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Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation
Accepted Manuscript Research Article Hepatocyte-specific deletion of IL1-RI attenuatesliver injury by blocking IL-1 driven autoinflammation Nadine Gehrke, Nadine Hövelmeyer, Ari Waisman, Beate K. Straub, Julia Weinmann-Menke, Marcus A. Wörns, Peter R. Galle, Jörn M. Schattenberg PII: DOI: Reference:
S0168-8278(18)30020-5 https://doi.org/10.1016/j.jhep.2018.01.008 JHEPAT 6827
To appear in:
Journal of Hepatology
Received Date: Revised Date: Accepted Date:
23 January 2017 5 December 2017 10 January 2018
Please cite this article as: Gehrke, N., Hövelmeyer, N., Waisman, A., Straub, B.K., Weinmann-Menke, J., Wörns, M.A., Galle, P.R., Schattenberg, J.M., Hepatocyte-specific deletion of IL1-RI attenuatesliver injury by blocking IL-1 driven autoinflammation, Journal of Hepatology (2018), doi: https://doi.org/10.1016/j.jhep.2018.01.008
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Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation
Nadine Gehrke1, Nadine Hövelmeyer2, Ari Waisman2, Beate K. Straub3, Julia Weinmann-Menke1, Marcus A. Wörns1, Peter R. Galle1, Jörn M. Schattenberg1* 1
I. Department of Medicine,
2
Institute for Molecular Medicine and
3
Institute of
Pathology, University Medical Center Mainz, Germany.
Corresponding author: Jörn M. Schattenberg University Medical Center of the Johannes Gutenberg University I. Department of Medicine Langenbeckstraße 1 55131 Mainz, Germany Telephone: +49 6131 176074 Telefax: +49 6131 17477282 E-Mail: [email protected]
Running title: IL-1RI in hepatocytes amplifies liver injury. Keywords: interleukin-1; interleukin-1 receptor type 1; acute liver failure; acute on chronic liver failure; therapy; liver disease; inflammation; neutrophils; NALP3 inflammasome; caspase 1; pyroptosis; NF-kappaB; JNK; TLR4; TNF-alpha Electronic word count: 5896 Number of figures and tables: 8 figures Conflict of interest statement: The authors declare no conflict of interest. Financial support statement: JMS received funding from the Deutsche Krebshilfe (grant number 93199) and support of intramural funds of the Johannes Gutenberg University Mainz. NG received funding from the Mainzer Wissenschaftsstiftung (MWS). 1
Authors contributions: NG
acquisition, analysis and interpretation of data, statistical analysis, preparation of figures and tables, drafting of the manuscript
NH
technical and material support, preparation of figures, critical revision of the manuscript for important intellectual content
AW
providing of the IL-1R1flox/flox mice, technical and material support, preparation of figures, critical revision of the manuscript for important intellectual content
BKS histological analyses and interpretation, preparation of figures, critical revision of the manuscript for important intellectual content JW
material support, critical revision of the manuscript for important intellectual content
MAW critical revision of the manuscript for important intellectual content PRG material support, critical revision of the manuscript for important intellectual content JMS study concept, design and supervision, analysis and interpretation of data, preparation of figures and tables, drafting of the manuscript, obtained funding
Figure 1A was prepared by NH, AW and NG. Figure 2C was prepared by BKS and NG. The data in all other figures and tables presented were generated by NG with the help of technical assistance and were assembled by NG and JMS.
2
Abstract:
Background & Aims: Interleukin (IL)-1-type cytokines including IL-1alpha, IL-1beta and interleukin-1 receptor antagonist (IL-1Ra) are among the most potent molecules of the innate immune system and exert biological activities through the ubiquitously expressed interleukin-1 receptor type 1 (IL-1RI). The role of IL-1RI in hepatocytes during acute liver failure (ALF) remains undetermined. Methods: IL-1RI during ALF was investigated using a novel transgenic mouse model exhibiting deletion of all signaling-capable IL-1R isoforms in hepatocytes (IL-1RIHep-/-). Results: ALF induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was significantly attenuated in IL-1RIHep-/- mice leading to a reduced mortality. Conditional deletion of IL-1RI decreased activation of injurious c-Jun N-terminal kinases (JNK)/c-Jun signaling, activated nuclear factor-kappaB (NF-kappaB) p65, inhibited extracellularsignal regulated kinase (ERK) and prevented caspase 3-mediated apoptosis. Moreover, IL-1RIHep-/- mice exhibited reduced local and systemic inflammatory cytokine and chemokine levels, especially TNF-alpha, IL-1alpha/beta, IL-6, CCchemokine ligand 2 (CCL2), C-X-C motif ligand 1 (CXCL-1) and CXCL-2, and a reduced neutrophil recruitment into the hepatic tissue in response to injury. NALP3 inflammasome expression and caspase 1 activation were suppressed in the absence of the hepatocellular IL-1RI. Inhibition of IL-1RI using IL-1ra (anakinra) attenuated the severity of liver injury, while IL-1alpha administration exaggerated it. These effects were lost ex vivo and at later time points, supporting a role of IL-1RI in inflammatory signal amplification during acute liver injury. Conclusion: IL-1RI in hepatocytes plays a pivotal role in an IL-1-driven auto-amplification of cell death and inflammation in the onset of ALF.
3
Lay Summary: Acute liver injury which can cause lethal liver failure is medicated by a class of proteins called cytokines. Among these, interleukin-1 (IL-1) and the corresponding receptor IL-1RI play a prominent role in the immune system, but their role in the liver is undetermined. In the current study, a novel mouse model with defective IL-1RI in liver cells was studied. Mice lacking this receptor in liver cells were protected from cell death to a certain extent. This protection occurred only in the presence of other, neighboring cells arguing for the involvement of proteins derived from these cells. This effect is called paracrine signaling and the current study has for the first time shown that the IL-1RI receptor on hepatocytes is involved in acute liver failure in this context. The approved drug anakinra – which blocks IL-1RI – had the same effect supporting the proposed mechanism of action. The findings of this study suggest new treatment options for patients with acute liver failure by blocking defined signals of the immune system.
4
Introduction: Acute liver failure (ALF) and acute-on-chronic liver failure (ACLF) both possess a high mortality with limited treatment options and an altered inflammatory response has been implicated in their pathophysiology.1,
2
Despite obvious
differences in the etiology, both conditions exhibit activation of immune mechanisms that augment inflammation in the sequel of the initial insult and drive a lethal loss of hepatic function from increased cell death. In order to improve the poor prognosis of patients with ALF and ACLF mechanistic studies that address the underlying pathomechanisms are urgently required with the aim to identify potential, selective immune-modulatory therapies. The interleukin (IL)-1 family members IL-1alpha and IL-1beta are key proinflammatory mediators and signal through the cell surface interleukin-1 receptor type 1 (IL-1RI).3 Upon binding of IL-1alpha/beta IL-1RI recruits - in complex with its receptor accessory protein (IL-1RacP) - the cytosolic adapter molecule myeloid differentiation primary response gene 88 (MyD88) and through an intracellular signaling complex IL-1R-associated kinases (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6).4 These in turn activate transforming growth factor beta-activated kinase 1 (TAK1) and mitogen-activated protein kinases (MAPK) that are involved in cellular survival, e.g. c-Jun terminal kinases (JNK) and extracellular
signal-regulated
kinase
1/2
(ERK),
as
well
as
down-stream
transcriptions factors that contribute to hepatic inflammation.5 The role of IL-1 signaling in ALF is controversial and differs between studied models. Aggravation of ALF from acetaminophen (APAP) related to IL-1 signaling is supported by observations in transgenic mice, studies using recombinant human IL1ra anakinra to block the effects of IL-1alpha/beta6, 7 and studies exploring the role of Toll-like receptors (TLRs).8 In particular, IL-1alpha, but not IL-1beta, released from 5
Kupffer cells following hepatocellular injury seems to amplify hepatic inflammation.6 IL-1beta has received special attention, as NACHT, LRR, and PYD domainscontaining protein 3 (NALP3/NLRP3) inflammasome-mediated maturation of IL-1beta through caspase 1 has been implied in different liver disease models.9-11 On the other hand, genetic disruption of IL-1R targeting Il1r112 failed to protect from a lethal dose of D-galactosamine (D-GalN) and lipopolysaccharide (LPS)13, while it protected from acetaminophen (APAP)-induced liver injury and inflammation. Others have not observed an alteration of injury when IL-1beta was given to C57Bl/6 mice in addition to APAP.14 Moreover, major controversies regarding cell-type specific functions of IL1 signaling and especially the functional role of IL-1RI in hepatocytes remain unanswered.8 Recently a single-nucleotide polymorphisms (SNPs) in the Il1 gene cluster was linked to the severity of ACLF in clinical cohorts.15 Based on the central involvement of IL-1 in the regulation of inflammation, we tested the hypothesis, that the hepatic IL-1RI is centrally involved in the pathophysiology of inflammatory liver failure in a novel transgenic mouse model with hepatocyte-restricted deletion of all IL1R isoforms.
6
Materials and Methods: Animal model and primary hepatocytes The targeting strategy for Il1r1 gene disruption has been recently described.16 In conditional IL-1R1 mouse mutant (IL-1R1flox/flox) exon 5 of the Il1r1 gene, encoding amino-acids 166-222 and corresponding to half of the Ig-like C2 type 2 region that encodes parts of the extracellular binding region of full length IL-1R1 and truncated IL-1R3, was flanked by loxP sites leading to a transcriptional frame shift and inactivation of the two functional Il1r1 gene transcripts after Cre-mediated recombination. Hepatocyte-specific deletion of all signaling IL-1R isoforms was achieved by crossing IL-1R1flox/flox mice C57Bl6 with mice expressing Crerecombinase under an albumin promoter resulting in albumin-cre:IL-1R1flox/flox mice. Mice carrying homozygous floxed IL-1R1 and albumin-cre (IL-1RIHep-/-) were compared to their cre-negative control littermates, which are referred to as wild type (wt) mice. Genotypic identification was carried out by PCR as previously described for IL-1R1-/- mice and Cre genotyping.16 All animals were held and bred with free access to food and water at 12h light/dark cycles at the animal facility of the University Medical Center Mainz, according to the criteria outlined by the “Guide for the Care and Use of Laboratory Animals”. Studies were approved by the committee for experimental animal research (Landesuntersuchungsamt Rheinland-Pfalz) and were designed according to the ARRIVE guidelines.17 Isolation of primary hepatocytes from males and females by collagen perfusion and in vitro experiments are detailed in the supplementary materials.
Model of acute liver injury Acute liver injury was induced in mice male aged 10-12 weeks by i.p. injection of D-GalN (0.75mg/g, from D-(+)-galactosamine hydrochloride G1639, Carl Roth, 7
Karlsruhe, Germany) and LPS (2.5µg/g or 0.01µg/g from Escherichia coli Serotype 026:B6, L-8274, Sigma-Aldrich, Hamburg, Germany) according to published protocols.18, 19 Age-matched controls received saline injections. When indicated, mice received an i.p. injection of recombinant mouse (rm) IL-1alpha CF protein (1µg/mouse), rmIL-1beta CF protein (1µg/mouse, both R&D Systems, Minneapolis, MN, USA), or Kineret (anakinra, IL-1ra, 50µg/g bodyweight, Swedish Orphan Biovitrum AB, Stockholm, Sweden). Blood and liver tissue were harvested at 4h or 6h after D-GalN/LPS administration for evaluation of liver injury.
Serological analysis Serum was obtained by cardiac puncture and alanine amino transferase (ALT), aspartate amino transferase (AST) and lactate dehydrogenase (LDH) were measured using standard analyzer (Hitachi 917, Roche, Mannheim, Germany).
Quantitative real-time PCR Isolation of total RNA from snap frozen liver tissue, cDNA synthesis and qRTPCR were performed as previously described.20 All samples were performed in duplicates. Roche LightCycler software (LightCycler 480 Software Release 1.5.0) was used to perform advanced analysis relative quantification using the 2(-∆∆C(T)) method. Expression data were normalized to the housekeeping gene Gapdh (Qiagen, Hilden, Germany) and the mean of untreated wild type cells/saline-treated wild type mice was considered 1. Primer sequences (all Eurofins Genomics, Ebersberg, Germany) are detailed in the supplementary materials.
Protein isolation, immunoblotting and NF-kappaB p65 activity
8
Proteins were isolated and separated as previously described.20 Antibodies employed are detailed in the supplementary methods. Densitometric analyses of western blots were performed using ImageJ software. Representative western blots with densitometric analysis out of three independently performed experiments are shown. NF-kappaB p65 activity was measured in duplicates using the TransAM NFkappaB Family Kit (Active Motif, Carlsbad, CA, USA).
Quantitative analysis of cytokines and chemokines Serum levels of TNF-alpha, IL-1alpha, IL-6, IFN-gamma and CCL2 were measured by cytometric bead array (CBA) Mouse Inflammation Kit (BD Biosciences, Heidelberg,
Germany)
and
FlowCytomix
Mouse
Th1/Th2
10plex
(BenderMedSystems, Vienna, Austria) using a BD FACS Canto II flow cytometer (BD Biosciences). Analysis was performed by FCAP ArrayTMv3 analysis software (Soft Flow, St. Louis Park, MN, USA) and FlowCytomix Pro 2.2 (BenderMedSystems). IL1beta was analyzed by Mouse IL-1beta ELISA Ready-SET-Go! (eBioscience, San Diego, CA, USA). IL-6, TNF-alpha and IL-1alpha proteins in hepatocyte culture supernatants were measured by Mouse IL-6, TNF-alpha and IL-1alpha ELISA MAXTM (BioLegend, San Diego, CA, USA).
Isolation and analysis of intrahepatic leukocytes Intrahepatic leukocytes were isolated as previously described and subjected to FACS (all antibodies from BioLegend) and qRT-PCR analyses.21
Determination of caspase 1 activity Caspase 1 activity was determined in whole liver tissue in duplicate experiments (lysis buffer: 20mM Tris/HCl pH 8.0, 5mM EDTA, 0.5% Triton X, 9
cOmplete Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA)). 50µl of tissue lysate (4mg/ml) were used with a assay mixture containing 50µl 2x reaction buffer (50mM HEPES pH 7.5, 100mM NaCl, 20% glycerol, 0.1% 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 10mM DTT) and 5µl 4mM chromogenic peptide substrate Ac-YVAD-pNA (Santa Cruz Biotechnology) and incubated at 37°C for 2h without light. Cleavage was monitored colorimetrically at 405nm.
Statistical analysis Values are given as mean ± standard error of the mean (SEM). The F-test was used to verify the assumption of equal variances, and two-tailed Student’s t-test was used to determine statistical significance. Survival times were analyzed by KaplanMeier curves with p values assessed with log-rank (Mantel-Cox) test using GraphPad Prism (GraphPad Software, Inc, La Jolla, CA, USA).
10
Results: Analysis of IL-1RI in hepatocytes
To study the functional role of the IL-1RI and the loss of IL-1 mediated signaling in hepatocytes in vivo, we generated hepatocyte-specific IL-1RI knockout mice (IL-1RIHep-/-) using the cre-loxP system under control of the albumin promoter (Figure 1A). qRT-PCR analyses confirmed a 99.5% reduction of Il1r1 mRNA expression in primary hepatocytes derived from IL-1RIHep-/- mice at the age of 10-12 weeks, whereas the Il1r1 mRNA expression in other liver resident cells was unchanged (Figure 1B). Immunoblotting indicated a significant reduction of IL-1RI protein in IL-1RIHep-/- hepatocytes compared to the wild type (Figure 1C). In vitro stimulation of IL-1RIHep-/- hepatocytes with rmIL-1alpha failed to induce Il1r1 and the IL-1 target genes Il1a, Il1rn, Il6 and Ccl2 (Figure 1D), while LPS was able to induce transcript levels of these genes irrespective of IL-1RI. Likewise, the secretion of IL-6 and TNF-alpha from cultured IL-1RIHep-/- hepatocytes in the presence of rmIL-1alpha was impaired, while LPS induced the secretion of these cytokines in cultured hepatocytes irrespective of the genotype (Figure 1E). Thus, we were able to establish a hepatocytes-restricted model with functional loss of IL-1RI signaling without affecting downstream mechanisms. Conditional deletion of the IL-1RI in hepatocytes prevents liver injury and caspase activation from D-GalN/LPS
Conditional knockout of IL-1RI in hepatocytes did not produce a spontaneous phenotype compared to wild type littermates up until the age of 6 months (Supplementary Table 1A-B, Figure 2B-C). Next, D-GalN/LPS was employed to examine the functional role of IL-1RI in hepatocytes during ALF. D-GalN/LPS
11
induced an up-regulation of IL-1RI in the hepatic tissue of wild type mice that was not seen in IL-1RIHep-/- mice (Figure 2A). Strikingly, conditional ablation of the IL-1RI in hepatocytes significantly ameliorated liver injury and cell death. Compared to the wild type ALT and AST activities as well as LDH levels were significantly decreased in IL1RIHep-/- mice at 4h (Figure 2B and Supplementary Figure 1). Liver histology showed decreased inflammation, a reduction in the number of polymorphnuclear cells (Figure 2C and Supplementary Table 2) and reduced activation of caspase 3 (Figure 2D and E). The overall mortality in this model was high irrespective of the genotype (Supplementary Figure 2) and over time liver injury increased and was not significantly different between the two genotypes (Supplementary Figure 3A). Using D-GalN and a low-dose of LPS, mortality in IL-1RIHep-/- was significantly decreased (median survival IL-1RIHep-/- vs. wt: 8.75 vs. 7.17 p<0.03; Figure 2F). Thus the functional loss of IL-1RI in hepatocytes is critical for caspase-dependent cell death and survival from ALF induced by D-GalN/LPS. Hepatocyte-specific deletion of the IL-1RI inhibits JNK/c-Jun activation through increased NF-kappaB p65 activity
TNF-alpha-induced cell death in hepatocytes involves prolonged activation of JNK.22 In IL-1RIHep-/- mice phosphorylation of the p46 and p54 JNK isoforms and activation of the downstream transcription factor c-Jun were significantly decreased in response to D-GalN/LPS at 4h without affecting levels of total JNK, respectively cJun protein (Figure 3A-B). Furthermore, D-GalN/LPS-treated wild type mice exhibited increased levels of phospho-ERK compared to IL-1RIHep-/- mice, while changes in p38 MAPK were less pronounced with no differences between genotypes (Figure 3C).
12
NF-kappaB can act both pro- or anti-inflammatory in response to LPS, and controls survival of hepatocytes during inflammation. Accumulating evidence has shown that NF-kappaB exerts cytoprotective effects by suppressing JNK activation 23
. In response to D-GalN/LPS expression of the p65 (RelA) protein decreased
significantly in the wild type, while it remained unaffected in IL-1RIHep-/- mice (Figure 3D). In parallel, IL-1RIHep-/- mice retained phosphorylation of p65 at serine residue 536 (Figure 3D) and exhibited a significant higher NF-kappaB p65 binding using a functional assay at 4h (Figure 3E). Taken together, these results indicate that IL-1RI affects the activity of NF-kappaB and inflammation-related MAPKs. Deletion of IL-1RI protected hepatocytes from the deleterious loss of NF-kappaB p65 activity from DGalN/LPS. The severity of liver injury aggravated over time and the protection observed in IL-1RIHep-/- mice was lost at 6h (Supplementary Figure 3A-C). The role of IL-1RI in TNF-alpha-mediated cell death in vitro and in vivo
In contrast to the findings in vivo, isolated hepatocytes treated with actinomycin D (ActD) and TNF-alpha showed comparable JNK activation and cell death at 24h (Figure 4A). Cellular injury was partially inhibited by the pan-caspase inhibitor zVAD, the caspase 1 inhibitor Ac-YVAD-cmk and the JNK inhibitor SP600125 with no difference between genotypes. As these data suggest that TNFalpha-induced cell death of hepatocytes in vitro does not depend on IL-1RI signaling, we tested whether the addition of exogenous IL-1RI ligands boosts hepatocellular death. Interestingly rmIL-1alpha - but not rmIL-1beta - aggravated TNF-alphainduced cell death in wild type but not IL-1RIHep-/- hepatocytes (Figure 4B). In line with these findings, exogenously administered rmIL-1alpha acted as an inflammatory amplifier during ALF in vivo. Injection of rmIL-1alpha following application of DGalN/LPS significantly augmented liver injury in wild type mice, while rmIL-1beta did 13
not (Figure 4C). These data demonstrate that other inflammatory mediators and/or immune cells are required to trigger TNF-alpha-mediated hepatocyte injury in vivo. Hepatic and systemic cytokines and chemokines are modulated by IL-1RI on hepatocytes Next inflammatory and chemotactic cytokines were examined in vivo. Following D-GalN/LPS injection, TNF-alpha, IL-1alpha, IL-1beta, IL-6, IFN-gamma and the monocyte-attractant CCL2 increased in the serum at 4h (Figure 5A). This increase was markedly more pronounced in the wild type, suggesting that the extent of liver injury could be related to the cytokine response. In liver tissue expression of Il1a, Il1b, Il6, Ifng and Ccl2, but not Tnf transcript levels were likewise attenuated in IL-1RIHep-/- mice in response D-GalN/LPS (Figure 5B). Cxcl1 and Cxcl2 mRNA encoding the neutrophil-attractant chemokine (C-X-C motif) ligand 1 (CXCL1) and 2 (CXCL2) were also significantly lower in IL-1RIHep-/- mice. These data suggest that loss of IL-1RI in hepatocytes blunts the inflammatory and chemotactic cytokines response following liver injury. Neutrophil recruitment during D-GalN/LPS injury depends on IL-1RI signaling in hepatocytes Decreased cytokine levels could be associated with reduced leukocyte migration into the liver following D-GalN/LPS injection. At baseline, IL-1RIHep-/- mice and wild type littermates did not differ with respect to the number and composition of intrahepatic immune cells (data not shown). Following D-GalN/LPS leukocyte and especially neutrophil recruitment was detectable, that was largely abolished in the absence of the hepatocellular IL-1RI. Accordingly, the D-GalN/LPS-induced increase of hepatic Ptprc, Elane and Mpo gene expression encoding the leukocyte-antigen 14
CD45, the neutrophil expressed elastase (ELANE) and myeloperoxidase (MPO) was significantly attenuated in IL-1RIHep-/- mice (Figure 6A). Quantification of living intrahepatic CD45+Ly-6G+ cells by FACS revealed a significant increase that was only seen in wild type mice, while the increase of CD45+F4/80+ macrophages occurred independently of the genotype (Figure 6B). Moreover, transcript levels of Tlr4 and Myd88 (Figure 6C) and TLR4 protein levels (Figure 6D) in hepatic tissue were comparable. NALP3-dependent caspase 1 activity is reduced in IL-1RIHep-/- mice
Both IL-1alpha as well as IL-1beta provide a positive feed-forward stimulation involving the IL-1RI and control their own precursors, the synthesis of inflammasome components as well as other inflammatory cytokines by post-translational processing and can amplify inflammation.24 In hepatocytes, in which inflammasome-dependent caspase 1 activation was described10,
25
, both IL-1 molecules are potent priming
signals for the induction of Nlrp3 and Casp1 mRNA encoding NLRP3 and caspase 1 (Supplementary Figure 4A-B). Based on the previous observations, it was assumed that IL-1RI-deficiency leads to the inhibition of an auto- resp. paracrine signaling loop that involves inflammasome activation. The hepatic mRNA levels of Nlrp3 and Casp1 were markedly increased at 4h after D-GalN/LPS in wild type mice and significantly lower in IL-1RIHep-/- mice (Figure 7A). Accordingly, immunoblotting and caspase 1 enzyme assays revealed D-GalN/LPS-induced caspase 1 activation only in the wild type (Figure 7B and C). Also, increasing gasdermin D and the cytotoxic N-terminal p30 fragment - that is produced by caspase 1 activation - was detectable only in wild type mice (Figure 7D). Specificity of the p30 protein band was confirmed by using J774 macrophages, in which caspase 1-mediated pyroptosis was induced upon activation of the NALP3 inflammasome by LPS plus ATP or nigericin (Supplementary 15
Figure 5A-C). Moreover, rmIL-1beta addition to LPS-primed primary hepatocyte cultures resulted in a significant increase of IL-1alpha protein secretion after exposure to ATP that was not detectable in IL-1RI-deficient hepatocytes (Figure 7E), while LDH release ex-vivo was not different (Supplementary Figure 5D). In conclusion, the results show that IL-1RI-deficiency in hepatocytes affects NLRP3dependent pathways, limits pyroptotic cell death and the release of inflammatory mediators like which augment hepatic inflammation. Modulation of hepatotoxicity by inhibitory IL-1ra.
Our data implicate that the IL-1RI plays a critical role in the onset of ALF and thus presents a potential therapeutic target. Therefore, we tested whether blocking IL-1 signaling by treatment with the recombinant human IL-1 receptor antagonist (IL1ra) anakinra is capable of modulating liver injury from D-GalN/LPS. Administration of IL-1ra in doses of 50µg/g bodyweight 30min prior to D-GalN/LPS application prevented liver injury in wild type mice evident by significantly decreased ALT and AST after 4h (Figure 8A). Pretreatment with 100µg or 10µg/g IL-1ra was equally respectively less effective (data not shown). Application of anakinra following the injection of D-GalN/LPS failed to rescue the liver phenotype (Figure 8A). While overall survival was not prolonged by a single injection of IL-1ra (Figure 8B), repeated application of IL-1ra showed a trend towards prolonged median survival time without reaching significance (median survival: D-GalN/LPS vs. D-GalN/LPS +IL-1ra pre vs. D-GalN/LPS +IL-1ra pre and post: 8.10 vs. 7.41 vs. 9.75h). These results indicate that blockade of IL-1 signaling confers protection against DGalN/LPS-induced hepatotoxicity; however its effects are strongly dependent on the time point and dose of administration.
16
Discussion Acute liver failure (ALF) exhibits a high mortality and therapeutic options are still limited. Recent studies proposed a role of IL-1 signaling in the pathogenesis of ALF
8
and have identified macrophage-derived IL-1alpha as a critical modulator in
APAP-induced hepatotoxicity.6 The role of IL-1RI in hepatocytes remained controversial. We employed a conditional interleukin-1 receptor 1 (IL-1RI) knock-out model exhibiting deletion of exon 5 and thus all signaling-capable IL-1R isoforms specifically in hepatocytes to further explore its role in the pathophysiology of liver disease. IL-1RI deficiency was evident both at the level of mRNA and protein expression in hepatocytes and functional assays ex vivo. Importantly, the current model allowed to specifically assess the role of IL-1RI in hepatocytes, while retaining intact cell surface expression of IL-1RI in non-parenchymal cells. Up to the age of 6 months no spontaneous phenotypical differences were observed. These mice differ from the published IL-1RI knockout model that exhibits a deletion of exon 1 and 2 and retained residual IL-1 signaling capacity.26 The central findings in the current study are a marked reduction of liver injury, caspase and MAPK activation in mice lacking the IL-1RI in hepatocytes and reduced mortality from D-GalN/LPS. These effects appear to be secondary to reduced release of cytokines and chemokines in vivo and are accompanied by increased activity of p65 NF-kappaB. In the absence of IL-1RI reduced levels of TNF-alpha, IL-1alpha, IL1beta, IL-6, IFN-gamma, CCL2, CXCL-1 and CXCL-2, were detectable both in the serum, as well as hepatic tissue. Interestingly, exogenous IL-1alpha but not IL-1beta, was able to augment TNF-alpha-mediated liver injury in wild type mice. This effect was also observed ex vivo arguing for an amplification of TNF-alpha-mediated liver injury by IL-1RI. Importantly, TLR4 and the response to TLR4 ligands were not impaired in IL-1RI knockout hepatocytes. Therefore, we assume that IL-1alpha and 17
to a lesser extent IL-1beta - released from dying hepatocytes as well as activated immune cells - ultimately contribute to the activation of the IL-1RI signaling pathway in hepatocytes and promote a pro-injurious, inflammatory response. Although the current study did not investigate differential signal transduction of IL-1alpha and IL-1beta, it is well established that both IL-1 molecules trigger inflammation through IL-1RI involving MyD88 activation and culminates in NFkappaB-induced transcription of inflammatory genes including their own precursors; inflammasome components and chemokines.24 This promotes chemotaxis and influx of neutrophils in the liver in response to injury, which was significantly suppressed in the hepatocyte-specific knockout mice. The ability of IL-1RI to trigger neutrophilic infiltration is not restricted to the hepatic tissue, but has also been observed in other inflammatory models.27 This type of immune cell activation has been termed sterile inflammation.28 A comparable effect of neutrophils during sterile inflammation has been described in a variety of liver injury models and hepatic fibrosis.29 Our data suggest that inhibition of IL-1RI in hepatocytes attenuates the attraction of neutrophils during cellular injury and thereby prevents ‘collateral damage’ that would further augment cell death. Interestingly, macrophages did not seem to be involved to the same extent in the current analysis. Previous studies have shown that IL-1RI is required for APAP-induced liver injury and neutrophilic inflammation but not for monocyte recruitment.8 As discussed above, no immediate involvement of IL-1beta was noticeable in the current model and other studies have linked IL-1beta to later stages of inflammation, which are characterized by the retention of macrophages, but not neutrophils.8, 28 The NALP3 inflammasome and specifically caspase 1 activation play an important in the regulation of inflammatory cell death - a process called pyroptosis and does regulate inflammatory and immune reactions in response to dying cells.30 18
Interestingly, caspase 1-knockout mice, which are incapable of producing IL-1alpha, IL-1beta and IL-18, are resistant to LPS-induced sepsis 31, while loss of IL-1beta or IL18 is not sufficient to prevent LPS-induced injury.32 In the IL-1RI hepatocyte-knockout model, the induction of NALP3 inflammasome components, caspase 1 activation and gasdermin D cleavage were significantly suppressed. Although the specific contribution of NALP3-dependent caspase 1 activation in hepatocytes versus nonparenchymal liver cells during ALF and the role of pyroptotic hepatocyte death have not yet been fully explored, our data suggests a synergy between IL-1 and LPS signaling pathways - including NALP3 inflammasome effector mechanisms - which is lost in absence of IL-1RI. As previous studies have suggested an inflammasomemediated release of danger signals to stimulate immune cells in non-alcoholic steatohepatitis (NASH), the findings in the IL-1RI knockout mice could have a broader implication in the pathophysiology of liver disease.10 A comparable mechanism of inflammatory signal amplification involving IL-1RI has recently also been proposed for alveolar macrophages.33 The importance of immune cell activation was confirmed in ex vivo analysis, when caspase 1 inhibition using a molecular inhibitor reduced cell death from TNF-alpha/ActD irrespective of the underlying genotype. The current data strongly suggests, that the IL-1RI signal in hepatocytes is a critical coactivator in vivo that promotes NALP3 inflammasome activation, as it has previously been observed in human PBMCs.34 In conclusion, the current study suggests that IL-1RI in hepatocytes acts to amplify acute, inflammatory liver injury through immune cell recruitment and activation with amplification of hepatocellular death. These findings underline the importance of IL-1 signaling as potential therapeutic target in acute liver disease and blocking IL-1RI signaling could be an approach to improve the poor overall outcome in acute liver failure - if the timing is right. 19
Acknowledgements: Irina Wagner, Christine Waldmann, Sonja Hoch-Kraft (I. Department of Medicine) and Claudia Braun (Core Facility Immunohistochemistry, University Medical Center Mainz, Germany) contributed with excellent technical assistance.
20
References: [1] Bernal W, Jalan R, Quaglia A, Simpson K, Wendon J, Burroughs A. Acute-on-chronic liver failure. Lancet 2015;386:1576-1587. [2] Wu Z, Han M, Chen T, Yan W, Ning Q. Acute liver failure: mechanisms of immunemediated liver injury. Liver Int 2010;30:782-794. [3] Di Paolo NC, Shayakhmetov DM. Interleukin 1[alpha] and the inflammatory process. Nat Immunol 2016;17:906-913. [4] Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL, et al. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 2000;408:111-115. [5] Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitindependent kinase of MKK and IKK. Nature 2001;412:346-351. [6] Zhang C, Feng J, Du J, Zhuo Z, Yang S, Zhang W, et al. Macrophage-derived IL1alpha promotes sterile inflammation in a mouse model of acetaminophen hepatotoxicity. Cell Mol Immunol 2017. [7] Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 1999;18:65-73. [8] Chen CJ, Kono H, Golenbock D, Reed G, Akira S, Rock KL. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med 2007;13:851-856. [9] Baroja-Mazo A, Martin-Sanchez F, Gomez AI, Martinez CM, Amores-Iniesta J, Compan V, et al. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 2014;15:738-748. [10] Csak T, Ganz M, Pespisa J, Kodys K, Dolganiuc A, Szabo G. Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology 2011;54:133-144. [11] Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology 2014;59:898-910. [12] Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, et al. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 2009;119:305-314. [13] Glaccum MB, Stocking KL, Charrier K, Smith JL, Willis CR, Maliszewski C, et al. Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J Immunol 1997;159:3364-3371. [14] Williams CD, Farhood A, Jaeschke H. Role of caspase-1 and interleukin-1beta in acetaminophen-induced hepatic inflammation and liver injury. Toxicol Appl Pharmacol 2010;247:169-178. [15] Alcaraz-Quiles J, Titos E, Casulleras M, Pavesi M, López-Vicario C, Rius B, et al. Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute-on-chronic liver failure. Hepatology 2017;65:202-216. [16] Abdulaal WH, Walker CR, Costello R, Redondo-Castro E, Mufazalov IA, Papaemmanouil A, et al. Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system. European Journal of Immunology 2016;46:912-918. [17] Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J Pharmacol Pharmacother 2010;1:94-99. [18] Zhao E, Ilyas G, Cingolani F, Choi JH, Ravenelle F, Tanaka KE, et al. Pentamidine blocks hepatotoxic injury in mice. Hepatology 2017;66:922-935. [19] Urbanik T, Koehler BC, Wolpert L, Elssner C, Scherr AL, Longerich T, et al. CYLD deletion triggers nuclear factor-kappaB-signaling and increases cell death resistance in murine hepatocytes. World J Gastroenterol 2014;20:17049-17064. [20] Schattenberg JM, Zimmermann T, Worns M, Sprinzl MF, Kreft A, Kohl T, et al. Ablation of c-FLIP in hepatocytes enhances death-receptor mediated apoptosis and toxic liver injury in vivo. Journal of hepatology 2011;55:1272-1280.
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[21] Kohl T, Gehrke N, Schad A, Nagel M, Worns MA, Sprinzl MF, et al. Diabetic liver injury from streptozotocin is regulated through the caspase-8 homolog cFLIP involving activation of JNK2 and intrahepatic immunocompetent cells. Cell death & disease 2013;4:e712. [22] Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ. Tumor necrosis factorinduced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006;281:15258-15267. [23] Schwabe RF, Brenner DA. Mechanisms of Liver Injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways. Am J Physiol Gastrointest Liver Physiol 2006;290:G583-589. [24] Schroder K, Tschopp J. The inflammasomes. Cell 2010;140:821-832. [25] Petrasek J, Bala S, Csak T, Lippai D, Kodys K, Menashy V, et al. IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice. The Journal of clinical investigation 2012;122:3476-3489. [26] Qian J, Zhu L, Li Q, Belevych N, Chen Q, Zhao F, et al. Interleukin-1R3 mediates interleukin-1-induced potassium current increase through fast activation of Akt kinase. Proc Natl Acad Sci U S A 2012;109:12189-12194. [27] Berda-Haddad Y, Robert S, Salers P, Zekraoui L, Farnarier C, Dinarello CA, et al. Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin1alpha. Proc Natl Acad Sci U S A 2011;108:20684-20689. [28] Rider P, Carmi Y, Guttman O, Braiman A, Cohen I, Voronov E, et al. IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol 2011;187:4835-4843. [29] Koyama Y, Brenner D. Liver inflammation and fibrosis. J Clin Invest 2017;127:55-64. [30] Galluzzi L, López-Soto A, Kumar S, Kroemer G. Caspases Connect Cell-Death Signaling to Organismal Homeostasis. Immunity 2016;44:221-231. [31] Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, et al. Mice deficient in IL1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 1995;80:401-411. [32] Fantuzzi G, Zheng H, Faggioni R, Benigni F, Ghezzi P, Sipe JD, et al. Effect of endotoxin in IL-1 beta-deficient mice. J Immunol 1996;157:291-296. [33] He X, Qian Y, Li Z, Fan EK, Li Y, Wu L, et al. TLR4-Upregulated IL-1beta and IL-1RI Promote Alveolar Macrophage Pyroptosis and Lung Inflammation through an Autocrine Mechanism. Sci Rep 2016;6:31663. [34] Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 2009;183:787791.
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Figure Legends: Figure 1: Conditional deletion of the IL-1RI in hepatocytes. (A) Generation of mice with targeted disruption of exon 5 of the Il1r1 gene in IL-1RI. (B) qRT-PCR analyses (n=6 mice/genotype ±SEM) and (C) immunoblotting in hepatocyte lysates on the expression of IL-1RI. (D) qRT-PCR of Il1r1, Il1a, Il1b, Il1rn, Il6 and Ccl2 gene expression in knockout (IL-1RIHep-/-) and wild type (wt) mice following treatment with recombinant mouse (rm)IL-1alpha protein (10ng/ml) or LPS (10µg/ml). (E) IL-6 and TNF-alpha in hepatocyte culture supernatants assayed by ELISA. Numerical data in mean ±SEM of three independent experiments performed in duplicate readings. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test.
Figure 2: Liver injury and caspase-dependent cell death from D-GalN/LPS in IL1RIHep-/- mice IL-1RIHep-/- and wt mice were treated with D-GalN/LPS (0.75mg/g) and LPS (2.5 µg/g) or PBS and liver injury was assessed after 4h. (A) IL-1RI expression in whole liver tissue by immunoblotting, (B) ALT and AST (n=11-13 D-GalN/LPS-treated mice; n=5 PBS-treated controls per genotype ±SEM), (C) H&E staining (scale bar: 1000µm), (D) immunohistochemistry (scale bar: 400µm), and (E) immunoblotting of cleaved caspase 3. (F) Kaplan-Meier curve following D-GalN (0.75mg/g) and LPS (0.01µg/g) in n=9-11 mice/group. Representative blots with densitometric analyses, histology and immunohistochemistry are shown. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A, B, E) and log-rank test (F).
Figure 3: MAP kinases and NF-kappaB p65 signaling in IL-1RIHep-/- mice. Immunoblot analysis of liver tissue at 4h after D-GalN/LPS for total (A) JNK, (B) cJun, (C) ERK, p38, and (D) NF-kappaB p65 protein with densitometric analyses. (E) 23
NF-kappaB p65 activity was assessed by functional binding assay from n=8-10 DGalN/LPS-treated and n=5 PBS-treated mice per genotype ±SEM. * p<.05 ** p<.01, *** p<.001 using two-tailed Student’s t-test (A-E).
Figure 4: Mechanisms of TNF-alpha-induced cell death in vitro and in vivo. Primary hepatocytes were treated ex vivo with ActD (200ng/ml) and TNF-alpha (100µg/ml) to induce TNF-alpha-receptor driven cell death. Cotreatment with pancaspase inhibitor zVAD (50µM), JNK inhibitor SP600125 (100µM), NF-kappaB inhibitor BAY-11-7082 (10µM), or caspase 1 inhibitor Ac-YVAD-cmk (100µM) was performed 1h before ActD/TNF-alpha treatment. When indicated rmIL-1alpha or rmIL-1beta protein (10ng/ml) was added 1h after ActD/TNF-alpha treatment. (A-B) Cell viability at 24h was assessed by MTT colorimetric assay. Results are expressed as relative cell viability ±SEM from three independent, triplicate experiments. (C) Transaminases were measured at 4h (n=4-10 mice/group ±SEM) in the presence or absence of rmIL-1alpha or rmIL-1beta (1µg per mouse) as indicated in C57BL/6J mice 0.5h post D-GalN/LPS challenge. * p <.05, ** p <.01, *** p <.001 according to a two-tailed Student’s t-test (A-C).
Figure 5: Hepatic and systemic cytokine and chemokine levels. (A) Serum concentration and (B) relative hepatic mRNA expression of inflammatory cytokines and chemokines at 4h after D-GalN/LPS in IL-1RIHep-/- and wt mice. Data from n=8-13 D-GalN/LPS-treated mice and n=5 PBS-treated controls per genotype ±SEM. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A-B).
Figure 6: Decreased neutrophil influx in the absence of IL-1RI in hepatocytes.
24
(A) Hepatic gene expression levels of Ptprc, Elane and Mpo were determined by qRT-PCR, and (B) living intrahepatic CD45+ immune cells were analyzed by FACS at 4h. Relative counts of intrahepatic leukocyte subsets were quantitated by gating on CD45+Ly-6G+ for neutrophils and CD45+F4/80+ for macrophages. (C) Tlr4 and Myd88 mRNA and (D) protein expression of TLR4 (representative western blot with densitometric analysis). In A and C n=8-10 D-GalN/LPS-treated and n=5 PBS-treated controls per genotype ±SEM; in B n=7mice/group ±SEM. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test.
Figure 7: NALP3 inflammasome and caspase 1 activation during D-GalN/LPSinduced acute liver failure.
(A) Gene expression levels of Nlrp3 and Casp1 analyzed by qRT-PCR (n=8-10 DGalN/LPS-treated mice; n=5 PBS-treated per genotype ±SEM). (B) Levels of cleaved and total caspase 1 protein determined by immunoblotting and (C) caspase 1 activity using the Ac-YVAD-pNA substrate (n=8-10 D-GalN/LPS-treated; n=5 PBS-treated controls per genotype ±SEM). (D) Western blot analyses of pro-Gasdermin D and Gasdermin D p30 expression with representative immunoblots and corresponding densitometric analysis. (*) Non-specific binding. (E) IL-1alpha secretion in hepatocytes primed with LPS (10µg/ml; 24h) +/- rmIL-1beta (10ng/ml; 23h) and stimulated with ATP (5mM; 0.5h) was determined by ELISA from three independent experiments (means ±SEM). * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A-E).
Figure 8: IL-1ra alleviates liver injury from D-GalN/LPS.
25
(A) Effect of recombinant interleukin-1 receptor antagonist (50µg/g IL-1ra i.p., anakinra) pre- (0.5h before) or post-treatment with D-GalN/LPS on ALT and AST at 4h (n=4-10 C57BL/6J /group ±SEM). (B) Kaplan-Meier using n=10-15 mice/group pretreated (0.5h) or treated both pre- and post-DGalN/LPS (0.5h pre and 2.5h post). * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A), or log-rank test (B).
26
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Danger signal 1 PAMPs/DAMPs including IL-1/b
TNF
IL-1 IL-1b
released from activated Kupffer cells
PRRs
released from activated Kupffer cells and injured/dying hepatocytes
IL-1RI
including TLR4
Danger signal 2 DAMPs including ATP, ROS
TNFR NF-B JNK
Upregulation of inflammatory genes encoding IL-1RI, pro-IL-1/b, NLRP3 etc.
ERK
NLRP3
ASC
Pro-Caspase 1
c-Jun
Caspase 3
Apoptosis Hepatocyte
Caspase 1 Pro-IL-1/b, TNF, IL-6, CCL2, CXCL-1/2 etc.
Pyroptosis?
DAMPs
IL-1 IL-1b Immune cell recruitment
Highlights • Hepatocytes-specific deletion of IL-1RI does not produce a spontaneous phenotype • Loss of IL-1RI in hepatocytes ameliorates D-GalN/LPS-induced liver injury and improves survival • Inflammatory signal amplification in acute liver failure involves liver-infiltrating neutrophils • NALP-3 dependent caspase 1 activation amplifies hepatic inflammation through IL1RI
27
S0168-8278(18)30020-5 https://doi.org/10.1016/j.jhep.2018.01.008 JHEPAT 6827
To appear in:
Journal of Hepatology
Received Date: Revised Date: Accepted Date:
23 January 2017 5 December 2017 10 January 2018
Please cite this article as: Gehrke, N., Hövelmeyer, N., Waisman, A., Straub, B.K., Weinmann-Menke, J., Wörns, M.A., Galle, P.R., Schattenberg, J.M., Hepatocyte-specific deletion of IL1-RI attenuatesliver injury by blocking IL-1 driven autoinflammation, Journal of Hepatology (2018), doi: https://doi.org/10.1016/j.jhep.2018.01.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation
Nadine Gehrke1, Nadine Hövelmeyer2, Ari Waisman2, Beate K. Straub3, Julia Weinmann-Menke1, Marcus A. Wörns1, Peter R. Galle1, Jörn M. Schattenberg1* 1
I. Department of Medicine,
2
Institute for Molecular Medicine and
3
Institute of
Pathology, University Medical Center Mainz, Germany.
Corresponding author: Jörn M. Schattenberg University Medical Center of the Johannes Gutenberg University I. Department of Medicine Langenbeckstraße 1 55131 Mainz, Germany Telephone: +49 6131 176074 Telefax: +49 6131 17477282 E-Mail: [email protected]
Running title: IL-1RI in hepatocytes amplifies liver injury. Keywords: interleukin-1; interleukin-1 receptor type 1; acute liver failure; acute on chronic liver failure; therapy; liver disease; inflammation; neutrophils; NALP3 inflammasome; caspase 1; pyroptosis; NF-kappaB; JNK; TLR4; TNF-alpha Electronic word count: 5896 Number of figures and tables: 8 figures Conflict of interest statement: The authors declare no conflict of interest. Financial support statement: JMS received funding from the Deutsche Krebshilfe (grant number 93199) and support of intramural funds of the Johannes Gutenberg University Mainz. NG received funding from the Mainzer Wissenschaftsstiftung (MWS). 1
Authors contributions: NG
acquisition, analysis and interpretation of data, statistical analysis, preparation of figures and tables, drafting of the manuscript
NH
technical and material support, preparation of figures, critical revision of the manuscript for important intellectual content
AW
providing of the IL-1R1flox/flox mice, technical and material support, preparation of figures, critical revision of the manuscript for important intellectual content
BKS histological analyses and interpretation, preparation of figures, critical revision of the manuscript for important intellectual content JW
material support, critical revision of the manuscript for important intellectual content
MAW critical revision of the manuscript for important intellectual content PRG material support, critical revision of the manuscript for important intellectual content JMS study concept, design and supervision, analysis and interpretation of data, preparation of figures and tables, drafting of the manuscript, obtained funding
Figure 1A was prepared by NH, AW and NG. Figure 2C was prepared by BKS and NG. The data in all other figures and tables presented were generated by NG with the help of technical assistance and were assembled by NG and JMS.
2
Abstract:
Background & Aims: Interleukin (IL)-1-type cytokines including IL-1alpha, IL-1beta and interleukin-1 receptor antagonist (IL-1Ra) are among the most potent molecules of the innate immune system and exert biological activities through the ubiquitously expressed interleukin-1 receptor type 1 (IL-1RI). The role of IL-1RI in hepatocytes during acute liver failure (ALF) remains undetermined. Methods: IL-1RI during ALF was investigated using a novel transgenic mouse model exhibiting deletion of all signaling-capable IL-1R isoforms in hepatocytes (IL-1RIHep-/-). Results: ALF induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was significantly attenuated in IL-1RIHep-/- mice leading to a reduced mortality. Conditional deletion of IL-1RI decreased activation of injurious c-Jun N-terminal kinases (JNK)/c-Jun signaling, activated nuclear factor-kappaB (NF-kappaB) p65, inhibited extracellularsignal regulated kinase (ERK) and prevented caspase 3-mediated apoptosis. Moreover, IL-1RIHep-/- mice exhibited reduced local and systemic inflammatory cytokine and chemokine levels, especially TNF-alpha, IL-1alpha/beta, IL-6, CCchemokine ligand 2 (CCL2), C-X-C motif ligand 1 (CXCL-1) and CXCL-2, and a reduced neutrophil recruitment into the hepatic tissue in response to injury. NALP3 inflammasome expression and caspase 1 activation were suppressed in the absence of the hepatocellular IL-1RI. Inhibition of IL-1RI using IL-1ra (anakinra) attenuated the severity of liver injury, while IL-1alpha administration exaggerated it. These effects were lost ex vivo and at later time points, supporting a role of IL-1RI in inflammatory signal amplification during acute liver injury. Conclusion: IL-1RI in hepatocytes plays a pivotal role in an IL-1-driven auto-amplification of cell death and inflammation in the onset of ALF.
3
Lay Summary: Acute liver injury which can cause lethal liver failure is medicated by a class of proteins called cytokines. Among these, interleukin-1 (IL-1) and the corresponding receptor IL-1RI play a prominent role in the immune system, but their role in the liver is undetermined. In the current study, a novel mouse model with defective IL-1RI in liver cells was studied. Mice lacking this receptor in liver cells were protected from cell death to a certain extent. This protection occurred only in the presence of other, neighboring cells arguing for the involvement of proteins derived from these cells. This effect is called paracrine signaling and the current study has for the first time shown that the IL-1RI receptor on hepatocytes is involved in acute liver failure in this context. The approved drug anakinra – which blocks IL-1RI – had the same effect supporting the proposed mechanism of action. The findings of this study suggest new treatment options for patients with acute liver failure by blocking defined signals of the immune system.
4
Introduction: Acute liver failure (ALF) and acute-on-chronic liver failure (ACLF) both possess a high mortality with limited treatment options and an altered inflammatory response has been implicated in their pathophysiology.1,
2
Despite obvious
differences in the etiology, both conditions exhibit activation of immune mechanisms that augment inflammation in the sequel of the initial insult and drive a lethal loss of hepatic function from increased cell death. In order to improve the poor prognosis of patients with ALF and ACLF mechanistic studies that address the underlying pathomechanisms are urgently required with the aim to identify potential, selective immune-modulatory therapies. The interleukin (IL)-1 family members IL-1alpha and IL-1beta are key proinflammatory mediators and signal through the cell surface interleukin-1 receptor type 1 (IL-1RI).3 Upon binding of IL-1alpha/beta IL-1RI recruits - in complex with its receptor accessory protein (IL-1RacP) - the cytosolic adapter molecule myeloid differentiation primary response gene 88 (MyD88) and through an intracellular signaling complex IL-1R-associated kinases (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6).4 These in turn activate transforming growth factor beta-activated kinase 1 (TAK1) and mitogen-activated protein kinases (MAPK) that are involved in cellular survival, e.g. c-Jun terminal kinases (JNK) and extracellular
signal-regulated
kinase
1/2
(ERK),
as
well
as
down-stream
transcriptions factors that contribute to hepatic inflammation.5 The role of IL-1 signaling in ALF is controversial and differs between studied models. Aggravation of ALF from acetaminophen (APAP) related to IL-1 signaling is supported by observations in transgenic mice, studies using recombinant human IL1ra anakinra to block the effects of IL-1alpha/beta6, 7 and studies exploring the role of Toll-like receptors (TLRs).8 In particular, IL-1alpha, but not IL-1beta, released from 5
Kupffer cells following hepatocellular injury seems to amplify hepatic inflammation.6 IL-1beta has received special attention, as NACHT, LRR, and PYD domainscontaining protein 3 (NALP3/NLRP3) inflammasome-mediated maturation of IL-1beta through caspase 1 has been implied in different liver disease models.9-11 On the other hand, genetic disruption of IL-1R targeting Il1r112 failed to protect from a lethal dose of D-galactosamine (D-GalN) and lipopolysaccharide (LPS)13, while it protected from acetaminophen (APAP)-induced liver injury and inflammation. Others have not observed an alteration of injury when IL-1beta was given to C57Bl/6 mice in addition to APAP.14 Moreover, major controversies regarding cell-type specific functions of IL1 signaling and especially the functional role of IL-1RI in hepatocytes remain unanswered.8 Recently a single-nucleotide polymorphisms (SNPs) in the Il1 gene cluster was linked to the severity of ACLF in clinical cohorts.15 Based on the central involvement of IL-1 in the regulation of inflammation, we tested the hypothesis, that the hepatic IL-1RI is centrally involved in the pathophysiology of inflammatory liver failure in a novel transgenic mouse model with hepatocyte-restricted deletion of all IL1R isoforms.
6
Materials and Methods: Animal model and primary hepatocytes The targeting strategy for Il1r1 gene disruption has been recently described.16 In conditional IL-1R1 mouse mutant (IL-1R1flox/flox) exon 5 of the Il1r1 gene, encoding amino-acids 166-222 and corresponding to half of the Ig-like C2 type 2 region that encodes parts of the extracellular binding region of full length IL-1R1 and truncated IL-1R3, was flanked by loxP sites leading to a transcriptional frame shift and inactivation of the two functional Il1r1 gene transcripts after Cre-mediated recombination. Hepatocyte-specific deletion of all signaling IL-1R isoforms was achieved by crossing IL-1R1flox/flox mice C57Bl6 with mice expressing Crerecombinase under an albumin promoter resulting in albumin-cre:IL-1R1flox/flox mice. Mice carrying homozygous floxed IL-1R1 and albumin-cre (IL-1RIHep-/-) were compared to their cre-negative control littermates, which are referred to as wild type (wt) mice. Genotypic identification was carried out by PCR as previously described for IL-1R1-/- mice and Cre genotyping.16 All animals were held and bred with free access to food and water at 12h light/dark cycles at the animal facility of the University Medical Center Mainz, according to the criteria outlined by the “Guide for the Care and Use of Laboratory Animals”. Studies were approved by the committee for experimental animal research (Landesuntersuchungsamt Rheinland-Pfalz) and were designed according to the ARRIVE guidelines.17 Isolation of primary hepatocytes from males and females by collagen perfusion and in vitro experiments are detailed in the supplementary materials.
Model of acute liver injury Acute liver injury was induced in mice male aged 10-12 weeks by i.p. injection of D-GalN (0.75mg/g, from D-(+)-galactosamine hydrochloride G1639, Carl Roth, 7
Karlsruhe, Germany) and LPS (2.5µg/g or 0.01µg/g from Escherichia coli Serotype 026:B6, L-8274, Sigma-Aldrich, Hamburg, Germany) according to published protocols.18, 19 Age-matched controls received saline injections. When indicated, mice received an i.p. injection of recombinant mouse (rm) IL-1alpha CF protein (1µg/mouse), rmIL-1beta CF protein (1µg/mouse, both R&D Systems, Minneapolis, MN, USA), or Kineret (anakinra, IL-1ra, 50µg/g bodyweight, Swedish Orphan Biovitrum AB, Stockholm, Sweden). Blood and liver tissue were harvested at 4h or 6h after D-GalN/LPS administration for evaluation of liver injury.
Serological analysis Serum was obtained by cardiac puncture and alanine amino transferase (ALT), aspartate amino transferase (AST) and lactate dehydrogenase (LDH) were measured using standard analyzer (Hitachi 917, Roche, Mannheim, Germany).
Quantitative real-time PCR Isolation of total RNA from snap frozen liver tissue, cDNA synthesis and qRTPCR were performed as previously described.20 All samples were performed in duplicates. Roche LightCycler software (LightCycler 480 Software Release 1.5.0) was used to perform advanced analysis relative quantification using the 2(-∆∆C(T)) method. Expression data were normalized to the housekeeping gene Gapdh (Qiagen, Hilden, Germany) and the mean of untreated wild type cells/saline-treated wild type mice was considered 1. Primer sequences (all Eurofins Genomics, Ebersberg, Germany) are detailed in the supplementary materials.
Protein isolation, immunoblotting and NF-kappaB p65 activity
8
Proteins were isolated and separated as previously described.20 Antibodies employed are detailed in the supplementary methods. Densitometric analyses of western blots were performed using ImageJ software. Representative western blots with densitometric analysis out of three independently performed experiments are shown. NF-kappaB p65 activity was measured in duplicates using the TransAM NFkappaB Family Kit (Active Motif, Carlsbad, CA, USA).
Quantitative analysis of cytokines and chemokines Serum levels of TNF-alpha, IL-1alpha, IL-6, IFN-gamma and CCL2 were measured by cytometric bead array (CBA) Mouse Inflammation Kit (BD Biosciences, Heidelberg,
Germany)
and
FlowCytomix
Mouse
Th1/Th2
10plex
(BenderMedSystems, Vienna, Austria) using a BD FACS Canto II flow cytometer (BD Biosciences). Analysis was performed by FCAP ArrayTMv3 analysis software (Soft Flow, St. Louis Park, MN, USA) and FlowCytomix Pro 2.2 (BenderMedSystems). IL1beta was analyzed by Mouse IL-1beta ELISA Ready-SET-Go! (eBioscience, San Diego, CA, USA). IL-6, TNF-alpha and IL-1alpha proteins in hepatocyte culture supernatants were measured by Mouse IL-6, TNF-alpha and IL-1alpha ELISA MAXTM (BioLegend, San Diego, CA, USA).
Isolation and analysis of intrahepatic leukocytes Intrahepatic leukocytes were isolated as previously described and subjected to FACS (all antibodies from BioLegend) and qRT-PCR analyses.21
Determination of caspase 1 activity Caspase 1 activity was determined in whole liver tissue in duplicate experiments (lysis buffer: 20mM Tris/HCl pH 8.0, 5mM EDTA, 0.5% Triton X, 9
cOmplete Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA)). 50µl of tissue lysate (4mg/ml) were used with a assay mixture containing 50µl 2x reaction buffer (50mM HEPES pH 7.5, 100mM NaCl, 20% glycerol, 0.1% 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 10mM DTT) and 5µl 4mM chromogenic peptide substrate Ac-YVAD-pNA (Santa Cruz Biotechnology) and incubated at 37°C for 2h without light. Cleavage was monitored colorimetrically at 405nm.
Statistical analysis Values are given as mean ± standard error of the mean (SEM). The F-test was used to verify the assumption of equal variances, and two-tailed Student’s t-test was used to determine statistical significance. Survival times were analyzed by KaplanMeier curves with p values assessed with log-rank (Mantel-Cox) test using GraphPad Prism (GraphPad Software, Inc, La Jolla, CA, USA).
10
Results: Analysis of IL-1RI in hepatocytes
To study the functional role of the IL-1RI and the loss of IL-1 mediated signaling in hepatocytes in vivo, we generated hepatocyte-specific IL-1RI knockout mice (IL-1RIHep-/-) using the cre-loxP system under control of the albumin promoter (Figure 1A). qRT-PCR analyses confirmed a 99.5% reduction of Il1r1 mRNA expression in primary hepatocytes derived from IL-1RIHep-/- mice at the age of 10-12 weeks, whereas the Il1r1 mRNA expression in other liver resident cells was unchanged (Figure 1B). Immunoblotting indicated a significant reduction of IL-1RI protein in IL-1RIHep-/- hepatocytes compared to the wild type (Figure 1C). In vitro stimulation of IL-1RIHep-/- hepatocytes with rmIL-1alpha failed to induce Il1r1 and the IL-1 target genes Il1a, Il1rn, Il6 and Ccl2 (Figure 1D), while LPS was able to induce transcript levels of these genes irrespective of IL-1RI. Likewise, the secretion of IL-6 and TNF-alpha from cultured IL-1RIHep-/- hepatocytes in the presence of rmIL-1alpha was impaired, while LPS induced the secretion of these cytokines in cultured hepatocytes irrespective of the genotype (Figure 1E). Thus, we were able to establish a hepatocytes-restricted model with functional loss of IL-1RI signaling without affecting downstream mechanisms. Conditional deletion of the IL-1RI in hepatocytes prevents liver injury and caspase activation from D-GalN/LPS
Conditional knockout of IL-1RI in hepatocytes did not produce a spontaneous phenotype compared to wild type littermates up until the age of 6 months (Supplementary Table 1A-B, Figure 2B-C). Next, D-GalN/LPS was employed to examine the functional role of IL-1RI in hepatocytes during ALF. D-GalN/LPS
11
induced an up-regulation of IL-1RI in the hepatic tissue of wild type mice that was not seen in IL-1RIHep-/- mice (Figure 2A). Strikingly, conditional ablation of the IL-1RI in hepatocytes significantly ameliorated liver injury and cell death. Compared to the wild type ALT and AST activities as well as LDH levels were significantly decreased in IL1RIHep-/- mice at 4h (Figure 2B and Supplementary Figure 1). Liver histology showed decreased inflammation, a reduction in the number of polymorphnuclear cells (Figure 2C and Supplementary Table 2) and reduced activation of caspase 3 (Figure 2D and E). The overall mortality in this model was high irrespective of the genotype (Supplementary Figure 2) and over time liver injury increased and was not significantly different between the two genotypes (Supplementary Figure 3A). Using D-GalN and a low-dose of LPS, mortality in IL-1RIHep-/- was significantly decreased (median survival IL-1RIHep-/- vs. wt: 8.75 vs. 7.17 p<0.03; Figure 2F). Thus the functional loss of IL-1RI in hepatocytes is critical for caspase-dependent cell death and survival from ALF induced by D-GalN/LPS. Hepatocyte-specific deletion of the IL-1RI inhibits JNK/c-Jun activation through increased NF-kappaB p65 activity
TNF-alpha-induced cell death in hepatocytes involves prolonged activation of JNK.22 In IL-1RIHep-/- mice phosphorylation of the p46 and p54 JNK isoforms and activation of the downstream transcription factor c-Jun were significantly decreased in response to D-GalN/LPS at 4h without affecting levels of total JNK, respectively cJun protein (Figure 3A-B). Furthermore, D-GalN/LPS-treated wild type mice exhibited increased levels of phospho-ERK compared to IL-1RIHep-/- mice, while changes in p38 MAPK were less pronounced with no differences between genotypes (Figure 3C).
12
NF-kappaB can act both pro- or anti-inflammatory in response to LPS, and controls survival of hepatocytes during inflammation. Accumulating evidence has shown that NF-kappaB exerts cytoprotective effects by suppressing JNK activation 23
. In response to D-GalN/LPS expression of the p65 (RelA) protein decreased
significantly in the wild type, while it remained unaffected in IL-1RIHep-/- mice (Figure 3D). In parallel, IL-1RIHep-/- mice retained phosphorylation of p65 at serine residue 536 (Figure 3D) and exhibited a significant higher NF-kappaB p65 binding using a functional assay at 4h (Figure 3E). Taken together, these results indicate that IL-1RI affects the activity of NF-kappaB and inflammation-related MAPKs. Deletion of IL-1RI protected hepatocytes from the deleterious loss of NF-kappaB p65 activity from DGalN/LPS. The severity of liver injury aggravated over time and the protection observed in IL-1RIHep-/- mice was lost at 6h (Supplementary Figure 3A-C). The role of IL-1RI in TNF-alpha-mediated cell death in vitro and in vivo
In contrast to the findings in vivo, isolated hepatocytes treated with actinomycin D (ActD) and TNF-alpha showed comparable JNK activation and cell death at 24h (Figure 4A). Cellular injury was partially inhibited by the pan-caspase inhibitor zVAD, the caspase 1 inhibitor Ac-YVAD-cmk and the JNK inhibitor SP600125 with no difference between genotypes. As these data suggest that TNFalpha-induced cell death of hepatocytes in vitro does not depend on IL-1RI signaling, we tested whether the addition of exogenous IL-1RI ligands boosts hepatocellular death. Interestingly rmIL-1alpha - but not rmIL-1beta - aggravated TNF-alphainduced cell death in wild type but not IL-1RIHep-/- hepatocytes (Figure 4B). In line with these findings, exogenously administered rmIL-1alpha acted as an inflammatory amplifier during ALF in vivo. Injection of rmIL-1alpha following application of DGalN/LPS significantly augmented liver injury in wild type mice, while rmIL-1beta did 13
not (Figure 4C). These data demonstrate that other inflammatory mediators and/or immune cells are required to trigger TNF-alpha-mediated hepatocyte injury in vivo. Hepatic and systemic cytokines and chemokines are modulated by IL-1RI on hepatocytes Next inflammatory and chemotactic cytokines were examined in vivo. Following D-GalN/LPS injection, TNF-alpha, IL-1alpha, IL-1beta, IL-6, IFN-gamma and the monocyte-attractant CCL2 increased in the serum at 4h (Figure 5A). This increase was markedly more pronounced in the wild type, suggesting that the extent of liver injury could be related to the cytokine response. In liver tissue expression of Il1a, Il1b, Il6, Ifng and Ccl2, but not Tnf transcript levels were likewise attenuated in IL-1RIHep-/- mice in response D-GalN/LPS (Figure 5B). Cxcl1 and Cxcl2 mRNA encoding the neutrophil-attractant chemokine (C-X-C motif) ligand 1 (CXCL1) and 2 (CXCL2) were also significantly lower in IL-1RIHep-/- mice. These data suggest that loss of IL-1RI in hepatocytes blunts the inflammatory and chemotactic cytokines response following liver injury. Neutrophil recruitment during D-GalN/LPS injury depends on IL-1RI signaling in hepatocytes Decreased cytokine levels could be associated with reduced leukocyte migration into the liver following D-GalN/LPS injection. At baseline, IL-1RIHep-/- mice and wild type littermates did not differ with respect to the number and composition of intrahepatic immune cells (data not shown). Following D-GalN/LPS leukocyte and especially neutrophil recruitment was detectable, that was largely abolished in the absence of the hepatocellular IL-1RI. Accordingly, the D-GalN/LPS-induced increase of hepatic Ptprc, Elane and Mpo gene expression encoding the leukocyte-antigen 14
CD45, the neutrophil expressed elastase (ELANE) and myeloperoxidase (MPO) was significantly attenuated in IL-1RIHep-/- mice (Figure 6A). Quantification of living intrahepatic CD45+Ly-6G+ cells by FACS revealed a significant increase that was only seen in wild type mice, while the increase of CD45+F4/80+ macrophages occurred independently of the genotype (Figure 6B). Moreover, transcript levels of Tlr4 and Myd88 (Figure 6C) and TLR4 protein levels (Figure 6D) in hepatic tissue were comparable. NALP3-dependent caspase 1 activity is reduced in IL-1RIHep-/- mice
Both IL-1alpha as well as IL-1beta provide a positive feed-forward stimulation involving the IL-1RI and control their own precursors, the synthesis of inflammasome components as well as other inflammatory cytokines by post-translational processing and can amplify inflammation.24 In hepatocytes, in which inflammasome-dependent caspase 1 activation was described10,
25
, both IL-1 molecules are potent priming
signals for the induction of Nlrp3 and Casp1 mRNA encoding NLRP3 and caspase 1 (Supplementary Figure 4A-B). Based on the previous observations, it was assumed that IL-1RI-deficiency leads to the inhibition of an auto- resp. paracrine signaling loop that involves inflammasome activation. The hepatic mRNA levels of Nlrp3 and Casp1 were markedly increased at 4h after D-GalN/LPS in wild type mice and significantly lower in IL-1RIHep-/- mice (Figure 7A). Accordingly, immunoblotting and caspase 1 enzyme assays revealed D-GalN/LPS-induced caspase 1 activation only in the wild type (Figure 7B and C). Also, increasing gasdermin D and the cytotoxic N-terminal p30 fragment - that is produced by caspase 1 activation - was detectable only in wild type mice (Figure 7D). Specificity of the p30 protein band was confirmed by using J774 macrophages, in which caspase 1-mediated pyroptosis was induced upon activation of the NALP3 inflammasome by LPS plus ATP or nigericin (Supplementary 15
Figure 5A-C). Moreover, rmIL-1beta addition to LPS-primed primary hepatocyte cultures resulted in a significant increase of IL-1alpha protein secretion after exposure to ATP that was not detectable in IL-1RI-deficient hepatocytes (Figure 7E), while LDH release ex-vivo was not different (Supplementary Figure 5D). In conclusion, the results show that IL-1RI-deficiency in hepatocytes affects NLRP3dependent pathways, limits pyroptotic cell death and the release of inflammatory mediators like which augment hepatic inflammation. Modulation of hepatotoxicity by inhibitory IL-1ra.
Our data implicate that the IL-1RI plays a critical role in the onset of ALF and thus presents a potential therapeutic target. Therefore, we tested whether blocking IL-1 signaling by treatment with the recombinant human IL-1 receptor antagonist (IL1ra) anakinra is capable of modulating liver injury from D-GalN/LPS. Administration of IL-1ra in doses of 50µg/g bodyweight 30min prior to D-GalN/LPS application prevented liver injury in wild type mice evident by significantly decreased ALT and AST after 4h (Figure 8A). Pretreatment with 100µg or 10µg/g IL-1ra was equally respectively less effective (data not shown). Application of anakinra following the injection of D-GalN/LPS failed to rescue the liver phenotype (Figure 8A). While overall survival was not prolonged by a single injection of IL-1ra (Figure 8B), repeated application of IL-1ra showed a trend towards prolonged median survival time without reaching significance (median survival: D-GalN/LPS vs. D-GalN/LPS +IL-1ra pre vs. D-GalN/LPS +IL-1ra pre and post: 8.10 vs. 7.41 vs. 9.75h). These results indicate that blockade of IL-1 signaling confers protection against DGalN/LPS-induced hepatotoxicity; however its effects are strongly dependent on the time point and dose of administration.
16
Discussion Acute liver failure (ALF) exhibits a high mortality and therapeutic options are still limited. Recent studies proposed a role of IL-1 signaling in the pathogenesis of ALF
8
and have identified macrophage-derived IL-1alpha as a critical modulator in
APAP-induced hepatotoxicity.6 The role of IL-1RI in hepatocytes remained controversial. We employed a conditional interleukin-1 receptor 1 (IL-1RI) knock-out model exhibiting deletion of exon 5 and thus all signaling-capable IL-1R isoforms specifically in hepatocytes to further explore its role in the pathophysiology of liver disease. IL-1RI deficiency was evident both at the level of mRNA and protein expression in hepatocytes and functional assays ex vivo. Importantly, the current model allowed to specifically assess the role of IL-1RI in hepatocytes, while retaining intact cell surface expression of IL-1RI in non-parenchymal cells. Up to the age of 6 months no spontaneous phenotypical differences were observed. These mice differ from the published IL-1RI knockout model that exhibits a deletion of exon 1 and 2 and retained residual IL-1 signaling capacity.26 The central findings in the current study are a marked reduction of liver injury, caspase and MAPK activation in mice lacking the IL-1RI in hepatocytes and reduced mortality from D-GalN/LPS. These effects appear to be secondary to reduced release of cytokines and chemokines in vivo and are accompanied by increased activity of p65 NF-kappaB. In the absence of IL-1RI reduced levels of TNF-alpha, IL-1alpha, IL1beta, IL-6, IFN-gamma, CCL2, CXCL-1 and CXCL-2, were detectable both in the serum, as well as hepatic tissue. Interestingly, exogenous IL-1alpha but not IL-1beta, was able to augment TNF-alpha-mediated liver injury in wild type mice. This effect was also observed ex vivo arguing for an amplification of TNF-alpha-mediated liver injury by IL-1RI. Importantly, TLR4 and the response to TLR4 ligands were not impaired in IL-1RI knockout hepatocytes. Therefore, we assume that IL-1alpha and 17
to a lesser extent IL-1beta - released from dying hepatocytes as well as activated immune cells - ultimately contribute to the activation of the IL-1RI signaling pathway in hepatocytes and promote a pro-injurious, inflammatory response. Although the current study did not investigate differential signal transduction of IL-1alpha and IL-1beta, it is well established that both IL-1 molecules trigger inflammation through IL-1RI involving MyD88 activation and culminates in NFkappaB-induced transcription of inflammatory genes including their own precursors; inflammasome components and chemokines.24 This promotes chemotaxis and influx of neutrophils in the liver in response to injury, which was significantly suppressed in the hepatocyte-specific knockout mice. The ability of IL-1RI to trigger neutrophilic infiltration is not restricted to the hepatic tissue, but has also been observed in other inflammatory models.27 This type of immune cell activation has been termed sterile inflammation.28 A comparable effect of neutrophils during sterile inflammation has been described in a variety of liver injury models and hepatic fibrosis.29 Our data suggest that inhibition of IL-1RI in hepatocytes attenuates the attraction of neutrophils during cellular injury and thereby prevents ‘collateral damage’ that would further augment cell death. Interestingly, macrophages did not seem to be involved to the same extent in the current analysis. Previous studies have shown that IL-1RI is required for APAP-induced liver injury and neutrophilic inflammation but not for monocyte recruitment.8 As discussed above, no immediate involvement of IL-1beta was noticeable in the current model and other studies have linked IL-1beta to later stages of inflammation, which are characterized by the retention of macrophages, but not neutrophils.8, 28 The NALP3 inflammasome and specifically caspase 1 activation play an important in the regulation of inflammatory cell death - a process called pyroptosis and does regulate inflammatory and immune reactions in response to dying cells.30 18
Interestingly, caspase 1-knockout mice, which are incapable of producing IL-1alpha, IL-1beta and IL-18, are resistant to LPS-induced sepsis 31, while loss of IL-1beta or IL18 is not sufficient to prevent LPS-induced injury.32 In the IL-1RI hepatocyte-knockout model, the induction of NALP3 inflammasome components, caspase 1 activation and gasdermin D cleavage were significantly suppressed. Although the specific contribution of NALP3-dependent caspase 1 activation in hepatocytes versus nonparenchymal liver cells during ALF and the role of pyroptotic hepatocyte death have not yet been fully explored, our data suggests a synergy between IL-1 and LPS signaling pathways - including NALP3 inflammasome effector mechanisms - which is lost in absence of IL-1RI. As previous studies have suggested an inflammasomemediated release of danger signals to stimulate immune cells in non-alcoholic steatohepatitis (NASH), the findings in the IL-1RI knockout mice could have a broader implication in the pathophysiology of liver disease.10 A comparable mechanism of inflammatory signal amplification involving IL-1RI has recently also been proposed for alveolar macrophages.33 The importance of immune cell activation was confirmed in ex vivo analysis, when caspase 1 inhibition using a molecular inhibitor reduced cell death from TNF-alpha/ActD irrespective of the underlying genotype. The current data strongly suggests, that the IL-1RI signal in hepatocytes is a critical coactivator in vivo that promotes NALP3 inflammasome activation, as it has previously been observed in human PBMCs.34 In conclusion, the current study suggests that IL-1RI in hepatocytes acts to amplify acute, inflammatory liver injury through immune cell recruitment and activation with amplification of hepatocellular death. These findings underline the importance of IL-1 signaling as potential therapeutic target in acute liver disease and blocking IL-1RI signaling could be an approach to improve the poor overall outcome in acute liver failure - if the timing is right. 19
Acknowledgements: Irina Wagner, Christine Waldmann, Sonja Hoch-Kraft (I. Department of Medicine) and Claudia Braun (Core Facility Immunohistochemistry, University Medical Center Mainz, Germany) contributed with excellent technical assistance.
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References: [1] Bernal W, Jalan R, Quaglia A, Simpson K, Wendon J, Burroughs A. Acute-on-chronic liver failure. Lancet 2015;386:1576-1587. [2] Wu Z, Han M, Chen T, Yan W, Ning Q. Acute liver failure: mechanisms of immunemediated liver injury. Liver Int 2010;30:782-794. [3] Di Paolo NC, Shayakhmetov DM. Interleukin 1[alpha] and the inflammatory process. Nat Immunol 2016;17:906-913. [4] Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL, et al. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 2000;408:111-115. [5] Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitindependent kinase of MKK and IKK. Nature 2001;412:346-351. [6] Zhang C, Feng J, Du J, Zhuo Z, Yang S, Zhang W, et al. Macrophage-derived IL1alpha promotes sterile inflammation in a mouse model of acetaminophen hepatotoxicity. Cell Mol Immunol 2017. [7] Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 1999;18:65-73. [8] Chen CJ, Kono H, Golenbock D, Reed G, Akira S, Rock KL. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med 2007;13:851-856. [9] Baroja-Mazo A, Martin-Sanchez F, Gomez AI, Martinez CM, Amores-Iniesta J, Compan V, et al. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 2014;15:738-748. [10] Csak T, Ganz M, Pespisa J, Kodys K, Dolganiuc A, Szabo G. Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology 2011;54:133-144. [11] Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology 2014;59:898-910. [12] Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, et al. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 2009;119:305-314. [13] Glaccum MB, Stocking KL, Charrier K, Smith JL, Willis CR, Maliszewski C, et al. Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J Immunol 1997;159:3364-3371. [14] Williams CD, Farhood A, Jaeschke H. Role of caspase-1 and interleukin-1beta in acetaminophen-induced hepatic inflammation and liver injury. Toxicol Appl Pharmacol 2010;247:169-178. [15] Alcaraz-Quiles J, Titos E, Casulleras M, Pavesi M, López-Vicario C, Rius B, et al. Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute-on-chronic liver failure. Hepatology 2017;65:202-216. [16] Abdulaal WH, Walker CR, Costello R, Redondo-Castro E, Mufazalov IA, Papaemmanouil A, et al. Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system. European Journal of Immunology 2016;46:912-918. [17] Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J Pharmacol Pharmacother 2010;1:94-99. [18] Zhao E, Ilyas G, Cingolani F, Choi JH, Ravenelle F, Tanaka KE, et al. Pentamidine blocks hepatotoxic injury in mice. Hepatology 2017;66:922-935. [19] Urbanik T, Koehler BC, Wolpert L, Elssner C, Scherr AL, Longerich T, et al. CYLD deletion triggers nuclear factor-kappaB-signaling and increases cell death resistance in murine hepatocytes. World J Gastroenterol 2014;20:17049-17064. [20] Schattenberg JM, Zimmermann T, Worns M, Sprinzl MF, Kreft A, Kohl T, et al. Ablation of c-FLIP in hepatocytes enhances death-receptor mediated apoptosis and toxic liver injury in vivo. Journal of hepatology 2011;55:1272-1280.
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[21] Kohl T, Gehrke N, Schad A, Nagel M, Worns MA, Sprinzl MF, et al. Diabetic liver injury from streptozotocin is regulated through the caspase-8 homolog cFLIP involving activation of JNK2 and intrahepatic immunocompetent cells. Cell death & disease 2013;4:e712. [22] Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ. Tumor necrosis factorinduced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006;281:15258-15267. [23] Schwabe RF, Brenner DA. Mechanisms of Liver Injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways. Am J Physiol Gastrointest Liver Physiol 2006;290:G583-589. [24] Schroder K, Tschopp J. The inflammasomes. Cell 2010;140:821-832. [25] Petrasek J, Bala S, Csak T, Lippai D, Kodys K, Menashy V, et al. IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice. The Journal of clinical investigation 2012;122:3476-3489. [26] Qian J, Zhu L, Li Q, Belevych N, Chen Q, Zhao F, et al. Interleukin-1R3 mediates interleukin-1-induced potassium current increase through fast activation of Akt kinase. Proc Natl Acad Sci U S A 2012;109:12189-12194. [27] Berda-Haddad Y, Robert S, Salers P, Zekraoui L, Farnarier C, Dinarello CA, et al. Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin1alpha. Proc Natl Acad Sci U S A 2011;108:20684-20689. [28] Rider P, Carmi Y, Guttman O, Braiman A, Cohen I, Voronov E, et al. IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol 2011;187:4835-4843. [29] Koyama Y, Brenner D. Liver inflammation and fibrosis. J Clin Invest 2017;127:55-64. [30] Galluzzi L, López-Soto A, Kumar S, Kroemer G. Caspases Connect Cell-Death Signaling to Organismal Homeostasis. Immunity 2016;44:221-231. [31] Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, et al. Mice deficient in IL1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 1995;80:401-411. [32] Fantuzzi G, Zheng H, Faggioni R, Benigni F, Ghezzi P, Sipe JD, et al. Effect of endotoxin in IL-1 beta-deficient mice. J Immunol 1996;157:291-296. [33] He X, Qian Y, Li Z, Fan EK, Li Y, Wu L, et al. TLR4-Upregulated IL-1beta and IL-1RI Promote Alveolar Macrophage Pyroptosis and Lung Inflammation through an Autocrine Mechanism. Sci Rep 2016;6:31663. [34] Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 2009;183:787791.
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Figure Legends: Figure 1: Conditional deletion of the IL-1RI in hepatocytes. (A) Generation of mice with targeted disruption of exon 5 of the Il1r1 gene in IL-1RI. (B) qRT-PCR analyses (n=6 mice/genotype ±SEM) and (C) immunoblotting in hepatocyte lysates on the expression of IL-1RI. (D) qRT-PCR of Il1r1, Il1a, Il1b, Il1rn, Il6 and Ccl2 gene expression in knockout (IL-1RIHep-/-) and wild type (wt) mice following treatment with recombinant mouse (rm)IL-1alpha protein (10ng/ml) or LPS (10µg/ml). (E) IL-6 and TNF-alpha in hepatocyte culture supernatants assayed by ELISA. Numerical data in mean ±SEM of three independent experiments performed in duplicate readings. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test.
Figure 2: Liver injury and caspase-dependent cell death from D-GalN/LPS in IL1RIHep-/- mice IL-1RIHep-/- and wt mice were treated with D-GalN/LPS (0.75mg/g) and LPS (2.5 µg/g) or PBS and liver injury was assessed after 4h. (A) IL-1RI expression in whole liver tissue by immunoblotting, (B) ALT and AST (n=11-13 D-GalN/LPS-treated mice; n=5 PBS-treated controls per genotype ±SEM), (C) H&E staining (scale bar: 1000µm), (D) immunohistochemistry (scale bar: 400µm), and (E) immunoblotting of cleaved caspase 3. (F) Kaplan-Meier curve following D-GalN (0.75mg/g) and LPS (0.01µg/g) in n=9-11 mice/group. Representative blots with densitometric analyses, histology and immunohistochemistry are shown. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A, B, E) and log-rank test (F).
Figure 3: MAP kinases and NF-kappaB p65 signaling in IL-1RIHep-/- mice. Immunoblot analysis of liver tissue at 4h after D-GalN/LPS for total (A) JNK, (B) cJun, (C) ERK, p38, and (D) NF-kappaB p65 protein with densitometric analyses. (E) 23
NF-kappaB p65 activity was assessed by functional binding assay from n=8-10 DGalN/LPS-treated and n=5 PBS-treated mice per genotype ±SEM. * p<.05 ** p<.01, *** p<.001 using two-tailed Student’s t-test (A-E).
Figure 4: Mechanisms of TNF-alpha-induced cell death in vitro and in vivo. Primary hepatocytes were treated ex vivo with ActD (200ng/ml) and TNF-alpha (100µg/ml) to induce TNF-alpha-receptor driven cell death. Cotreatment with pancaspase inhibitor zVAD (50µM), JNK inhibitor SP600125 (100µM), NF-kappaB inhibitor BAY-11-7082 (10µM), or caspase 1 inhibitor Ac-YVAD-cmk (100µM) was performed 1h before ActD/TNF-alpha treatment. When indicated rmIL-1alpha or rmIL-1beta protein (10ng/ml) was added 1h after ActD/TNF-alpha treatment. (A-B) Cell viability at 24h was assessed by MTT colorimetric assay. Results are expressed as relative cell viability ±SEM from three independent, triplicate experiments. (C) Transaminases were measured at 4h (n=4-10 mice/group ±SEM) in the presence or absence of rmIL-1alpha or rmIL-1beta (1µg per mouse) as indicated in C57BL/6J mice 0.5h post D-GalN/LPS challenge. * p <.05, ** p <.01, *** p <.001 according to a two-tailed Student’s t-test (A-C).
Figure 5: Hepatic and systemic cytokine and chemokine levels. (A) Serum concentration and (B) relative hepatic mRNA expression of inflammatory cytokines and chemokines at 4h after D-GalN/LPS in IL-1RIHep-/- and wt mice. Data from n=8-13 D-GalN/LPS-treated mice and n=5 PBS-treated controls per genotype ±SEM. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A-B).
Figure 6: Decreased neutrophil influx in the absence of IL-1RI in hepatocytes.
24
(A) Hepatic gene expression levels of Ptprc, Elane and Mpo were determined by qRT-PCR, and (B) living intrahepatic CD45+ immune cells were analyzed by FACS at 4h. Relative counts of intrahepatic leukocyte subsets were quantitated by gating on CD45+Ly-6G+ for neutrophils and CD45+F4/80+ for macrophages. (C) Tlr4 and Myd88 mRNA and (D) protein expression of TLR4 (representative western blot with densitometric analysis). In A and C n=8-10 D-GalN/LPS-treated and n=5 PBS-treated controls per genotype ±SEM; in B n=7mice/group ±SEM. * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test.
Figure 7: NALP3 inflammasome and caspase 1 activation during D-GalN/LPSinduced acute liver failure.
(A) Gene expression levels of Nlrp3 and Casp1 analyzed by qRT-PCR (n=8-10 DGalN/LPS-treated mice; n=5 PBS-treated per genotype ±SEM). (B) Levels of cleaved and total caspase 1 protein determined by immunoblotting and (C) caspase 1 activity using the Ac-YVAD-pNA substrate (n=8-10 D-GalN/LPS-treated; n=5 PBS-treated controls per genotype ±SEM). (D) Western blot analyses of pro-Gasdermin D and Gasdermin D p30 expression with representative immunoblots and corresponding densitometric analysis. (*) Non-specific binding. (E) IL-1alpha secretion in hepatocytes primed with LPS (10µg/ml; 24h) +/- rmIL-1beta (10ng/ml; 23h) and stimulated with ATP (5mM; 0.5h) was determined by ELISA from three independent experiments (means ±SEM). * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A-E).
Figure 8: IL-1ra alleviates liver injury from D-GalN/LPS.
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(A) Effect of recombinant interleukin-1 receptor antagonist (50µg/g IL-1ra i.p., anakinra) pre- (0.5h before) or post-treatment with D-GalN/LPS on ALT and AST at 4h (n=4-10 C57BL/6J /group ±SEM). (B) Kaplan-Meier using n=10-15 mice/group pretreated (0.5h) or treated both pre- and post-DGalN/LPS (0.5h pre and 2.5h post). * p<.05, ** p<.01, *** p<.001 using two-tailed Student’s t-test (A), or log-rank test (B).
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Danger signal 1 PAMPs/DAMPs including IL-1/b
TNF
IL-1 IL-1b
released from activated Kupffer cells
PRRs
released from activated Kupffer cells and injured/dying hepatocytes
IL-1RI
including TLR4
Danger signal 2 DAMPs including ATP, ROS
TNFR NF-B JNK
Upregulation of inflammatory genes encoding IL-1RI, pro-IL-1/b, NLRP3 etc.
ERK
NLRP3
ASC
Pro-Caspase 1
c-Jun
Caspase 3
Apoptosis Hepatocyte
Caspase 1 Pro-IL-1/b, TNF, IL-6, CCL2, CXCL-1/2 etc.
Pyroptosis?
DAMPs
IL-1 IL-1b Immune cell recruitment
Highlights • Hepatocytes-specific deletion of IL-1RI does not produce a spontaneous phenotype • Loss of IL-1RI in hepatocytes ameliorates D-GalN/LPS-induced liver injury and improves survival • Inflammatory signal amplification in acute liver failure involves liver-infiltrating neutrophils • NALP-3 dependent caspase 1 activation amplifies hepatic inflammation through IL1RI
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