Claire Piquet-Pellorce

TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation.

Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.

Interleukin-33 overexpression is associated with liver fibrosis in mice and humans

Interleukin‐33 (IL‐33), the most recently identified member of the IL‐1 family, induces synthesis of T Helper 2 (Th2)‐type cytokines via its heterodimeric ST2/IL‐1RAcP receptor. Th2‐type cytokines play an important role in fibrosis; thus, we investigated the role of IL‐33 in liver fibrosis. IL‐33, ST2 and IL‐1RAcP gene expression was analysed in mouse and human normal (n= 6) and fibrotic livers (n= 28), and in human hepatocellular carcinoma (HCC; n= 22), using real‐time PCR. IL‐33 protein was detected in normal and fibrotic liver sections and in isolated liver cells using Western blotting and immunolocalization approaches. Our results showed that IL‐33 and ST2 mRNA was overproduced in mouse and human fibrotic livers, but not in human HCC. IL‐33 expression correlated with ST2 expression and also with collagen expression in fibrotic livers. The major sources of IL‐33 in normal liver from both mice and human beings are the liver sinusoidal endothelial cells and, in fibrotic liver, the activated hepatic stellate cells (HSC). Moreover, IL‐33 expression was increased in cultured HSC when stimulated by pro‐inflammatory cytokines. In conclusion, IL‐33 is strongly associated with fibrosis in chronic liver injury and activated HSC are a source of IL‐33.

Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening

The entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh‐7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down‐regulation of different factors implied in clathrin‐mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4‐kinase type III‐α (PI4KIIIα) prevented infection by HCVpp or by cell‐culture grown JFH‐1‐based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIIIα knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIIIβ in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME‐dependant entry route of HCV and identified 2 kinases, PI4KIIIα and β,as relevant potential therapeutic targets.—Trotard, M., Lepère‐Douard, C., Régeard, M., Piquet‐Pellorce, C., Lavillette, D., Cosset, F.‐L., Gripon, P., Le Seyec, J. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. FASEBJ. 23, 3780–3789 (2009). www.fasebj.org

Infection with Influenza Virus Induces IL-33 in Murine Lungs

IL-33, a novel IL-1 family member, is crucially expressed and involved in pulmonary diseases, but its regulation in viral diseases such as influenza A virus (IAV) remains unclear. This study aimed to characterize the expression and release of IL-33 in lungs of IAV-infected mice in vivo and in murine respiratory epithelial cells (MLE-15) in vitro. Our results provide evidence of up-regulation of IL-33 mRNA in IAV-infected murine lungs, compared with noninfected control mice. The overexpression of IL-33 was positively correlated with a significant increase in mRNA encoding the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6, and was also associated with an increase in IFN-β mRNA. A profound overexpression of IL-33 protein was evident in IAV-infected murine lungs and bronchoalveolar lavages of influenza-infected mice, compared with low concentrations in naive lungs in vivo. Immunolocalization highlighted the cellular expression of IL-33 in alveolar epithelial and endothelial cells, along with increased infiltrate cells in virus-infected lungs. Further in vitro experiments showed an induction of IL-33 transcript-in MLE-15 cells and human epithelial cells (A549) infected with different strains of IAV in comparison with noninfected cells. In conclusion, our findings evidenced a profound expression of IL-33 in lungs during both in vivo and in vitro IAV infections, suggesting a role for IL-33 in virus-induced lung infections.

CXCR7 is up-regulated in human and murine hepatocellular carcinoma and is specifically expressed by endothelial cells

Development of hepatocellular carcinoma (HCC) is a complex and progressive disease that involves cycles of liver cell death, inflammation, and tissue regeneration/remodelling. Chemokines and chemokine receptors play numerous and integral roles in the disease progression of HCC. Here we investigated the novel chemokine receptor CXCR7/RDC1 in HCC progression, its two known ligands CXCL12 and CXCL11, as well as the other CXCL12 receptor, CXCR4. Our results show that in a cohort of 408 human HCCs, CXCR7 and CXCL11 were significantly higher in tumours compared to normal liver controls (5- and 10-fold, respectively). Immunohistochemical (IHC) staining on human HCC sections confirmed that both CXCL11 and CXCR7 were much higher in cancer tissues. Furthermore, IHC staining revealed that CXCR7 protein was only expressed in endothelial cells whereas CXCL11 exhibited a much broader tissue expression. At the cellular level we observed that in vitro, human microvascular endothelial cells (HMEC-1) up-regulated CXCR7 under hypoxic and acidic pH conditions, which are well known characteristics of the HCC tumour micro-environment. As for its ligand, we observed that IFNγ robustly induced CXCL11 in hepatic stellate cells, hepatocytes, and HMEC-1s. In addition, in the mouse Diethylnitrosamine model of hepatocarcinogenesis we observed a very strong induction of CXCR7 and CXCL11 transcripts, confirming that CXCR7/CXCL11 up-regulation is conserved between human and mice liver cancer. Altogether, our results strongly support the hypothesis that the CXCL11/CXCR7 pathway is involved HCC progression.

Leukemia Inhibitory Factor Expression and Regulation within the Testis

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine known to control the proliferation and survival of stem cells including primordial germ cells and gonocytes. This led us to study the origin and regulation of testicular LIF. The LIF transcript was detected in the rat testis by RT-PCR from 13.5 days postcoitum until adulthood. LIF expression was investigated further in vitro in seven different highly purified testicular cell populations using RT-PCR and bioassays combined with neutralization experiments. LIF was found to be produced by peritubular cells and, to a much lesser extent, by the other testicular somatic cell types. No LIF was detected in meiotic and postmeiotic germ cell-conditioned medium, and only low levels of LIF were detected in spermatogonia-conditioned medium. Large amounts of bioactive LIF were measured in testicular lymph. While LIF production was greatly enhanced in presence of serum, lipopolysaccharide, and TNFalpha further increased this production in peritubular and Sertoli cells, and human CG enhanced Leydig cell LIF production. In conclusion, peritubular cells are the principal source of testicular LIF, probably accounting for its high concentration in the lymph. Given the proliferative effect of LIF on immature germ cells, we suggest that peritubular LIF plays an important role in the regulation of testicular function.

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)

IL‐33 and HMGB1 alarmins: sensors of cellular death and their involvement in liver pathology

‘Alarmins’ are a group of proteins or molecules that are released from cells during cellular demise to alert the host immune system. Two of them, Interleukin‐33 (IL‐33) and high‐mobility group box‐1 (HMGB1), share many similarities of cellular localization, functions and involvement in various inflammatory pathologies including hepatitis. The expressions of IL‐33 and HMGB1, and their receptors ST2 and receptor for advanced glycation end products (RAGE), are substantially up‐regulated during acute and chronic hepatitis. Recent data evidence a possible protective role of IL‐33/ST2 axis during liver injury. A contrast in expression of IL‐33 and HMGB1 alarmins were associated with type of hepatocellular death mediated by immune cells or hepato‐toxic agents. The massive release of active form of IL‐33 from hepatocytes may affect the recruitment and activation of its ST2‐positive target immune cells in the liver to confer its alarmin functions. This review highlights the emerging roles of alarmin proteins in various liver pathologies, by focusing on classical HMGB1 and a newly discovered alarmin, the IL‐33.

Crucial and Diverse Role of the Interleukin-33/ST2 Axis in Infectious Diseases

ABSTRACT Interleukin-33 (IL-33) has now emerged as a cytokine with diverse and pleiotropic functions in various infectious and inflammatory diseases. IL-33 is expressed by epithelial cells, endothelial cells, fibroblasts, and hepatocytes. The target cells of IL-33 are Th2 cells, basophils, dendritic cells, mast cells, macrophages, NKT cells, and nuocytes, newly discovered natural helper cells/innate lymphoid cells bearing the ST2 receptor. IL-33 has dual functions, both as a traditional cytokine and as a nuclear factor that regulates gene transcription. IL-33 functions as an “alarmin” released following cell death, as a biomarker, and as a vaccine adjuvant, with proinflammatory and protective effects during various infections. The exacerbated or protective role of the IL-33/ST2 axis during different infections is dependent upon the organ involved, type of infectious agent, whether the infection is acute or chronic, the invasiveness of the infectious agent, the host immune compartment, and cellular and cytokine microenvironments. In this review, we focus on recent advances in the understanding of the role of the IL-33/ST2 axis in various viral, bacterial, fungal, helminth, and protozoal infectious diseases gained from animal models and studies in human patients. The functional role of IL-33 and ST2 during experimentally induced infections has been summarized by accumulating the data for IL-33- and ST2-deficient mice or for mice exogenously administered IL-33. In summary, exploring the crucial and diverse roles of the IL-33/ST2 axis during infections may help in the development of therapeutic interventions for a wide range of infectious diseases.