Jérémie Gautheron

A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis

Non‐alcoholic fatty liver disease (NAFLD) represents the most common liver disease in Western countries and often progresses to non‐alcoholic steatohepatitis (NASH) leading ultimately to liver fibrosis and liver cancer. The occurrence of hepatocyte cell death—so far characterized as hepatocyte apoptosis—represents a fundamental step from benign steatosis toward progressive steatohepatitis. In contrast, the function of RIP3‐dependent “necroptosis” in NASH and NASH‐induced fibrosis is currently unknown. We show that RIP3 is upregulated in human NASH and in a dietary mouse model of steatohepatitis. RIP3 mediates liver injury, inflammation, induction of hepatic progenitor cells/activated cholangiocytes, and liver fibrosis through a pathway suppressed by Caspase‐8. This function of RIP3 is mediated by a positive feedback loop involving activation of Jun‐(N)‐terminal Kinase (JNK). Furthermore, RIP3‐dependent JNK activation promotes the release of pro‐inflammatory mediators like MCP‐1, thereby attracting macrophages to the injured liver and further augmenting RIP3‐dependent signaling, cell death, and liver fibrosis. Thus, RIP3‐dependent necroptosis controls NASH‐induced liver fibrosis. This pathway might represent a novel and specific target for pharmacological strategies in patients with NASH.

RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction.

AIMS Programmed necrosis (necroptosis) represents a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Like apoptosis, necroptosis is tightly regulated by distinct signalling pathways. A key regulatory role in programmed necrosis has been attributed to interactions of the receptor-interacting protein kinases, RIP1 and RIP3. However, the specific functional role of RIP3-dependent signalling and necroptosis in the heart is unknown. The aims of this study were thus to assess the significance of necroptosis and RIP3 in the context of myocardial ischaemia. METHODS AND RESULTS Immunoblots revealed strong expression of RIP3 in murine hearts, indicating potential functional significance of this protein in the myocardium. Consistent with a role in promoting necroptosis, adenoviral overexpression of RIP3 in neonatal rat cardiomyocytes and stimulation with TNF-α induced the formation of a complex of RIP1 and RIP3. Moreover, RIP3 overexpression was sufficient to induce necroptosis of cardiomyocytes. In vivo, cardiac expression of RIP3 was up-regulated upon myocardial infarction (MI). Conversely, mice deficient for RIP3 (RIP3(-/-)) showed a significantly better ejection fraction (45 ± 3.6 vs. 32 ± 4.4%, P < 0.05) and less hypertrophy in magnetic resonance imaging studies 30 days after experimental infarction due to left anterior descending coronary artery ligation. This was accompanied by a diminished inflammatory response of infarcted hearts and decreased generation of reactive oxygen species. CONCLUSION Here, we show that RIP3-dependent necroptosis modulates post-ischaemic adverse remodelling in a mouse model of MI. This novel signalling pathway may thus be an attractive target for future therapies that aim to limit the adverse consequences of myocardial ischaemia.

U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in ‘circulating miRNA’. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

Levels of circulating miR-133a are elevated in sepsis and predict mortality in critically ill patients.

Objective:Serum levels of microRNA have been proposed as biomarkers in various inflammatory diseases. However, up to now, their clinical relevance in critical illness and sepsis is unclear. Design:Single-center clinical study. Setting:Fourteen-bed medical ICU of the University Hospital Aachen, university laboratory research unit. Subjects and Patients:Experimental sepsis model in C57Bl/6 mice; 223 critically ill patients in comparison with 76 healthy volunteers. Interventions:We used the model of cecal pole ligation and puncture for induction of polymicrobial sepsis in mice and measured alterations in serum levels of six different microRNAs with a known function in inflammatory diseases upon induction of septic disease. These results from mice were translated into a large and well-characterized cohort of critically ill patients admitted to the medical ICU. Measurements and Main Results:Serum miR-133a was then measured in 223 critically ill patients (138 with sepsis and 85 without sepsis) and 76 controls and associated with disease severity, organ failure, and prognosis. Significant alterations of miR-133a, miR-150, miR-155, and miR-193b* were found in mice after cecal pole ligation and puncture–induced sepsis. Among all regulated microRNAs, miR-133a displayed the most prominent and concordant up-regulation in sepsis, and this microRNA was therefore chosen for further investigation in the human. Here, significantly elevated miR-133a levels were found in critically ill patients at ICU admission, when compared with healthy controls, especially in patients with sepsis. Correlation analyses revealed significant correlations of miR-133a with disease severity, classical markers of inflammation and bacterial infection, and organ failure. Strikingly, high miR-133a levels were predictive for an unfavorable prognosis and represented a strong independent predictor for both ICU and long-term mortality in critically ill patients. Conclusions:miR-133a serum levels were significantly elevated in critical illness and sepsis. High miR-133a levels were associated with the severity of disease and predicted an unfavorable outcome of critically ill patients.

Elevated miR-122 serum levels are an independent marker of liver injury in inflammatory diseases.

Serum concentrations of miR‐122 were proposed as a marker for various inflammatory diseases, but the mechanisms driving alterations in miR‐122 serum levels are unknown.

miR-30c and miR-193 are a part of the TGF-β-dependent regulatory network controlling extracellular matrix genes in liver fibrosis.

MicroRNAs (miRNAs) have recently emerged as novel regulators in liver fibrosis. miR‐30c and miR‐193 are involved in fibrotic remodeling processes and cancer development, respectively. This study aimed to explore the role of miR‐30c and miR‐193 in liver fibrosis.

IκB kinaseα/β control biliary homeostasis and hepatocarcinogenesis in mice by phosphorylating the cell‐death mediator receptor‐interacting protein kinase 1

The IκB‐Kinase (IKK) complex—consisting of the catalytic subunits, IKKα and IKKβ, as well as the regulatory subunit, NEMO—mediates activation of the nuclear factor κB (NF‐κB) pathway, but previous studies suggested the existence of NF‐κB‐independent functions of IKK subunits with potential impact on liver physiology and disease. Programmed cell death is a crucial factor in the progression of liver diseases, and receptor‐interacting kinases (RIPKs) exerts strategic control over multiple pathways involved in regulating novel programmed cell‐death pathways and inflammation. We hypothesized that RIPKs might be unrecognized targets of the catalytic IKK‐complex subunits, thereby regulating hepatocarcinogenesis and cholestasis. In this present study, mice with specific genetic inhibition of catalytic IKK activity in liver parenchymal cells (LPCs; IKKα/βLPC‐KO) were intercrossed with RIPK1LPC‐KO or RIPK3−/− mice to examine whether RIPK1 or RIPK3 might be downstream targets of IKKs. Moreover, we performed in vivo phospho‐proteome analyses and in vitro kinase assays, mass spectrometry, and mutagenesis experiments. These analyses revealed that IKKα and IKKβ—in addition to their known function in NF‐κB activation—directly phosphorylate RIPK1 at distinct regions of the protein, thereby regulating cell viability. Loss of this IKKα/β‐dependent RIPK1 phosphorylation in LPCs inhibits compensatory proliferation of hepatocytes and intrahepatic biliary cells, thus impeding HCC development, but promoting biliary cell paucity and lethal cholestasis. Conclusions: IKK‐complex subunits transmit a previously unrecognized signal through RIPK1, which is fundamental for the long‐term consequences of chronic hepatic inflammation and might have potential implications for future pharmacological strategies against cholestatic liver disease and cancer. (Hepatology 2016;64:1217‐1231)

RIPK1 Suppresses a TRAF2-Dependent Pathway to Liver Cancer

Receptor-interacting protein kinase 1 (RIPK1) represents an essential signaling node in cell death and inflammation. Ablation of Ripk1 in liver parenchymal cells (LPC) did not cause a spontaneous phenotype, but led to tumor necrosis factor (TNF)-dependent hepatocyte apoptosis and liver injury without affecting inducible nuclear factor κB (NF-κB) activation. Loss of Ripk1 induced the TNF-dependent proteasomal degradation of the E3-ligase, TNF receptor-associated factor 2 (TRAF2), in a kinase-independent manner, thereby activating caspase-8. Moreover, loss of both Ripk1 and Traf2 in LPC not only resulted in caspase-8 hyperactivation but also impaired NF-κB activation, promoting the spontaneous development of hepatocellular carcinoma. In line, low RIPK1 and TRAF2 expression in human HCCs was associated with an unfavorable prognosis, suggesting that RIPK1 collaborates with TRAF2 to inhibit murine and human hepatocarcinogenesis.

Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease

Pathogen‐ and injury‐related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver‐derived plasma protein, histidine‐rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver‐derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild‐type (WT) and Hrg−/− mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti‐inflammatory M2 subtype in Hrg−/− mice. Upon chronic liver damage induced by CCl4 or methionine‐choline‐deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg−/−, compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg−/− mice. Moreover, HRG‐deficient mice showed significantly enhanced hepatic vascularization by micro‐computed tomography and histology, corroborating proangiogenic activities of M2‐polarized liver macrophages. Purified HRG protein induced, but HRG‐deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg−/− mice transplanted with Hrg+/+ bone marrow, but not Hrg−/−‐transplanted Hrg+/+ mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up‐regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. Conclusions: Liver‐derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases. (Hepatology 2016;63:1310‐1324)

IKK-related genetic diseases: probing NF-κB functions in humans and other matters

The transcription factor NF-κB plays a key role in numerous physiological processes such as inflammation, immunity, cell proliferation or control of cell death. Its activation is tightly controlled by a kinase complex, IκB kinase (IKK), composed of three core proteins: IKK1/IKKα, IKK2/IKKβ and NEMO/IKKγ. The first two are structurally related kinases whereas the third one is a regulatory subunit exhibiting affinity for upstream activators modified by polyubiquitin chains. Over the years, several inherited diseases caused by mutations of each of the three subunits of IKK have been identified in humans together with diseases caused by mutations of several of its substrates. They are associated with very specific and complex phenotypes involving a broad range of abnormalities such as impaired innate and acquired immune response, perturbed skin development and defects of the central nervous system. Here, we summarize the diverse clinical, cellular and molecular manifestations of IKK-related genetic diseases and show that studying patient-related mutations affecting the IKK subunits and some of their substrates offers the opportunity to understand the various functions of NF-κB in humans, complementing studies performed with mouse models. This analysis also provides glimpses about putative functions of IKK subunits that may be NF-κB-independent.