Catherine Lucas-Clerc

NKT cells are required to induce high IL-33 expression in hepatocytes during ConA-induced acute hepatitis

Interleukin‐33 (IL‐33) is thought to be released during cellular death as an alarmin cytokine during the acute phase of disease, but its regulation in vivo is poorly understood. We investigated the expression of IL‐33 in two mouse models of acute hepatitis by administering either carbon tetrachloride (CCl4) or concanavalin A (ConA). IL‐33 was overexpressed in both models but with a stronger induction in ConA‐induced hepatitis. IL‐33 was weakly expressed in vascular and sinusoidal endothelial cells from normal liver and was clearly induced in CCl4‐treated mice. Surprisingly, we found that hepatocytes strongly expressed IL‐33 exclusively in the ConA model. CD1d knock‐out mice, which are deficient in NKT cells and resistant to ConA‐induced hepatitis, no longer expressed IL‐33 in hepatocytes following ConA administration. Interestingly, invariant NKT (iNKT) cells adoptively transferred into ConA‐treated CD1d KO mouse restored IL‐33 expression in hepatocytes. This strongly suggests that NKT cells are responsible for the induction of IL‐33 in hepatocytes.

TRAIL but not FasL and TNFα, regulates IL‐33 expression in murine hepatocytes during acute hepatitis

Interleukin (IL)‐33, a member of the IL‐1 cytokine family, positively correlates with acute hepatitis and chronic liver failure in mice and humans. IL‐33 is expressed in hepatocytes and is regulated by natural killer T (NKT) cells during concanavalin A (ConA)‐induced acute liver injury. Here, we investigated the molecular mechanisms underlying the expression of IL‐33 during acute hepatitis. The expression of IL‐33 and its regulation by death receptor pathways was investigated after the induction of ConA‐acute hepatitis in wildtype (WT), perforin−/−, tumor necrosis factor related apoptosis inducing ligand (TRAIL)−/−, and NKT cell‐deficient (CD1d−/−) mice. In addition, we used a model of acute liver injury by administering Jo2/Fas‐antibody or D‐galactosamine‐tumor necrosis factor alpha (TNFα) in WT mice. Finally, the effect of TRAIL on IL‐33 expression was assessed in primary cultured murine hepatocytes. We show that IL‐33 expression in hepatocytes is partially controlled by perforin during acute liver injury, but not by TNFα or Fas ligand (FasL). Interestingly, the expression of IL‐33 in hepatocytes is blocked during ConA‐acute hepatitis in TRAIL‐deficient mice compared to WT mice. In contrast, administration of recombinant murine TRAIL associated with ConA‐priming in CD1d‐deficient mice or in vitro stimulation of murine hepatocytes by TRAIL but not by TNFα or Jo2 induced IL‐33 expression in hepatocytes. The IL‐33‐deficient mice exhibited more severe ConA liver injury than WT controls, suggesting a protective effect of IL‐33 in ConA‐hepatitis. Conclusion: The expression of IL‐33 during acute hepatitis is dependent on TRAIL, but not on FasL or TNFα. (HEPATOLOGY 2012)

The chemical inhibitors of cellular death, PJ34 and Necrostatin-1, down-regulate IL-33 expression in liver

Interleukin-33 (IL-33), a cytokine belonging to the IL-1 family, is crucially involved in inflammatory pathologies including liver injury and linked to various modes of cell death. However, a link between IL-33 and necroptosis or programmed necrosis in liver pathology remains elusive. We 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. The liver immunohistopathology, flow cytometry, serum transaminase measurement, ELISA, and qPCR-based cytokine measurement were carried out. By using a chemical approach, we showed that pretreatment of mice with Necrostatin-1 (Nec-1) (inhibitor of RIPK1) and/or PJ34 (inhibitor of PARP-1) significantly protected mice against concanavalin A (ConA) liver injury (aspartate amino-transferase (AST)/alanine amino-transferase (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 rather 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 was 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 cocktail of cytokines or staurosporine in vitro. In conclusion, we evidenced that the Nec-1/PJ34 is a potent inhibitor of liver injury and 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.Key messageNecroptosis inhibitors can protect mice against liver injury induced by ConA or Poly(I:C).IL-33 expression in liver injury in vivo is inhibited by PJ34.IL-33 expression in hepatic stellate cells in vitro is inhibited by PJ34.Hepatocyte-specific IL-33 expression is down-regulated by Nec-1/PJ34 during hepatitis.IL-33 is a new marker of necroptosis-associated liver injuries.

Pathogenic Mouse Hepatitis Virus or Poly(I:C) Induce IL-33 in Hepatocytes in Murine Models of Hepatitis

The IL-33/ST2 axis is known to be involved in liver pathologies. Although, the IL-33 levels increased in sera of viral hepatitis patients in human, the cellular sources of IL-33 in viral hepatitis remained obscure. Therefore, we aimed to investigate the expression of IL-33 in murine fulminant hepatitis induced by a Toll like receptor (TLR3) viral mimetic, poly(I:C) or by pathogenic mouse hepatitis virus (L2-MHV3). The administration of poly(I:C) plus D-galactosamine (D-GalN) in mice led to acute liver injury associated with the induction of IL-33 expression in liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells (VEC), while the administration of poly(I:C) alone led to hepatocyte specific IL-33 expression in addition to vascular IL-33 expression. The hepatocyte-specific IL-33 expression was down-regulated in NK-depleted poly(I:C) treated mice suggesting a partial regulation of IL-33 by NK cells. The CD1d KO (NKT deficient) mice showed hepatoprotection against poly(I:C)-induced hepatitis in association with increased number of IL-33 expressing hepatocytes in CD1d KO mice than WT controls. These results suggest that hepatocyte-specific IL-33 expression in poly(I:C) induced liver injury was partially dependent of NK cells and with limited role of NKT cells. In parallel, the L2-MHV3 infection in mice induced fulminant hepatitis associated with up-regulated IL-33 expression as well as pro-inflammatory cytokine microenvironment in liver. The LSEC and VEC expressed inducible expression of IL-33 following L2-MHV3 infection but the hepatocyte-specific IL-33 expression was only evident between 24 to 32h of post infection. In conclusion, the alarmin cytokine IL-33 was over-expressed during fulminant hepatitis in mice with LSEC, VEC and hepatocytes as potential sources of IL-33.

RIPK1 protects from TNF- α -mediated liver damage during hepatitis

Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-α) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-α-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1K45A) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1LPC-KO), to characterize the role of RIPK1 and TNF-α in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1LPC-KO mice challenged with ConA, TNF-α triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-κB activation, as well as TNF-dependent, but canonical NF-κB-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases.

RIPK1 protects hepatocytes from Kupffer cells-mediated TNF-induced apoptosis in mouse models of PAMP-induced hepatitis

BACKGROUND & AIMS The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. METHODS Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1LPC-KO). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-α during LPS-mediated liver injury. RESULTS Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. CONCLUSIONS Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. LAY SUMMARY Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure.

Ablation of interaction between IL-33 and ST2+ regulatory T cells increases immune cell-mediated hepatitis and activated NK cell liver infiltration

The IL-33/ST2 axis plays a protective role in T-cell-mediated hepatitis, but little is known about the functional impact of endogenous IL-33 on liver immunopathology. We used IL-33-deficient mice to investigate the functional effect of endogenous IL-33 in concanavalin A (Con A)-hepatitis. IL-33(-/-) mice displayed more severe Con A liver injury than wild-type (WT) mice, consistent with a hepatoprotective effect of IL-33. The more severe hepatic injury in IL-33(-/-) mice was associated with significantly higher levels of TNF-α and IL-1β and a larger number of NK cells infiltrating the liver. The expression of Th2 cytokines (IL-4, IL-10) and IL-17 was not significantly varied between WT and IL-33(-/-) mice following Con A-hepatitis. The percentage of CD25(+) NK cells was significantly higher in the livers of IL-33(-/-) mice than in WT mice in association with upregulated expression of CXCR3 in the liver. Regulatory T cells (Treg cells) strongly infiltrated the liver in both WT and IL-33(-/-) mice, but Con A treatment increased their membrane expression of ST2 and CD25 only in WT mice. In vitro, IL-33 had a significant survival effect, increasing the total number of splenocytes, including B cells, CD4(+) and CD8(+) T cells, and the frequency of ST2(+) Treg cells. In conclusion, IL-33 acts as a potent immune modulator protecting the liver through activation of ST2(+) Treg cells and control of NK cells.

Chlordecone potentiates hepatic fibrosis in chronic liver injury induced by carbon tetrachloride in mice

Chronic liver damage due to viral or chemical agents leads to a repair process resulting in hepatic fibrosis. Fibrosis may lead to cirrhosis, which may progress to liver cancer or a loss of liver function, with an associated risk of liver failure and death. Chlordecone is a chlorinated pesticide used in the 1990s. It is not itself hepatotoxic, but its metabolism in the liver triggers hepatomegaly and potentiates hepatotoxic agents. Chlordecone is now banned, but it persists in soil and water, resulting in an ongoing public health problem in the Caribbean area. We assessed the probable impact of chlordecone on the progression of liver fibrosis in the population of contaminated areas, by developing a mouse model of chronic co-exposure to chlordecone and a hepatotoxic agent, carbon tetrachloride (CCl4). After repeated administrations of chlordecone and CCl4 by gavage over a 12-week period, we checked for liver damage in the exposed mice, by determining serum liver transaminase (AST, ALT) levels, histological examinations of the liver and measuring the expression of genes encoding extracellular matrix components. The co-exposure of mice to CCl4 and chlordecone resulted in significant increases in ALT and AST levels. Chlordecone also increased expression of the Col1A2, MMP-2, TIMP-1 and PAI-1 genes in CCl4-treated mice. Finally, we demonstrated, by quantifying areas of collagen deposition and alpha-SMA gene expression, that chlordecone potentiated the hepatic fibrosis induced by CCl4. In conclusion, our data suggest that chlordecone potentiates hepatic fibrosis in mice with CCl4-induced chronic liver injury.

Endogenous IL-33 has no effect on the progression of fibrosis during experimental steatohepatitis

Interleukin (IL)-33 has been recently reported to be strongly pro-fibrogenic in various models of liver disease. Our aim was to study the role of endogenous IL-33 in a diet-induced model of steatohepatitis. IL-33 deficient mice and wild type (WT) littermates received a high-fat diet (HFD), or a standard diet for 12 weeks. The HFD-induced steatohepatitis was associated with an upregulation of IL-33 transcripts and protein. An insulin tolerance test revealed lower systemic insulin sensitivity in IL-33-/—HFD mice than in WT-HFD mice. Nevertheless, IL-33 deficiency did not affect the severity of liver inflammation by histological and transcriptomic analyses, nor the quantity of liver fibrosis. Livers from HFD mice had more myeloid populations, markedly fewer NKT cells and higher proportion of ST2+ Treg cells and ST2+ type 2 innate lymphoid cells (ILC2), all unaffected by IL-33 deficiency. In conclusion, deficiency of endogenous IL-33 does not affect the evolution of experimental diet-induced steatohepatitis towards liver fibrosis.

Oncostatin M induces IL-33 expression in liver endothelial cells in mice and expands ST2+CD4+ lymphocytes

Interleukin (IL)-33 is crucially involved in liver pathology and drives hepatoprotective functions. However, the regulation of IL-33 by cytokines of the IL-6 family, including oncostatin M (OSM) and IL-6, is not well studied. The aim of the present study was to determine whether OSM mediates regulation of IL-33 expression in liver cells. Intramuscular administration in mice of an adenovirus encoding OSM (AdOSM) leads to increase in expression of OSM in muscles, liver, and serum of AdOSM-infected mice compared with control mice. The increase of circulating OSM markedly regulated mRNA of genes associated with blood vessel biology, chemotaxis, cellular death, induction of cell adhesion molecules, and the alarmin cytokine IL-33 in liver. Steady-state IL-33 mRNA was upregulated by OSM at an early phase (8 h) following AdOSM infection. At the protein level, the expression of IL-33 was significantly induced in liver endothelial cells [liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells] with a peak at 8 days post-AdOSM infection in mice. In addition, we found OSM-stimulated human microvascular endothelial HMEC-1 cells and human LSEC/TRP3 cells showed a significant increase in expression of IL-33 mRNA in a dose-dependent manner in cell culture. The OSM-mediated overexpression of IL-33 was associated with the activation/enrichment of CD4(+)ST2(+) cells in liver of AdOSM-infected mice compared with adenovirus encoding green fluorescent protein-treated control mice. In summary, these data suggest that the cytokine OSM regulates the IL-33 expression in liver endothelial cells in vivo and in HMEC-1/TRP3 cells in vitro and may specifically expand the target CD4(+)ST2(+) cells in liver.