Mice were treated with PJ34 intraperitoneally at 10 mg/kg body weight in PBS or PBS alone 1 hour before oral gavage of CCl4 in oil to induce acute hepatitis at a dose of 2.4 g/kg body weight. After 24 hours of the first injection, mice were treated with PJ34 and were sacrificed at 48 or 72 hours following CCl4 injection.
All mice were bred in specific pathogen-free conditions at the local animal house facilities. The study was conducted in compliance with French laws and the institution’s guidelines for animal welfare. All efforts were made to minimize suffering and the number of animals involved. The protocol was approved by the Comité Rennais d’Ethique en matière d’Expérimentation Animale, the county committee agreed by the Ministry of Research and Higher Education.
Histopathological, Biochemical, and Immunohistochemical Analysis
Histopathological (hematoxylin and eosin staining) and serum biochemical analyses, immunolocalization of IL-33, and the counting of IL-33-positive hepatocytes were performed as described earlier. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured according to the IFCC primary reference procedures. For histopathology, hematoxylin and eosin staining of liver tissues was carried out to assess liver injury. Paraformaldehyde-fixed and paraffin-embedded mouse liver sections followed by antigen retrieval were incubated with primary antibody against murine IL-33. Revelation of the primary antibody was carried out using HRP-conjugated rabbit polyclonal anti-goat secondary antibody followed by diaminobenzidine and hematoxylin coloration. The counting of IL-33-positive hepatocytes was carried out in at least 20 different microscopic fields corresponding to a 2.67-mm2 surface area using image analysis software. The truly IL-33-positive hepatocytes were counted on the basis of the diameter of cells and intensity of signals/degree of immunopositivity/staining by image analysis software.
In Vitro Hepatic Stellate Cell Stimulation and Western Blot
Human hepatic stellate cells were prepared by collagenase/pronase digestion of liver using a perfusion system and subsequent fractionation of the heterogeneous cell suspension on a continuous density gradient with Percoll. The viability of hepatic stellate cells was greater than 90% as estimated by Trypan blue test. Cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. The cells were stimulated by a cocktail of cytokines containing IL-1β, IL-6, TNF-α, and interferon-γ or with staurosporine for 24 hours. The effect of varying concentrations of PJ34 was tested upon activated hepatic stellate cells. Protein was extracted from control and stimulated cells for Western blot analysis, and detection of IL-33 and β-actin proteins was carried out as previously described. The ratio of IL-33 to β-actin band intensity was measured by imager analysis.
RNA Isolation and RT-qPCR
Total RNA was extracted from mice livers using TRIzol reagent. First-strand complementary DNA was synthesized using SuperScript II Reverse Transcriptase. The cDNA or RT amplification was further verified by PCR amplification using the housekeeping gene GAPDH. The protocol and conditions for RT-PCR and qPCR were the same as reported earlier by the laboratory using specific primers for 18S, IL-33, IL-6, IL-1β, IFN-γ, TNF-α, and CXCL1.
Cytokine Measurement
The mouse Th1/Th2 10plex sample kit was used to measure cytokines in sera according to the prescribed protocol of the supplier.
Isolation of Liver Immune Cells and Flow Cytometry Analysis
Liver immune cells were isolated as described earlier with a viability of greater than 95%. Cells were resuspended in staining buffer and preincubated with anti-CD16/32 to block non-specific binding. The cells were then labeled with appropriate fluorochrome-conjugated antibodies and reagents. The stained cells were analyzed on a flow cytometer, and data were analyzed by appropriate software. Dead cells and doublet cells were excluded on the basis of forward and side scatter.
Statistical Analysis
The results are representative of three independent experiments with five to seven animals in each experimental group, and data were expressed as means ± SEM. Mann-Whitney U test was used for comparison of control group parameters with the treatment group, and multiple group analysis was carried out by one-way ANOVA. The correlation between continuous variables was analyzed using statistical software. For all statistical analyses, a p-value less than 0.05 was considered significant.
Results
Pretreatment with Nec-1 and PJ34 protected mice against ConA-induced liver injury, as evidenced by reduced serum AST and ALT levels and decreased histopathological damage. The protective effect was associated with a significant reduction in hepatocyte-specific IL-33 expression. In contrast, the expression of most systemic cytokines, except for IL-6, or the activation of liver immune cells was not altered by the chemical inhibitors. However, there was an increased infiltration of neutrophils in the liver. During Poly(I:C)-induced acute hepatitis, liver injury and hepatocyte-specific IL-33 expression were also inhibited by PJ34, but PJ34 did not provide protection in CCl4-induced liver injury. In vitro, PJ34 down-regulated the protein expression of IL-33 in activated hepatic stellate cells stimulated by a cocktail of cytokines or staurosporine.
In conclusion, these findings demonstrate that Nec-1 and PJ34 are potent inhibitors of liver injury and that their administration down-regulates hepatocyte-specific IL-33 expression in the liver in vivo and in hepatic stellate cells in vitro. This suggests that IL-33 may serve as a possible readout of necroptosis-involved liver pathologies.