Keywords: Cytokine, Acute Hepatitis, RIP kinase, RIPK1, TNF-α, LPS, CpG-DNA, PAMPs, DAMPs, Liver, Inflammation, cell death Acute on chronic liver failure, AST: aspartate aminotransferase, ALT: alanine amino-transferase, Cl 2 MBP: dichloromethylene bisphosphonate, Cl-Caspase3: Cleaved-Caspase3, DAMP: Damage Associated Molecular Pattern, DC: dendritic cells, D-GalN: D-Galactosamine, DR: death receptor, ETA: Etanercept, H&E: hematoxylin and eosin coloration, IHC: Immunohistochemistry, i Intraperitoneal, KC: kupffer cells, LPS: lipopolysaccharide, LSEC: Liver sinusoïdal endothelial cells, NASH: nonalcoholic fatty liver hepatitis, ODN: oligodeoxynucleotide, PAMP: Pathogen associated molecular patterns, p.i.: Post injection, RIPK: receptor interacting protein kinase, TLR: toll like receptor, TNF: tumor necrosis factor

1, the Région Bretagne, the "Ligue contre le cancer, comités du grand Ouest". a.

ABSTRACT (227 words)

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 making use of mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1 LPC-KO ). Administration of clodronate encapusated-liposome 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 hepatocyte to apoptosis and to liver damages following single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition.

INTRODUCTION

The liver is constantly exposed to bacterial Pathogen Associated Molecular Patterns (PAMPs) such as unmethylated CpG-DNA motifs or lipopolysaccharides (LPS) originating from the microbiota of the gastrointestinal tract. In physiological condition, the intestinal barrier prevents the translocation of large amount of bacterial byproducts to the liver [1]. The low quantities of bacterial debris reaching the liver are efficiently cleared by phagocytic hepatic cells, which avoids induction of inflammation and harmful response [2]. In chronic liver diseases of steatosic or alcoholic origin, this intestine barrier function is damaged [3] [4] resulting in an abnormal elevation of PAMPs in the liver and impairing liver homeostasis [5]. PAMPs increase in the liver is one of the most common factor responsible for the outbreak of acute hepatitis on chronic liver failure background (ACLF for acute on chronic liver failure) [6].

According to the European Association for the Study of Liver Disease (EASL) and the

Asian Pacific Association for the Study of the Liver (APASL), ACLF is an acute deterioration of pre-existing chronic liver diseases, and as a consequence, can provoke high short-term mortality. Thus, ACLF is, today, one of the most challenging fields in hepatology [START_REF] Sarin | Acute-on-chronic liver failure: terminology, mechanisms and management[END_REF]. However, the molecular mechanisms responsible for the liver failure are incompletely understood.

Due to its anatomical link with the gut, the liver plays a key role in the bacterial clearance, which explains why ~80% of macrophages are in the liver [5] [START_REF] Heymann | Immunology in the liver -from homeostasis to disease[END_REF]. These liver resident macrophages, named Kupffer cells (KC) efficiently phagocytose bacteria and eliminate endotoxins in order to avoid systemic bacterial infection [5]. In addition to phagocytosis, KC are among the first cells to initiate the inflammatory response through the sensing of microbial products by the toll like receptors (TLRs) expressed at their surface or within their endosomes [START_REF] Heymann | Immunology in the liver -from homeostasis to disease[END_REF]. For example CpG-DNA and LPS respectively activate TLR9 and TLR4. Activation of TLRs contributes to ACLF by triggering an inflammatory response. Thus, LPS has been shown to contribute to cirrhosis, autoimmune hepatitis, primary biliary cirrhosis, alcoholic and fatty liver diseases both in humans and mouse models [START_REF] Jirillo | The role of the liver in the response to LPS: experimental and clinical findings[END_REF] [10] [START_REF] Inokuchi | Toll-like receptor 4 mediates alcohol-induced steatohepatitis through bone marrow-derived and endogenous liver cells in mice[END_REF] [5]. Among proinflammatory cytokines released by LPS-activated-KC, TNF-α has emerged as a key factor in the inflammatory process, as levels of soluble TNF receptors correlate with endotoxin influx and liver injury [START_REF] Trebicka | Endotoxin and tumor necrosis factor-receptor levels in portal and hepatic vein of patients with alcoholic liver cirrhosis receiving elective transjugular intrahepatic portosystemic shunt[END_REF] [START_REF] Spahr | Soluble TNF-R1, but not tumor necrosis factor alpha, predicts the 3-month mortality in patients with alcoholic hepatitis[END_REF]. In mice, LPS intraperitoneal administration induces a systemic inflammatory response that can lead to death by septic shock, without inducing liver injury [START_REF] Galanos | Galactosamine-induced sensitization to the lethal effects of endotoxin[END_REF]. However, administration of LPS in a dietary NASH model or in combination with the hepatotoxic D-Galactosamine (D-GalN) in mice induces hepatocyte apoptosis in a TNF-α -dependent manner [START_REF] Kudo | Lipopolysaccharide triggered TNFalpha-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model[END_REF] [START_REF] Tiegs | Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice[END_REF]. D-GalN is metabolized into an active metabolite exclusively in hepatocytes and inhibits transcription by hepatic uridine nucleotide depletion [START_REF] Decker | Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death[END_REF]. Thus, it has been widely described that in the liver, NF-κB or transcription inhibition sensitizes hepatocytes to TNF-α -mediated apoptosis [START_REF] Leist | Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest[END_REF] [16] [START_REF] Geisler | Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro[END_REF]. However, due to the pleiotropic role of TNF-α, anti-TNF-α therapeutic strategies in patients with alcoholic hepatitis are associated with an increased risk of infection and mortality [START_REF] Naveau | A doubleblind randomized controlled trial of infliximab associated with prednisolone in acute alcoholic hepatitis[END_REF] [START_REF] Boetticher | A randomized, doubleblinded, placebo-controlled multicenter trial of etanercept in the treatment of alcoholic hepatitis[END_REF]. The Receptor Interacting Protein Kinase 1 (RIPK1) plays key roles in the signaling of several death receptors (DR) such as TNFR1 by deciding the cell fate between survival and death [START_REF] Pasparakis | Necroptosis and its role in inflammation[END_REF]. It has been recently reported that RIPK1 is implicated in epithelial cell death in intestine in TNFα and bacterial dependent manner [START_REF] Takahashi | RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis[END_REF]. As its role in the liver is poorly understood, we address here the potential role of RIPK1 in hepatocyte death induced by PAMPs and relayed by TNF-α, taking advantage of a knockout mouse line invalidated for the RIPK1 expressed in liver parenchymal cells (Ripk1 LPC-KO ).

MATERIAL AND METHODS

Animals and treatment protocols

Ripk1 LPC-KO mouse model has been previously developed [START_REF] Filliol | RIPK1 protects from TNF-α mediated liver damage during hepatitis[END_REF]. LPS (Sigma-Aldrich, #L6761) diluted at 2 µg/mL in PBS was administrated by intraperitoneal (i.p.) route

Genotyping

Genotyping was routinely performed using DNA extracted from tails with Nucleospin Tissue kit (Macherey Nagel, #740952), and PCR with couple of primers for Alfp-Cre gene. Forward and reverse primers sequences were 5'-GCC TGC ATT ACC GGT CGA TGC AAC GA-3' and 5'-GTG GCA GAT GGC GCG GCA ACA CCA TT-3', respectively.

Preparation of liposomes-encapsulated Cl 2 MBP (Lip-Cl 2 MBP)

Liposomes-encapsulated Cl 2 MBP were prepared according to Rooijen [START_REF] Van Rooijen | Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications[END_REF]. In brief, 86 mg of phosphatidylcholine (Sigma-Aldrich) and 9 mg of cholesterol (Sigma-Aldrich) were dissolved in chloroform in a round bottom flask. After 4 h on a gentle rotation on a rotary evaporator at 37°C and under reduced pressure, the thin film formed was resuspended with 10 mL of PBS, and 1 g of Cl 2 MBP (Sigma-Aldrich).

After vigorous shaking for 1 min, liposomes in the saturated Cl 2 MBP solution were extruded through 1 µm diameter pores, followed by centrifugation at 10,000 g for 1 h. The Lip-Cl 2 MBP were washed 3 times with PBS with centrifugations at 22,000 g during 30 min, and resuspended with 4 mL of PBS. The size measurement of Lip-Cl 2 MBP was checked by Dynamic Light Scattering (DLS) technology (Zeta Sizer Nanoseries, Malvern) and as expected, 95 % of the liposome had 1 µm diameter.

The Lip-Cl 2 MBP solution was diluted by half in PBS just before injection.

Histopathological and biochemical studies

Fragments of mouse livers were fixed in 4% paraformaldehyde and embedded in paraffin for IHC and hematoxylin and eosin (H&E). For histopathology, H&E staining of liver tissues was carried out to investigate liver injury. Serum ALT and AST transaminases were measured according to the IFCC primary reference procedures using Olympus AU2700 Autoanalyser ® (Olympus Optical, Tokyo, Japan).

Immunolocalization in liver tissues

For immunolocalization of cleaved caspase-3 (Cell Signaling) or myeloperoxidase (MPO) (Dako, #A0398) in liver tissues, paraffin-embedded mouse liver sections (5 µm) were dried 1 h at 58°C, followed by antigen retrieval and incubated with primary antibody (Cell Signaling, 9661S) in a Ventana automated instrument (Ventana Medical Systems, USA). Revelation of primary antibody was carried out using horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, USA) and DAB substrate kit (Ventana, #760-124). Slides were then counterstained with hematoxylin.

TUNEL analysis was performed on paraffin-embedded mouse liver sections (5 µm), incubated after antigen retrieval with a mix, composed of terminal transferase (Roche, #3333566011) and digoxigenin-11-UTP (Roche, #1558706) followed by HRP-anti-digoxigenin (Ventana, #760-4822). Revelation was used according to the manufacturer with the Discovery Rhodamine kit (Ventana #760-233) followed by nucleus labelling with DAPI.

All paraffin-embedded mouse liver sections were scanned with a digital slide scanner (Hamamatsu, Nanozoomer 2.0-RS) and files were analysis with the NDP viewer software.

TNF-α and F4/80 immunofluorescence staining were performed on cryosections of mouse liver tissues (7 µm). After fixation with 4 % paraformaldehyde and NH 4 Cl treatment, liver slides were blocked with 4 % of BSA and then incubated with rabbitanti-murine TNF-α (Abcam, #ab6671) and rat-anti-murine-F4/80 (eBioscience, #47-4801-80) at 4°C overnight. Revelation of primary antibodies was carried out using DyLight 649-conjugated anti-Rabbit and DyLight 549 anti-rat IgG secondary antibodies (Jackson ImmunoResearch Laboratories). Nuclei were stained with Hoechst (Invitrogen, H3570). Slides were observed with a fluorescence microscope (Nikon's Eclipse Ni-E).

Isolation of liver F4/80 positive cells and RNA analysis.

Liver from PBS or LPS treated mice was collected 1.5 h post-injection and crushed on a 70 µm filter. Liver immune cells were isolated after sedimentation and cell fractionation on a 35 % Percoll layer and red blood cells were lysed with the ammonium-chloride-potassium (ACK) buffer. In order to isolate F4/80 positive cells, we used the MagniSort Mouse F4/80 positive selection kit (eBioScience, #8802-6863-74) and followed the manufacturer protocol. Quickly, liver immune cells were incubated with anti-F4/80 antibodies and biotin-magnetic beads. To eliminate F4/80 negative cells, samples were incubated 3 times in a magnet separated by washes steps. Next, F4/80 positive cells were pelleted and stored dry at -80°C before RNA extraction.

In order to measure cell transcripts, messenger RNA (mRNA) from 50 000 F4/80 positive cells were extracted with the NucleoSpin RNA XS kit (Macherey Nagel, #740902). Next, first-strand cDNA was synthesized using the SuperScript TM II Reverse Transcriptase (Applied Biosystems, #4368813) and real-time quantitative PCR was performed using the fluorescent dye SYBR Green with the double-strand specific SYBR ® Green system (Applied Biosystems, #4367659), TNF-α (Fwd: 5′-TAG CTC CCA GAA AAG CAA GC-3′ ; Rev: 5′-TTT TCT GGA GGG AGA TGT GG-3′) and 18S (Fwd: 5′-CGC CGC TAG AGG TGA AAT TC-3′ ; Rev: 5′-TTG GCA AAT GCT TTC GCT C-3′) primers and the CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad). Each measurement was performed in triplicate. The relative gene expression was normalized against the 18S gene expression. The PBS-treated mice served as reference for mRNA expression (control mRNA level was arbitrarily set at 1).

Protein extraction and western blotting

Mouse liver specimens were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4; 1 % Triton X-100; 25 mM HEPES; 150 mM NaCl; 0,2 % SDS; 5 mM MgCl 2 ), complemented with 1 mM Na 3 VO 4 ; 1 mM NaF and proteases inhibitors (Roche, #04 693 132 001), using UltraTurax. For protein extraction from primary hepatocytes, cell cultures were lysed with RIPA complemented with proteases and phosphatases inhibitors one day after seeding. After 40 min in ice, samples were centrifuged at 13,000 g. Proteins from supernatant were assayed with the Bradford method (BioRad) and then separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked with non-fat milk or BSA 3-5 % in TBS (20 mM Tris, 137 mM NaCl) during 1-2 h and incubated overnight with primary antibody anti-cleaved Caspase-3 (Cell Signaling 9661S), anti-actin (Sigma A3854), anti-TNF-α (Abcam, ab6671) or anti-RIPK1 (Cell Signaling, 3493) at 4°C, and then with secondary goat anti-rabbit immunoglobulins/HRP antibody (Dako, P0448). Protein-antibody complexes were revealed by enhanced chemiluminescence (Millipore) and ImageQuant LAS-4000 mini imager analysis (GE-Healthcare).

Serum cytokine immunoassay by flow cytometry

Murine TNF-α, IL-6 and MIP-1α/CCL3 cytokines were quantified by bead-based immunoassays according to manufacturer protocol, using a filter plate and a vacuum filtration system for washing steps (BioLegend's LEGENDPLEX, multi-analyte flow assay kit). Samples were analyzed on a LSR Fortessa cytometer (BD Biosciences).

Statistical analysis

Data were expressed as means +/-SEM for all mice treated similarly. Kruskal-Wallis one-way analysis of variance (ANOVA) was performed, and mean differences between experimental groups were assessed using the non-parametric Mann-

RESULTS

Deficiency of RIPK1 in liver parenchymal cells sensitizes to liver damage induced by PAMPs

To investigate the role of RIPK1 in the liver, we used the Ripk1 LPC-KO strain mice which presented a specific deletion of RIPK1 in liver parenchymal cells (Supplementary Figure 1). As described previously [START_REF] Filliol | RIPK1 protects from TNF-α mediated liver damage during hepatitis[END_REF], these mice, as well as their Ripk1 fl/fl littermates are viable in adulthood and displayed normal serum transaminases levels suggested that deficiency of RIPK1 in steady state does not induce hepatolysis (Fig. 1A). Next, we evaluated acute effects on the liver of CpG-DNA or LPS administration in Ripk1 fl/fl and Ripk1 LPC-KO mice by monitoring serum levels of ALT and AST, as well as liver histology by H&E and TUNEL staining. Whereas Ripk1 fl/fl mice did not exhibit increase in serum transaminase levels, sera from Ripk1 LPC-KO mice revealed significant increase of ALT and AST after administration of CpG-DNA and severe hepatolysis after low dose of LPS (0.4 µg/20 g of body weight) injection (Fig. 1A). In line with these results, liver sections of Ripk1 fl/fl showed unaffected structures, while tissue damages, revealed by HES coloration, and associated with TUNEL positive cells and cleaved caspase-3 (Clcaspase 3) staining, a marker of apoptosis induction, were observed in liver sections from Ripk1 LPC-KO mice exposed to CpG-DNA (Fig. 1B). These were even more significant in Ripk1 LPC-KO mice subjected to LPS administration (Fig 1C).

In order to evaluate whether RIPK1 deficiency had any impact on the systemic inflammatory response, we measured the levels of serum pro-inflammatory cytokines in CpG-DNA or LPS injected mice. Administration of CpG-DNA triggered a quick release (1.5 h pi) of TNF-α and IL-6. Whereas TNF-α returned at its basal level 8.5 h post-injection, serum IL-6 quantities remained high. However, no difference was observed between Ripk1 fl/fl and Ripk1 LPC-KO mice (Fig. 2A). We next evaluated the same cytokines but also the chemokines MCP1/CCL2 and MIP-1α/CCL3 in sera from LPS injected mice. Interestingly, absence of RIPK1 did not affect the early (1.5 h p.i.) release of IL-6, CCL2 and CCL3 but weakly increased serum TNF-α levels, and led to a significant sustained expression of TNF-α, CCL2 and MIP-1α/CCL3 at the time of apoptosis induction (8.5 h p.i.) (Fig. 2B). In the same manner, PAMPs injection triggered TNF-α, CCL2 and CCL3 liver transcription. However, Ripk1 LPC-KO mice more expressed these cytokines 8.5 h after PAMPs administration which correlated with liver damage (Supplementary figure 2). Moreover, H&E coloration and

Depletion of Kupffer cells reduces LPS-mediated hepatitis in Ripk1 LPC-KO mice

Kupffer cells are widely involved in LPS-mediated inflammation [START_REF] Heymann | Immunology in the liver -from homeostasis to disease[END_REF]. In order to evaluate their contribution to the severe liver damage induced by LPS in Ripk1 LPC-KO mice, we specifically depleted mice from their macrophages by treating them for two days with liposome-encapsulated Cl 2 MBP (Lip-Cl 2 MBP) [START_REF] Van Rooijen | Elimination, blocking, and activation of macrophages: three of a kind?[END_REF]. In order to confirm macrophage depletion, including in the hepatic tissue, liver sections were stained with anti-F4/80, a well-known marker of murine macrophage population [START_REF] Austyn | F4/80, a monoclonal antibody directed specifically against the mouse macrophage[END_REF]. While abundant F4/80 positive cells were detected in the liver of sham Ripk1 LPC-KO mice (LPS challenged controls pretreated with vehicle [PBS] in place of Lip-Cl 2 MBP), the labelling was dramatically reduced in the liver of LPS-challenged animals pretreated with Lip-Cl 2 MBP (Supplementary figure 3A). This proved the effectiveness of our pretreatment. Accordingly, the early LPS-mediated increased in serum levels of CCL3 and IL-6, two cytokines mainly produced by macrophages, were significantly reduced in mice pretreated with Lip-Cl 2 MBP (Supplementary figure 3B). Interestingly, macrophage depletion greatly rescued the liver damage induced by LPS in Ripk1 LPC- transaminase levels (Fig. 3A), which was associated with a reduction of necrosis areas, of TUNEL positive cells as well as of apoptotic cells (Fig. 3B and3C).

TNF-α produced by Kupffer cells mediates LPS-induced liver damage in

Ripk1 LPC-KO mice

Our results demonstrated a key role of Kupffer cells during LPS-mediated liver injury in Ripk1 LPC-KO mice. Macrophages are the main source of the TNF-α produced in the body and RIPK1 deficient cells such as embryonic fibroblasts, thymocytes or keratinocytes [START_REF] Kelliher | The death domain kinase RIP mediates the TNF-induced NF-kappaB signal[END_REF] [29], as well as intestinal organoids [START_REF] Takahashi | RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis[END_REF] have been reported to be sensitized to TNF-α -mediated apoptosis. We therefore speculated that TNF-α, known to be mainly produced by Kupffer cells in response to LPS sensing [START_REF] Bautista | Elimination of macrophages by liposomeencapsulated dichloromethylene diphosphonate suppresses the endotoxin-induced priming of Kupffer cells[END_REF] [31], was responsible for the apoptotic death of the RIPK1 deficient hepatocytes. In accordance with this idea, we found that TNF-α was quickly released in Ripk1 LPC-KO and Ripk1fl/fl littermates following LPS injection (Fig. 2B), and that depletion of macrophages reduced by more than 90% the serum concentration of TNF-α, 1.5 h after LPS administration in Ripk1 LPC-KO mice (Fig. 4A). Moreover isolation of F4/80 positive cells from Ripk1 fl/fl and Ripk1 LPC-KO mice, 1.5 h after PBS or LPS treatment showed that LPS induced an upregulation of the TNF-α mRNA in these cells, at similar levels in both mouse strains (Fig. 4B). In addition, cellular staining of TNF-α and F4/80 on liver sections of Ripk1 LPC-KO mice pre-treated or not with Lip-Cl 2 MBP demonstrated that liver macrophages are the major source of TNF-α produced in the liver after LPS challenge (Fig. 4C, orange arrows). Some single TNF positive cells with no F4/80 labelling were detected. They may correspond to F4/80 negative cells, such as sinusoidal liver cells, able to produce TNF at lesser extend (Fig. 4B, white arrows).

As we demonstrated that macrophage depletion in Ripk1 LPC-KO mice prevented from both a TNF-α burst and severe liver damages, we investigated the role of TNF-α in our model by pre-treating Ripk1 LPC-KO mice for 1 h with Etanercept, a TNF-α decoy receptor widely used clinically in humans, before LPS injection. Remarkably, we found that TNF-α blockade fully protected Ripk1 LPC-KO mice from LPS-induced liver injury, as demonstrated by the dramatic reduction of serum transaminase levels (Fig. 5A), the absence of necrotic areas, and the reduction of TUNEL and Cl-caspase 3 positive cells in the livers of Ripk1 LPC-KO mice (Fig. 5B). Western blot analysis confirmed the presence of TNF-α release and Caspase 3 activation in the livers of Ripk1 fl/fl and Ripk1 LPC-KO mice after LPS injection, which were avoided by the Etanercept pre-treatment (Fig. 5C).

DISCUSSION

Hepatocyte death is a key trigger of liver disease progression [START_REF] Luedde | Cell death and cell death responses in liver disease: mechanisms and clinical relevance[END_REF]. During chronic hepatitis, acute hepatocyte death can occur and causes ACLF, and induces quick and high mortality rate in patients [START_REF] Sarin | Acute-on-chronic liver failure: terminology, mechanisms and management[END_REF]. One of its main cause is the elevation of gutderived-microbacterial products in the liver [6]. PAMPs activate TLR receptors, expressed at the surface of innate immune cells, such as KC, inducing cytokine release which promotes liver inflammation and hepatocyte death, eventually leading to liver failure [START_REF] Heymann | Immunology in the liver -from homeostasis to disease[END_REF]. Elevation of bacterial products in the liver can be mimic by LPS or CpG-DNA injection in mice. In healthy liver, their administration induces a quick delivery of cytokines without triggering liver injury. However, during ACLF, inflammatory context of chronic hepatitis sensitizes hepatocytes, and elevation of PAMPs in the liver triggers acute hepatocyte death and liver failure [START_REF] Jirillo | The role of the liver in the response to LPS: experimental and clinical findings[END_REF] [5] [10] [START_REF] Inokuchi | Toll-like receptor 4 mediates alcohol-induced steatohepatitis through bone marrow-derived and endogenous liver cells in mice[END_REF]. In many studies, treatment with D-GalN was used to investigate endotoxin mediated liver injury. D-GalN leads to uridine depletion that sensitizes hepatocyte to LPSinduced apoptosis by inhibiting survival proteins transcription [START_REF] Decker | Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death[END_REF]. Here, we showed that in Ripk1 LPC-KO mice, administration of LPS or CpG-DNA, without adding D-GalN, was sufficient to induce strong acute liver injury associated with hepatocyte apoptosis. Previously, an identical phenotype was observed in conditional or inducible LPC-KO mice for NEMO, TAK1 or RelA [START_REF] Kondylis | NEMO Prevents Steatohepatitis and Hepatocellular Carcinoma by Inhibiting RIPK1 Kinase Activity-Mediated Hepatocyte Apoptosis[END_REF] [34] [START_REF] Geisler | Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro[END_REF]. However, whereas these mice presented hepatolysis at steady state, RIPK1 seems dispensable for liver homeostasis as we and others previously showed that Ripk1 LPC-KO adult mice are viable and do not present abnormal hepatocyte death or proliferation [START_REF] Kondylis | NEMO Prevents Steatohepatitis and Hepatocellular Carcinoma by Inhibiting RIPK1 Kinase Activity-Mediated Hepatocyte Apoptosis[END_REF] [START_REF] Filliol | RIPK1 protects from TNF-α mediated liver damage during hepatitis[END_REF]. LPS injection induced a quick release of cytokines in bloodstream that was poorly or not affected by RIPK1 deficiency in LPC. However, whereas at latter time, serum cytokine levels were almost back to their basal levels in Ripk1 fl/fl mice, their Ripk1 LPC- KO littermates presented a sustained inflammation associated with inflammatory cells infiltration. This possibly resulted from the release of damage associated molecular pattern (DAMPs) issued from dead hepatocytes. As KC are important source of cytokine delivery after LPS administration, and as it has been observed that PAMP recognition by KC is a key step in the induction of liver injury during chronic alcoholic, NASH or acute hepatitis induced by D-GalN in mice [START_REF] Adachi | Risk factors for intrahepatic recurrence in human small hepatocellular carcinoma[END_REF] [36] [START_REF] Spruss | Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice[END_REF] [10] [START_REF] Uesugi | Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice[END_REF], we next investigated the role of KC in LPS-mediated liver damage in Ripk1 LPC-KO mice.

Liposome-encapsulated Cl 2 MBP is an efficient method to deplete macrophages and to study the role of cytokines induced by LPS stimulation [START_REF] Van Rooijen | Elimination, blocking, and activation of macrophages: three of a kind?[END_REF]. Indeed, in contrast to other methods (such as gadolinium chloride, silica or dextran sulfate), liposomes do not stimulate production of cytokines by macrophages [START_REF] Van Rooijen | Elimination, blocking, and activation of macrophages: three of a kind?[END_REF]. Depletion of KC in the liver was associated with a strong reduction of hepatolysis in Ripk1 LPC-KO mice challenged with LPS. Whereas RIPK1 has been shown to regulate TLR signaling [START_REF] Meylan | RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation[END_REF], our data revealed an indirect role of LPS to induce hepatocyte death in Ripk1 LPC-KO mice. Thus, we suggest that, in Ripk1 LPC-KO mice, LPS activated TLR4, mainly present on KC, which induced the release of cytokines deleterious for hepatocytes. In accordance with others, we found an important reduction of serum TNF-α level after LPS challenge in mice previously depleted from their macrophages [30] [31], suggesting a deleterious role of TNF-α in Ripk1 LPC-KO mice. Moreover TNF-α release by LPS-activated KC has been shown to play a key role during acute and chronic liver diseases in different mice models, where it was shown to promote fibrosis and hepatolysis [START_REF] Kudo | Lipopolysaccharide triggered TNFalpha-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model[END_REF] [16] [START_REF] Miura | Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2[END_REF]. In the same way, we showed that TNF-α inhibition with Etanercept fully protected hepatocytes from the apoptosis induced by LPS in Ripk1 LPC-KO . The low hepatolysis observed after LPS treatment in Ripk1 LPC-KO mice previously depleted from their macrophages can be probably attributed to the small detected quantities of TNF-α, which may originate from the few surviving macrophages which escaped from Lip-Cl 2 MBP pretreatment, and/or from other liver cells.

Previously, we showed that RIPK1 plays a key protective role in murine hepatocytes when TNF signaling is triggered by concanavalin A, a model of T-cells induced hepatitis [START_REF] Filliol | RIPK1 protects from TNF-α mediated liver damage during hepatitis[END_REF] (see extended discussion). These supported our herein data showing that physiological production of TNF-α by LPS-activated KC led to hepatocyte apoptosis in Ripk1 LPC-KO mice. The role of soluble and transmembrane TNF-α in LPSmediated hepatitis is currently discussed [START_REF] Olleros | Soluble TNF, but not membrane TNF, is critical in LPS-induced hepatitis[END_REF][START_REF] Yang | Kupffer-cell-expressed transmembrane TNF-alpha is a major contributor to lipopolysaccharide and D-galactosamine-induced liver injury[END_REF]. As Etanercept blocks both forms [START_REF] Tracey | Tumor necrosis factor antagonist mechanisms of action: a comprehensive review[END_REF], we cannot distinguish the more active one in our experiments. However, although no conclusions can be drawn from our experimental model on tm-TNF involvement, we can conclude that its soluble form was sufficient to promote liver injury. Indeed, LPS triggered high release of soluble TNF-α in the bloodstream and single soluble recombinant TNF-α injection is able alone to trigger acute hepatocyte apoptosis in RipK1 LPC-KO mice [START_REF] Filliol | RIPK1 protects from TNF-α mediated liver damage during hepatitis[END_REF]. TNF-α can generate intracellular signaling through interaction either with TNFR1 or with TNFR2. However, TNFR1 seems to play a predominant role in the liver [START_REF] Liedtke | The role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer[END_REF]. Thus, in the TNF-α/D-GalN hepatitis model, the deficiency of TNFR1 in liver parenchymal cells is sufficient to fully protect animals against liver damages [START_REF] Wroblewski | Opposing role of tumor necrosis factor receptor 1 signaling in T cell-mediated hepatitis and bacterial infection in mice[END_REF]. Taking into account the different aspects discussed above, we proposed a schematic model on the vital role played by RIPK1 to maintain liver homeostasis under elevated PAMPs conditions (Fig. 6).

In conclusion, the sensitivity of Ripk1 LPC-KO mice to PAMPs such as LPS and CpG-DNA, could be used as a model to test some TLR inhibitors or probiotics and antibiotics which are currently investigated to reduce inflammatory response induced by high bacterial translocation in the liver [5]. Moreover, this sensitivity of RIPK1 LPC-KO mice to PAMPs highlights the importance of the preservation of the RIPK1 function to maintain the liver homeostasis in pathological condition. Thus, analysis of Ripk1 gene polymorphism or RIPK1 expression could be helpful in the management of patients with chronic hepatitis and in the prediction of their risk to develop ACLF. Western-blot analysis of RIPK1 and β-actin, in protein extracts issued (A) from the liver, (B) or from primary hepatocyte cultures (PH) of WT or Ripk1 LPC-KO mice.

Extended discussion

The concanavalin A (ConA) model mimics some features of auto-immune or viral hepatitis in mice. Indeed, this lectin activates T-lymphocytes and more specifically NKT subset in an antigen-independent manner [2,3]. Thus, hepatocyte death is induced in a DR-dependent manner, also requiring the production of TNF-α, IFN-γ and IL-4. Closer to physiopathologic conditions, PAMPs administration allows to study the role of innate immune cells activation, such as Kupffer cells, in the development of hepatitis. Inflammation induced in this condition is important to control bacterial burden but in return it can provoke collateral tissue damage which is a cause of ACLF [4]. In WT animals, ConA induced a complex inflammatory environment which leads to hepatocyte lysis whereas single administration of PAMPs alone is ineffective. Altogether, these characteristics underline the complementarity of both models. The exacerbated liver injury associated with a sustained inflammation (certainly promotes by the release of DAMPs issued from dead hepatocytes) observed in Ripk1 LPC-KO mice during ConA [5] or PAMPs challenging highlights the importance of RIPK1 as a protector of hepatocytes during immune-cells stimulated conditions and TNF-signaling. These new data enlarge our knowledges on the RIPK1 platform functions in hepatocytes which were until now questioned as recently underlined by Dara and colleagues [6].

(

  20 µg/kg body weight and 200 µL/20 g). Unmethylated CpG oligodeoxynucleotide (ODN) (Invivogen, ODN 2395) diluted at 3.5 mg/mL in PBS was administrated by i.p. route (2 mg/kg body weight and 200 µL/20 g). Etanercept (Pfizer) was administrated by i.p. route 1 h before LPS injection (10 mg/kg body weight and 200 µL/20g). Two injections of liposomes-encapsulated dichloromethylene bisphosphonate (Cl 2 MBP) (see below for the preparation) were administrated by i.p. route in mice. The first injection was given with 200 µL/20 g body weight, and the second, 24 h after the first, with 100 µL/20 g body weight. Mice were administrated with LPS or control (PBS) 48 h after first injection of liposomes encapsulated Cl 2 MBP and sacrificed 8.5 h after LPS injection. In all experiments, genetically modified mice were systematically compared to their littermates. Mature mice at 10-12 weeks of age were used for each experiments. Animals were housed in individually ventilated cages at the VIB Inflammation Research Center (Ghent, Belgium) in conventional animal facilities. All experiments on mice were conducted according to institutional, national and European animal regulations. In vivo protocols were approved by the ethics committee of Ghent University.

  Whitney U-test with the GraphPad Prism5 software. The significance is shown as follows: * p<0.05, ** p<0.01, *** p<0.001, Ripk1fl/fl mice versus Ripk1 LPC-KO mice. # p<0.05, ## p<0.01, ### p<0.001, mice treated with LPS or CpG-DNA versus mice treated with PBS.

  myeloperoxidase (MPO) labelling of liver sections showed higher infiltrations of inflammatory cells in Ripk1 LPC-KO mice after LPS administration (Fig 1B-1C and Fig 2C). Thus, whereas CpG-DNA triggered a moderate hepatolysis associated with low inflammation, LPS administration induced severe acute hepatocyte death which were associated with more extended pro-inflammatory cytokine expression and more important recruitment of inflammatory cells when RIPK1 were absent in liver parenchymal cells. Thus, to better understand the protective role of RIPK1 during hepatitis induced by PAMPs administration, we continued experiments with the LPS model.

Figure 1 .

 1 Figure 1. RIPK1 deficiency sensitized mice to unmethylated CpG-DNA and LPS

Figure 2 .

 2 Figure 2. RIPK1 deficiency promoted inflammation during PAMPs-induced

Figure 3 .

 3 Figure 3. Depletion of Kupffer cells reduced LPS mediated hepatitis in Ripk1 LPC- KO mice.

Figure 4 .

 4 Figure 4. Kupffer cells induced LPS mediated liver damage in RIPK1 mice in a

Figure 5 .

 5 Figure 5. LPS induced TNF-mediated liver damages in Ripk1 LPC-KO mice.

Figure 6 .

 6 Figure 6. Role of RIPK1 in PAMPs mediated hepatitis.

Acknowledgements

This work was supported by INSERM, The "Ministère de l'Education Nationale de la Recherche et de la Technologie", the University of Rennes 1, the "Région Bretagne" and the "Ligue contre le cancer", "comités du grand Ouest", the Institut National du

Supplementary figure 2.

Levels of hepatic TNF-α, MCP1/CCL2 and MIP-1α/CCL3 in Ripk1 fl/fl or Ripk1 LPC

Supplementary Methods

Primary hepatocytes Isolation and culture

Hepatocytes were isolated from adult C57BL/6 mice by the retrograde perfusion approach [1]. Mice were previously anesthetized by i.p injection of a ketamine (Imalgene TM , supplier) and xylazine (Rompun TM , supplier) cocktail (60 and 10 mg/kg of live body weight, respectively) diluted in PBS. After laparotomy, the liver, perfused through inferior vena cava, was first washed with solution I (8 g/L NaCl, 0.2 g/L KCl, 0.1 g/L Na 2 HPO 4 . 12H 2 O and 2.38 g/L Hepes, pH 7.6) at a 5.5 ml/min flow rate for 8-10 min. Then, the perfusion solution I was supplemented with 5 mM CaCl 2 .2H 2 O and 0.01% collagenases (Liberase TM TM Research Grade, Roche) for 5-7 minutes.

Hepatocytes were harvested after 3 centrifugations at 90 g for 1 min and an additional purification of viable cells on a percoll cushion. Cell suspensions, with over 90% viability, were seeded at a density of 0.1 x 10 6 cells/cm 2 in 24-well plates, previously coated with collagen type I (BD Biosciences, Le Pont de Claix, France), in Williams' E medium supplemented with 10% (vol/vol) fetal calf serum, 2 mM glutamine, 10 IU/mL penicillin, 10 µg/mL streptomycin and 5 µg/mL insulin. Seeding medium was removed after a 4 h-period and replaced by a similar supplemented Williams' E medium, except that fetal calf serum was replaced by 1 mg/mL bovine serum albumin, but with 1 µg/mL of Etanercept. Experimental protocol was conducted in compliance with French laws and the institution's guidelines for animal welfare (agreement of M. Samson # A3523840).

RNA analysis

Total RNA was extracted, from mouse liver tissues by using the NucleoSpin RNA II kit (Macherey Nagel #740955). First-strand cDNA was synthesized using the SuperScriptTM II Reverse Transcriptase (Applied Biosystems). Real-time quantitative PCR was performed using the fluorescent dye SYBR Green with the double-strand specific SYBR Green system (Applied Biosystems) and the CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad). cDNA was used as template for amplification with specific primer pairs : TNF-α (Fwd: 5′-TAGCTCCCAGAAAAGCAAGC-3′ ; Rev: 5′-TTTTCTGGAGGGAGATGTGG-3'), CCL2 (Fwd: 5'-GTCCCTGTCATGCTTCTGG-3' ; Rev: 5'-TGCTGGTGATCCTCTTGTAGC-3') and CCL3 (Fwd: 5'-CCTTGCTGTTCTTCTCTGTACC-3' ; Rev: 5'AAATGACACCTGGCTGGG-3"). Each measurement was performed in triplicate.

The relative gene expression was normalized against the 18S gene expression (Fwd:

5′-CGCCGCTAGAGGTGAAATTC-3′; Rev: 5′-TTGGCAAATGCTTTCGCTC-3′). The control mice in each treatment group served as a reference for messenger RNA (mRNA) expression (control mRNA level was arbitrarily set at 1).