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Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF

  • Cornelius Engelmann
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom

    Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany

    Medical Department, Division of Hepatology and Gastroenterology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Germany

    Berlin Institute of Health – Charité – Universitätsmedizin Berlin, Germany
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  • Abeba Habtesion
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom
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  • Mohsin Hassan
    Affiliations
    Medical Department, Division of Hepatology and Gastroenterology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Germany
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  • Annarein JC. Kerbert
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom
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  • Linda Hammerich
    Affiliations
    Medical Department, Division of Hepatology and Gastroenterology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Germany
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  • Simone Novelli
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom

    Department of Mechanical and Aerospace Engineering, Sapienza University of Rome, Rome, Italy
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  • Marco Fidaleo
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom

    Department of Biology and Biotechnology “C. Darwin”, University of Rome Sapienza, 00185 Rome, Italy
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  • Alexandra Philips
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom
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  • Nathan Davies
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom
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  • Sofia Ferreira-Gonzalez
    Affiliations
    MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh, United Kingdom
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  • Stuart J. Forbes
    Affiliations
    MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh, United Kingdom
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  • Thomas Berg
    Affiliations
    Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany
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  • Author Footnotes
    § Dr Andreola and Prof Jalan are joint senior authors.
    Fausto Andreola
    Footnotes
    § Dr Andreola and Prof Jalan are joint senior authors.
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom
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  • Author Footnotes
    § Dr Andreola and Prof Jalan are joint senior authors.
    Rajiv Jalan
    Correspondence
    Corresponding author. Address: Liver Failure Group, Institute for Liver & Digestive Health, Division of Medicine, University College London UCL, Rowland Hill Street, London, NW3 2PF, United Kingdom; Tel.: +44 02074332795.
    Footnotes
    § Dr Andreola and Prof Jalan are joint senior authors.
    Affiliations
    Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, United Kingdom

    European Foundation of the Study of Chronic Liver Failure, Barcelona, Spain
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  • Author Footnotes
    § Dr Andreola and Prof Jalan are joint senior authors.
Open AccessPublished:July 14, 2022DOI:https://doi.org/10.1016/j.jhep.2022.07.006

      Highlights

      • We hypothesised that a TLR4 inhibitor, TAK-242, might mitigate the negative effects of G-CSF in ACLF.
      • G-CSF alone increases mortality and promotes inflammation in rodent models of ACLF.
      • The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF.

      Background & Aims

      Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration.

      Methods

      Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model.

      Results

      In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration.

      Conclusion

      The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF.

      Lay summary

      Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

      Graphical abstract

      Keywords

      Introduction

      Acute-on-chronic liver failure (ACLF) is characterised by multi-organ failure in hospitalised patients with acute decompensation of cirrhosis.
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      are also increased as a result of infections and/or bacterial translocation from the gut into the systemic circulation. Binding of DAMPs and PAMPs to their cognate pattern recognition receptors, predominantly Toll-like receptor 4 (TLR4), triggers NF-κB-dependent transcription and secretion of pro-inflammatory cytokines, such as IL1β, IL6 and TNFα leading to a cascade of inflammation and cell death.
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      Targeted inhibition of TLR4 with a small molecule inhibitor, TAK-242, has been shown to reduce the short-term mortality of animals with ACLF
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      Lack of hepatic regeneration is a feature of ACLF and, indeed, hepatocyte proliferation is a positive predictor of survival.
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      To modulate hepatic regeneration in ACLF, granulocyte-colony stimulating factor (G-CSF), which mobilises bone marrow-derived stem and immune cells has been studied extensively in patients with ACLF. In several small, underpowered studies in patients with decompensated cirrhosis, alcoholic hepatitis and ACLF, G-CSF was shown to improve survival and was associated with mobilisation of CD34+ stem cells.
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      Granulocyte-Colony Stimulating Factor (G-CSF) to treat acute-on-chronic liver failure, a multicenter randomized trial (GRAFT study).
      Additionally, about twice the number of G-CSF-treated patients developed ACLF during follow-up compared with the placebo group. Moreover, 7 serious adverse reactions were observed in the G-CSF-treated group; in 5 this was related to aggravated organ failure.
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      Granulocyte-Colony Stimulating Factor (G-CSF) to treat acute-on-chronic liver failure, a multicenter randomized trial (GRAFT study).
      Previous single-centre trials, using various dosing regimens ranging from 5 to 26 days with doses of 5 μg/kg once daily to 300 μg twice daily, reported similar beneficial results independent of the treatment scheme. The fact that the 2 multicentre trials covered a similar spectrum with short high-dose treatment (15 μg/kg once daily
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      ) speaks against the argument that the lack of efficacy was dose or timing related.
      This study was designed to test the hypothesis that the lack of beneficial effect of G-CSF was possibly due to worsening of systemic inflammation through simultaneous release of bone marrow-derived inflammatory cells. We further hypothesised that this increased inflammation was through a TLR4-dependent mechanism, as previously shown in a rat model of partial hepatectomy.
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      The aims of this study were to determine whether the addition of TAK-242, a TLR4 antagonist, would prevent G-CSF-related aggravation of liver injury and enable hepatic regeneration. We also aimed to better define the underlying mechanisms of the interplay between G-CSF and TAK-242.

      Material and methods

      Study design

      This study explored the hypothesis in 2 mouse models of ACLF. All animals were included in the analysis unless specified in the figure legends. Animals were randomly assigned to experimental interventions, but no blinding was applied. All experiments were performed and reported according to the ARRIVE guidelines.
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      Animal models

      All animal experiments were performed in accordance with UK Home Office Animals (Scientific Procedures) Act 1986 (updated 2012) and a project license (No.14378) provided by the UK Home Office. After intervention, animals were monitored closely and prematurely sacrificed if pre-comatose and/or if the disease condition rapidly worsened. Animals in each group were terminated by exsanguination under general anaesthesia with isoflurane (2% isoflurane in oxygen, Piramal Healthcare, USA).

      Carbon tetrachloride-LPS model

      Male C57BL/6 mice (body weight: 30 g±4 g; age:8-10 weeks) were gavaged with carbon tetrachloride (CCl4 0.5 mg/ml dissolved in olive oil – dose 0.5 ml/kg bw) twice weekly for a total of 6 weeks to induce liver fibrosis, as described previously.
      • Carl D.E.
      • Ghosh S.S.
      • Gehr T.W.
      • Abbate A.
      • Toldo S.
      • Sanyal A.J.
      A model of acute kidney injury in mice with cirrhosis and infection.
      To mimic ACLF, LPS (Klebsiella pneumonia, Sigma, UK), dissolved in saline to a final concentration of 6.25 μg/μl was injected i.p to a final dose of 4 mg/kg. Therapeutic interventions with G-CSF (250 μg/kg, s.c.) and/or TAK-242 (10 mg/kg, i.p.) were started 1 hour after LPS injection and repeated either 22 hours after LPS injection in the 24-hour model or daily in the 5-day model, respectively. Animals were sacrificed 24 hours or 5 days after LPS injections and 2 hours after the last therapeutic intervention. Time points were chosen based on our previous studies where we observed that there was significant liver injury 24 hours after LPS injection in CCl4 pre-treated animals.
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      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      Liver regeneration and hepatocyte proliferation reaches its maximum earliest after 72 hours, with re-established liver mass after 7 days,
      • Michalopoulos G.K.
      Liver regeneration.
      so that the second time point was chosen at 5 days after LPS injection.
      The study groups were as follows: Vehicle (olive oil, saline) (n = 10); CCl4 (n = 10); CCl4+G-CSF (5 days n = 10); CCl4+LPS (24 hours n = 10; 5 day n = 6); CCl4+LPS+G-CSF (24 hours n = 10; 5 days n = 6); CCl4+LPS+TAK-242 (24 hours n = 4; 5 days n = 6); CCl4+LPS+TAK-242+G-CSF (24 hours n = 4; 5 days n = 10).
      Vehicle injections were performed with olive oil for CCl4, saline for LPS, saline for G-CSF and citric acid/NMP/Captisol for TAK-242.

      CCl4-Galactosamine model

      Male C57BL/6 mice (n = 8-10 per group) (body weight: 30 g±5 g; age: 8-10 weeks) were gavaged with CCl4 (0.5 mg/ml dissolved in olive oil – dose 0.5 ml/kg) twice weekly for a total of 6 weeks to induce liver fibrosis. Galactosamine (GalN, Sigma, UK), dissolved in saline, was injected i.p. (1,000 mg/kg) to induce liver injury. Therapeutic interventions with G-CSF (250 μg/kg, s.c.) and/or TAK-242 (10 mg/kg, i.p.) were started 1 hour after GalN injection and repeated after 22 hours and 46 hours post GalN injection. For RIPK1 inhibition, RIPA56 (3 mg/kg, i.p.) was given 1 hour after GalN and continued twice daily until animals were terminated.
      The study groups were as follows: Vehicle (n = 10); CCl4 (n = 10); CCl4+GalN (n = 8); CCl4+GalN+RIPA56 (n = 8); CCl4+GalN+G-CSF (n = 8); CCl4+GalN+TAK-242 (n = 8); CCl4+GalN+TAK-242+G-CSF (n = 8).
      Vehicle injections were performed with olive oil for CCl4, saline for GalN, saline for G-CSF and citric acid/NMP/Captisol for TAK-242.

      Sampling and storage

      Blood samples were taken from the right heart. Lithium heparin plasma was centrifuged 2,500 rpm for 10 min and stored at −80°C for later analysis. All tissues (liver, brain, kidneys) were snap frozen in liquid nitrogen and stored at -80°C for further analysis. In addition, for histological assessments, a sample of the organs was formalin-fixed (10% neutral buffered saline, Leica Biosystems, UK) for 24 hours before paraffin-embedding.

      Statistical analysis

      The sample size was calculated based on the results provided by Theocharis et al.
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      Granulocyte-colony stimulating factor administration ameliorates liver regeneration in animal model of fulminant hepatic failure and encephalopathy.
      showing an alanine aminotransferase (ALT) reduction from 3,674 U/L ± 450 to 2,450 U/L ± 225 after treatment with recombinant G-CSF in mice with thioacetamine-induced acute liver failure. We hypothesised conservatively an effect size of 15% in our model with alpha error of 0.05, providing a power of 80% for samples of 7 animals per group and 90% for samples of 10 animals per group. The sample size was confirmed by using data from an ACLF rat model of bile duct ligation and LPS injection where LPS injection increased ALT levels to 129.4 ± 33 U/L whilst TAK-242 pre-treatment reduced it to 66.2 ± 9.4 U/L.
      • Engelmann C.
      • Sheikh M.
      • Sharma S.
      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      By using an alpha error of 0.05, we calculated that 8 animals per group were needed for a power of 80% and 12 animals per group for a power of 90%. Finally, we aimed at having 10 animals per group unless stated otherwise.
      All statistical analyses were performed using SPSS 24 software (SPSS Inc., Chicago; IL). Group comparisons for continuous variables were performed by using Man-Whitney U test and for categorical variables by using Chi-squared test. If more than 2 groups had to be analysed, one-sided ANOVA with post hoc Tukey analyses for multiple comparison were used. A p value ≤0.05 was considered statistically significant. Graphs were prepared in Prism (GraphPad, USA) and figures compiled in Adobe Photoshop (Adobe Systems, USA).
      For further details regarding the materials and methods used, please refer to the CTAT table and supplementary information.

      Results

      G-CSF increases the mortality of rodents with ACLF

      To understand the pathophysiological basis of the lack of benefit and possible deleterious effect of G-CSF treatment that was observed in the German clinical trial (GRAFT study),
      • Engelmann C.
      • Herber A.
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      • Schiefke I.
      • Zipprich A.
      • et al.
      Granulocyte-Colony Stimulating Factor (G-CSF) to treat acute-on-chronic liver failure, a multicenter randomized trial (GRAFT study).
      we performed preclinical studies in murine models of ACLF. In the first model, mice were gavaged for 6 weeks with CCl4 and then injected with LPS to induce liver injury, as described previously
      • Engelmann C.
      • Sheikh M.
      • Sharma S.
      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      ,
      • Carl D.E.
      • Ghosh S.S.
      • Gehr T.W.
      • Abbate A.
      • Toldo S.
      • Sanyal A.J.
      A model of acute kidney injury in mice with cirrhosis and infection.
      (Fig. 1A). Six-week CCl4 administration induced bridging fibrosis (Fig. 1D). This model mimics the typical features consisting of multi-organ injury in response to administration of LPS and pre-existing chronic liver injury.
      • Engelmann C.
      • Sheikh M.
      • Sharma S.
      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      ,
      • Carl D.E.
      • Ghosh S.S.
      • Gehr T.W.
      • Abbate A.
      • Toldo S.
      • Sanyal A.J.
      A model of acute kidney injury in mice with cirrhosis and infection.
      Twenty-four hours after LPS injection these animals developed significant liver and kidney injury with high ALT and creatinine levels and extended areas of hepatocyte cell death (TUNEL staining) (Fig. 2, Fig. S1). Hepatic expression of CCL5, ICAM-1 and NGAL, as markers of inflammation and organ injury, also increased after LPS injection compared to vehicle (p <0.001) (Fig. S2).
      • Engelmann C.
      • Sheikh M.
      • Sharma S.
      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      Liver injury, measured by ALT levels and liver cell death (TUNEL), was decreased 5 days after LPS injection (Fig. S1).
      Figure thumbnail gr1
      Fig. 1Effect of G-CSF in an ACLF mouse model.
      (A) Animals were gavaged for 6-weeks with 0.5 ml/ml CCl4 to induce chronic liver injury. Thereafter, LPS (Klebsiella pneumoniae) was injected to induce multi-organ injury. G-CSF (250 μg/kg, s.c.) was injected 1 hour after LPS and continued for 5 days once daily (Vehicle n = 10, CCl4 n = 10, CCl4 + LPS n = 6, CCl4 + LPS + G-CSF n = 6). (B) In total, 66% of all animals died in the group treated with G-CSF alone. Survival in the other groups was 100%. (C) 5 days of G-CSF treatment increased hepatic macrophage infiltration (F4/80+) compared to animals with CCl4+LPS only (p <0.001). (D) This ACLF model was associated with significant collagen accumulation (Sirius Red staining, p <0.001 compared to vehicle) after 5-days CCl4. Group comparison was performed by one-way ANOVA and post hoc Tukey’s multiple comparison. p >0.05 was considered statistically significant. ACLF, acute-on-chronic liver failure; CCl4, carbon tetrachloride; G-CSF, granulocyte-colony stimulating factor; LPS, lipopolysaccharide. (This figure appears in color on the web.)
      Figure thumbnail gr2
      Fig. 2TAK-242 prevents the deleterious effect of G-CSF in the CCl4-LPS model.
      (A) C57B/6 mice were gavaged for 6-weeks with CCl4 0.5 mg/ml to induce chronic liver injury. Thereafter, LPS (Klebsiella pneumoniae) was injected to induce multi-organ injury. G-CSF (250 μg/kg, s.c.) was injected 1 hour after LPS and continued for 24 hours or 5 days once daily. TAK-242 was administered concurrently (10 mg/kg, i.p.) and continued once daily for 24 hours or 5 days. (B) G-CSF injection led to an increase of CD45+ circulating haematopoietic cells (n = 4 per group). (C) TAK-242 reduced LPS-driven liver cell death (TUNEL) (p <0.01) (n = 4 per group) and ALT levels (all animals per group). (D) LPS injection increased liver TNFa mRNA expression (>2 fold), whereas the additional G-CSF treatment led to an overexpression of IL-6 mRNA (>2 fold). Expression of both cytokines was reduced after injection of TAK-242 (n = 4 per group). (E) G-CSF treatment after LPS injection led to high numbers of circulating neutrophils and Ly6c-high activated monocytes. TAK-242 reduced the number of both cell subsets and promoted the occurrence of Ly6c-low monocytes in the blood (flow cytometry) (n = 4 per group). (F) TAK-242 decreased the amount of liver-infiltrating F4/80+ macrophages (p <0.001) and Ly6G+ neutrophils (p <0.05) (n = 4 per group). Image J was used for image quantification and group comparison was performed by one-way ANOVA and post hoc Tukey’s multiple comparison only among the following groups: CCl4+LPS, CCl4+LPS+G-CSF, CCl4+LPS+TAK-242, CCl4+LPS+G-CSF+TAK-242. p >0.05 was considered statistically significant. mRNA data is delineated as ΔΔCt value. ALT, alanine aminotransferase; CCl4, carbon tetrachloride; G-CSF, granulocyte-colony stimulating factor; LPS, lipopolysaccharide. (This figure appears in color on the web.)
      One-hour after LPS injection, mice were treated with G-CSF subcutaneously (250 μg/kg) once daily for 5 consecutive days. Twenty-four hours after the first G-CSF injection the number of mobilised CD45+ cells in the blood increased from a median of 321,900 cells/ml blood (CCl4+LPS) to 510,504 cells/ml (CCl4+LPS+G-CSF) (Fig. 2B). G-CSF therapy led to a mortality rate of 66% after 48 hours (Fig. 1B) and a high degree of liver fibrosis (Sirius Red) (p <0.001, compared to vehicle) (Fig. 1D), whereas all other animals without G-CSF treatment survived the 5-day follow-up. G-CSF administration was associated with increased infiltration of the liver with macrophages (F4/80+-DAB-positive areas [CCl4+LPS] 4.3 % ± 1.1 vs. 8.4 % ± 3.9 [CCl4+LPS+G-CSF]; p <0.001) (Fig. 1C) after 5 days of G-CSF treatment. Therefore, in this model, G-CSF aggravated ACLF-related mortality, which was associated with worsening of hepatic macrophage infiltration.

      TLR4 inhibition by TAK-242 prevents G-CSF-related mortality and inflammation

      As our previous studies have shown increased hepatocyte TLR4 expression being involved in organ sensitisation to LPS in cirrhosis and a reduction in the severity of inflammation with inhibition of TLR4 in models of ACLF,
      • Engelmann C.
      • Adebayo D.
      • Oria M.
      • De Chiara F.
      • Novelli S.
      • Habtesion A.
      • et al.
      Recombinant alkaline phosphatase prevents acute on chronic liver failure.
      we hypothesised that the deleterious effect of G-CSF in ACLF may be modulated favourably by TLR4 inhibition.
      • Fang H.
      • Liu A.
      • Sun J.
      • Kitz A.
      • Dirsch O.
      • Dahmen U.
      Granulocyte colony stimulating factor induces lipopolysaccharide (LPS) sensitization via upregulation of LPS binding protein in rat.
      We therefore used TAK-242, a small molecule TLR4-inhibitor, with or without G-CSF in the CCl4 – LPS model over 24 hours and 5 days (Fig. 2A).
      Administration of TAK-242 together with G-CSF reduced the mortality rate from 66%, in G-CSF-treated animals, to 0% (Fig. S10). Liver cell death was measured by TUNEL staining 24 hours after LPS injection. The median relative TUNEL-positive area in liver tissue increased from 0.7% (range 0.2–3.3) to 10.1% (range 0.3–22.1), which was not significantly different to the 7.8% (range 1.6-22.1) observed when G-CSF was injected after LPS. Treatment with TAK-242, alone or in combination with G-CSF, significantly reduced the TUNEL-positive area to 0.19% (range 0.06-0.55, p <0.01) and 0.27% (range 0.16-0.5, p <0.01), respectively (Fig. 2C), and reduced ALT levels (Fig. 2C). The STAT3 pathway reduces apoptosis through release of B-cell lymphoma 2 protein (BCL2), which antagonises BCL2-associated X protein (BAX). The protective effect of TAK-242/G-CSF was associated with activation of the STAT3 pathway (increased pSTAT3) (Fig. S3), possibly induced by IL-22 secretion (Fig. S2E), with increased expression of anti-apoptotic BCL2 and higher BCL2/BAX ratio (Fig. S3). Likewise, injury markers (such as Lipocalin/NGAL and IL-13) were increased with G-CSF but reduced if TAK-242 was added, as shown by the cytokine/chemokine protein arrays (Fig. S2B/C).
      Inflammatory response was assessed by measuring liver cytokine expression at both the mRNA and protein level. LPS injection led to >4-fold increase in hepatic TNFα mRNA expression, whereas treatment with G-CSF induced an increase in IL-6 mRNA expression level (>2 fold), which was markedly reduced by TAK-242, with or without G-CSF (Fig. 2D). G-CSF increased the liver protein expression of inflammatory markers such as CXCL9, LIF and Petraxin3/TSG-14 as shown by the protein array data from pooled liver lysates. These markers were reduced after TAK-242 injection (Fig. S2). TAK-242 treatment resulted in reduced hepatic infiltration of F4/80+ macrophages (DAB-positive area [%] – 7.6%±3.6 [CCl4+LPS+G-CSF] vs. 2.9%±1.2 [CCl4+LPS+G-CSF+TAK-242], p <0.001) and Ly6G+ neutrophils (Ly6G positive cells per bright field – 68.9±1.2 [CCl4+LPS+G-CSF] vs. 32.3±12.7 [CCl4+LPS+G-CSF+TAK-242], p <0.05) (Fig. 2F). High numbers of circulating neutrophils and Ly6c-high monocytes occurred in the blood after G-CSF therapy. Their numbers decreased with TAK-242 therapy and the combination of TAK-242 and G-CSF led to an increase of Ly6c-low monocytes (Fig. 2E). LPS injection was also associated with stellate cell activation (αSMA expression), which was significantly abrogated by TAK-242 (Fig. S4).
      Monocytes and macrophages are major sources of cytokine release in liver disease.
      • Krenkel O.
      • Tacke F.
      Liver macrophages in tissue homeostasis and disease.
      We therefore tested to what extent LPS and G-CSF modulate the cytokine response in PMA (phorbol 12-myristate-13-acetate)-activated (macrophage-like) and naïve THP1 cells (monocyte-like) in vitro. The experiments showed that G-CSF incubation of PMA-activated THP1 cells and naïve THP1 cells prior to LPS stimulation resulted in an aggravated cytokine response, especially upregulation of IL6 mRNA expression (THP1macrophages+LPS [10 ng/ml]: 6.2-fold upregulation vs. LPS (10 ng/ml)+G-CSF (100 ng/ml): 6.7-fold upregulation; THP1monocytes+LPS [10 ng/ml]: 60.2-fold upregulation vs. LPS [10 ng/ml]+G-CSF [100 ng/ml]: 71-fold upregulation [Fig. S5A]). Results were confirmed in primary peripheral blood mononuclear cells isolated from healthy donors and incubated with pooled plasma from 9 patients with ACLF (Table S3). These cells exhibited a strong upregulation of IL-1b after incubation with G-CSF prior to LPS, compared to cells incubated with LPS alone. TAK-242 prevented excess cytokine production in peripheral blood mononuclear cells with or without G-CSF (Fig. S5B). These in vitro findings are in keeping with hepatic IL6 levels, which increased 2.8-fold in G-CSF-treated animals (Fig. 2D). These changes, either with LPS alone or in combination with G-CSF, were completely prevented in the presence of TAK-242, clearly showing that TAK-242 prevents the overwhelming inflammatory response driven by LPS and G-CSF. The data help to explain the reduction in markers of organ injury in the ACLF animals treated with TAK-242/G-CSF.

      Hepatocellular senescence is a feature of ACLF, which is aggravated by G-CSF but prevented by TAK-242

      Although modulation of inflammation using TAK-242 abrogated the severity of liver injury in the ACLF animals treated with G-CSF and/or LPS, this data does not uncover the mechanism underlying failure of regeneration in ACLF. Hepatocellular senescence, an essentially state of cell cycle arrest, is a pathophysiological feature of end-stage liver disease that inhibits regeneration and adequate tissue repair, resulting in poor clinical outcomes.
      • Sen B.
      • Rastogi A.
      • Nath R.
      • Shasthry S.M.
      • Pamecha V.
      • Pandey S.
      • et al.
      Senescent hepatocytes in decompensated liver show reduced UPR(MT) and its key player, CLPP, attenuates senescence in vitro.
      Mediators of cell cycle arrest, such as p16 or p21, are expressed in response to tissue injury and transforming growth factor beta-1 (TGFb1) is a key mediator of the senescence-associated secretory phenotype (SASP), where a paracrine spread of injury-independent cellular senescence is observed throughout the liver tissue.
      • Bird T.G.
      • Muller M.
      • Boulter L.
      • Vincent D.F.
      • Ridgway R.A.
      • Lopez-Guadamillas E.
      • et al.
      TGFbeta inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.
      ,
      • Ferreira-Gonzalez S.
      • Lu W.Y.
      • Raven A.
      • Dwyer B.
      • Man T.Y.
      • O'Duibhir E.
      • et al.
      Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
      We therefore evaluated the effect of LPS and interventions with G-CSF and TAK-242 on markers of senescence.
      LPS injection in CCl4-treated animals led to a prolonged (up to 5 days post administration) upregulation of both p16 mRNA (1.9-fold compared to vehicles) and TGFb1 in the liver (Western Blot, optical density [OD]: vehicle = 0.67 vs. CCl4+LPS 24 hours = 0.99 vs. CCl4+LPS 5 days = 1.14). Additionally, hepatic protein levels of p21 (Western Blot: OD vehicle = 0.33 vs. CCl4+LPS 24 hours = 0.8 vs. CCl4+LPS 5 days = 1.14) and Serpin E1/PAI-1 (proteome profiler) (Fig. 3A/B; Fig. S2C) were also upregulated by LPS, indicating the importance of senescence in this model of ACLF. Liver expression of both markers (p21 and Serpin E1/PAI-1) were further exacerbated by G-CSF treatment (p21 Western Blot OD CCl4+LPS+G-CSF 24 hours = 0.97). The administration of TAK-242, with or without G-CSF, effectively mitigated against the expression of all senescence markers (p16, TGFb1, p21 and Serpin E1/PAI-1) (Fig. 3A/B; Fig. S2C), as shown by mRNA expression (qPCR) and protein expression from pooled liver lysates. p21 immunohistochemistry revealed that activation of the p53/p21 pathway occurred predominantly in hepatocytes. The number of p21-expressing hepatocytes increased after 24 hours from 2.8% ± 2.2 with LPS alone to 8.7% ± 3 with LPS+G-CSF although this effect diminished 5 days after therapy. TAK-242 added to G-CSF significantly reduced the number of p21-expressing (senescent) hepatocytes to 0.2% ± 0.1, 24 hours after LPS injection (p <0.001) (Fig. 3C). Therefore, the data suggest that LPS is a key driver of hepatocellular senescence in ACLF, which is further exacerbated by G-CSF. TLR4 inhibition with TAK-242 prevents this senescent phenotype.
      Figure thumbnail gr3
      Fig. 3Evidence of hepatocellular senescence in the rodent model of ACLF and the effect of G-CSF and TAK-242.
      (A) Increased hepatic p16 mRNA expression in CCl4 + LPS animals with or without treatment with G-CSF treatment, after 24 hours and 5 days. TLR4 inhibition with TAK-242 effectively reduced p16 mRNA expression at both time points (n = 4 per group). (B) Increased hepatic protein expression of p21 and TGFb1 in CCl4+LPS animals with or without G-CSF treatment, after 24 hours and 5 days. The OD (Western Blot liver lysates) increased from vehicle = 0.67 to CCl4+LPS 24 hours = 0.99 and CCl4 + LPS 5 days = 1.14. Hepatic protein levels of p21 were raised starting from vehicle = 0.33 to CCl4+LPS 24 hours = 0.8 and CCl4+LPS 5 days = 1.14. G-CSF exaggerated TGFb1 (OD 1.1) and p21 expression (OD 0.97) 24 hours after LPS injection. TLR4 inhibition with TAK-242 effectively reduced protein expression of p21 and TGFb1 at both time points (all individuals per group). Western Blot was repeated twice and a representative blot is shown here. (C) Liver immunohistochemistry demonstrated that p21 expression occurred predominantly in hepatocytes and could be prevented by TAK-242, with or without G-CSF (n = 3 per group). 24 hours after LPS injection, 2.8% ± 2.2 hepatocytes expressed p21, which was enhanced to 8.7% ± 3 by adding G-CSF. This effect diminished 5 days after therapy. TAK-242 added to G-CSF significantly reduced the number of p21-expressing (senescent) hepatocytes to 0.2% ± 0.1, 24 hours after LPS injection (p <0.001). Image J was used for image quantification and group comparison was performed by one-way ANOVA and post hoc Tukey’s multiple comparison only among the following groups: CCl4+LPS, CCl4+LPS+G-CSF, CCl4+LPS+TAK-242, CCl4+LPS+G-CSF+TAK-242. p >0.05 was considered statistically significant. mRNA data is delineated as ΔΔCt value and a greater than 2-fold change in expression was considered as biologically relevant. Western blots were performed with protein lysates pooled from all animals per group and results shown in optical densitiy (OD). ACLF, acute-on-chronic liver failure; CCl4, carbon tetrachloride; G-CSF, granulocyte-colony stimulating factor; LPS, lipopolysaccharide; OD, optical density. (This figure appears in color on the web.)

      Failure of regeneration in ACLF is synergistically abrogated by G-CSF and TAK-242

      G-CSF has been shown to exhibit regenerative properties in non-inflammatory (not endotoxin-driven) models of liver injury.
      • Piscaglia A.C.
      • Shupe T.D.
      • Oh S.H.
      • Gasbarrini A.
      • Petersen B.E.
      Granulocyte-colony stimulating factor promotes liver repair and induces oval cell migration and proliferation in rats.
      ,
      • Tsolaki E.
      • Athanasiou E.
      • Gounari E.
      • Zogas N.
      • Siotou E.
      • Yiangou M.
      • et al.
      Hematopoietic stem cells and liver regeneration: differentially acting hematopoietic stem cell mobilization agents reverse induced chronic liver injury.
      We therefore hypothesised that the excessive inflammation in ACLF, driven by LPS and/or G-CSF as observed above, may prevent G-CSF from acting as pro-regenerative agent and that the addition of TAK-242, which modulates inflammation, may overcome this.
      In the CCl4-LPS model, therapy with G-CSF for 5 days resulted in hepatocyte activation of Cyclin A2 (Cyclin A2-positive hepatocytes – CCl4+LPS 0.2% ± 0.2 vs. CCl4+LPS+G-CSF 1.4% ± 0.7, p <0.001) (Fig. 4), which is known to mediate cell proliferation by promoting cell cycle progression from S- to M-phase; nevertheless, no induction of proliferation was observed, as indicated by the lack of Ki67 expression (Ki67-positive hepatocytes – CCl4+LPS 0.1% ± 0.2 vs. CCl4+LPS+G-CSF 0.6% ± 0.2, n.s.) (Fig. 4). However, when G-CSF was given for 5 days in combination with TAK-242 (TAK-242+G-CSF), both markers for cell division significantly increased in hepatocytes (Cyclin A2 hepatocyte expression: 1.2% ± 0.7 [p <0.01 vs. TAK-242 single treatment]; Ki67 hepatocyte expression 1.2% ± 1 [p <0.05 vs. TAK-242 single treatment] (Fig. 4), thus suggesting that TAK-242 enhanced the pro-proliferative effect of G-CSF.
      Figure thumbnail gr4
      Fig. 4Evidence for induction of regeneration markers in hepatocytes induced by TAK-242/G-CSF.
      Immunofluorescence and histochemistry staining for Cyclin A2 and Ki67, mediators of cell cycle progression, in paraffin embedded liver tissue (n = 3 per group) (Cyclin A2 upper panel, Ki67 lower panel). In the 24-hour treatment model, G-CSF increased Cyclin A2-expressing hepatocytes (Cyclin A2-positive hepatocytes – CCl4+LPS: 0.2% ± 0.2 vs. CCl4+LPS+G-CSF: 1.4% ± 0.7). TAK-242 decreased Cyclin A2-positive cells without reaching significance. In the 5-day treatment model, compared to TAK-242 treatment alone, G-CSF in combination with TAK-242 significantly stimulated expression of Cyclin A2 (0.6% ± 0.5 vs. 1.2% ± 0.7; p <0.001) and Ki67 (0.6% ± 0.3 vs. 1.2% ± 1; p <0.05). Ki67 expression was also enhanced by G-CSF, 24-hrs after LPS injection (p <0.05 to CCl4+LPS). Image J was used for image quantification and group comparison was performed by one-way ANOVA and post-hoc Tukey’s multiple comparison only among the following groups: CCl4+LPS, CCl4+LPS+G-CSF, CCl4+LPS+TAK-242, CCl4+LPS+G-CSF+TAK-242. p >0.05 was considered statistically significant. CCl4, carbon tetrachloride; G-CSF, granulocyte-colony stimulating factor; LPS, lipopolysaccharide. (This figure appears in color on the web.)
      To provide further evidence that G-CSF exhibits a pro-proliferative effect in an environment with lower grade inflammation, we tested 5 days of G-CSF therapy after CCl4 administration without LPS. In this setting, G-CSF injection significantly increased the number of proliferating hepatocytes; Ki67 staining was observed in 4.1% ± 2.9 (p <0.001 to CCl4+LPS+G-CSF 5 days) and Cyclin A2 in 2.1% ± 1.3 (p <0.05 vs. CCl4+LPS+G-CSF 5 days) (Fig. S6).
      Protein expression of other regenerative markers was assessed in pooled liver lysates using the proteome array. TAK-242 alone, administered over 24 hours in the CCl4-LPS mice, reduced both hepatic markers of vascular regeneration (such as angiopoietin 2, proliferin and platelet-derived growth factor) and other markers involved in liver regeneration, such as IL-22, Flt3-ligand and IGFBP-1 (Fig. S2D&E). Adding G-CSF to TAK-242 markedly increased the liver protein expression of these pro-regenerative markers (Fig. S2D,E).
      To provide more details on the link between inflammation, senescence and regeneration, a multiplex immunofluorescence staining of the liver was performed (Fig. 5) including cell type markers (HNF4a – hepatocytes, CK19 – cholangiocytes) as well as Na-K ATPase for cell membrane delineation. After LPS injection and G-CSF treatment, areas of hepatocyte injury (HNF4+) indicated by cleaved caspase 3 positivity (p <0.01 compared to CCl4+LPS) and macrophage infiltration (Iba1+) (p <0.01 compared to CCl4+LPS) were surrounded by γH2AX-expressing hepatocytes, which are associated with senescence. The combination of TAK-242 and G-CSF abrogated liver injury (cleaved caspase 3 – p <0.001 when comparing CCl4+LPS+G-CSF with CCl4+LPS+G-CSF+TAK-242) and reduced inflammatory infiltration/response (Iba1 – p <0.01 comparison between CCl4+LPS+G-CSF and CCl4+LPS+G-CSF+TAK-242). Therapy with TAK-242 and G-CSF resulted in a decrease of senescent hepatocytes (γH2Ax – p <0.01 comparison between CCl4+LPS+G-CSF and CCl4+LPS+G-CSF+TAK-242) whilst hepatocyte expression of proliferation markers PCNA (p <0.001 comparison between CCl4+LPS and CCl4+LPS+G-CSF+TAK-242) and Ki67 (p <0.001 comparison between CCl4+LPS+G-CSF and CCl4+LPS+G-CSF+TAK-242) was promoted predominantly in the periportal region (Fig. 5).
      Figure thumbnail gr5
      Fig. 5Multiplex staining of liver tissue depicts localisation of injury, senescence and proliferation after G-CSF and TAK-242 in the CCl4 – LPS model.
      Multiplex immunofluorescence staining on one exemplary sample per group was performed to understand the regional interaction between tissue injury, inflammation and regenerative response. Tissues were sequentially stained for HNF4a (hepatocytes), CK19 (cholangiocytes), Na-K-ATPase (cell membrane), cleaved caspase 3 (cell death/injury), RIPK3 (necroptosis), γH2AX (senescence), Iba1 (macrophage), Cyclin A2 and Ki67 (cell cycle progression). After CCl4+LPS+G-CSF there were extended areas of injury with macrophage infiltration and high numbers of senescent and non-proliferating hepatocytes. After abrogation of tissue injury and reduced number of macrophages by TAK-242 and G-CSF there was an increase of proliferating hepatocytes, predominantly in the periportal region. Image processing was performed by Ilastik and CellProfiler was used for quantification. Group comparison was performed by one-way ANOVA and post hoc Tukey’s multiple comparison only among the following groups: CCl4+LPS, CCl4+LPS+G-CSF, CCl4+LPS+TAK-242, CCl4+LPS+G-CSF+TAK-242. p >0.05 was considered statistically significant. CCl4, carbon tetrachloride; G-CSF, granulocyte-colony stimulating factor; LPS, lipopolysaccharide. (This figure appears in color on the web.)
      These findings support the hypothesis that G-CSF requires an environment with reduced inflammation to exert its proliferative effects on hepatocytes and, therefore, TAK-242 allows G-CSF to exert its pro-regenerative capacities.

      TAK-242/G-CSF reduces tissue injury in CCl4 – GalN-treated mice

      The effect of G-CSF, with or without TAK-242, was then tested in a second model where GalN, instead of LPS, was used as a sterile toxic insult in mice treated for 6 weeks with CCl4 (Fig. 6).
      Figure thumbnail gr6
      Fig. 6Effect of TAK-242/G-CSF in an ACLF model with low-grade inflammation.
      (A) C57B/6 mice were gavaged for 6 weeks with CCl4 0.5 mg/ml to induce chronic liver injury (n = 8 per group). Thereafter, GalN was injected to induce a sterile liver injury. G-CSF (250 μg/kg, s.c.) was injected first 1 hour after GalN and continued for 48 hours. TAK-242 was given concurrently (10 mg/kg, i.p.) and continued once daily for 48 hours. (B) GalN injection induced a liver injury with high ALT levels, cell death (TUNEL) and RIPK3 expression (necroptosis). Liver injury was reduced by all treatment options but the combination therapy, TAK-242+G-CSF, was superior to the single compounds. (C) The treatment effect was associated with activation of the STAT3 pathway and a trend to increased anti-apoptotic BCL2 expression as shown by Western blot in pooled samples. Western blot was repeated twice and a representative blot is shown here. (D) GalN induced a regenerative response with proliferating hepatocytes but also hepatocytes in cell cycle arrest. G-CSF alone or in combination with TAK-242 reduced cell death and the subsequent regenerative response. TAK-242 alone maintained the amount of proliferating (Cyclin A2, Ki67) and senescent (p21) hepatocytes. Image J was used for image quantification and group comparison was performed by one-way ANOVA and post hoc Tukey’s multiple comparison only among the following groups: CCl4+LPS, CCl4+LPS+G-CSF, CCl4+LPS+TAK-242, CCl4+LPS+G-CSF+TAK-242. mRNA data is delineated as ΔΔCt value and a greater than 2-fold change in expression was regarded as biologically significant. Western blots were performed with protein lysates pooled from all animals per group and results shown in optical density (OD). ACLF, acute-on-chronic liver failure; CCl4, carbon tetrachloride; GalN, galactosamine; G-CSF, granulocyte-colony stimulating factor; LPS, lipopolysaccharide. (This figure appears in color on the web.)
      First, we explored the extent of liver injury induced by GalN on the background of CCl4 and how this was modulated by G-CSF ± TAK-242. GalN injection induced a significant liver injury with high ALT levels (27.3 U/L [range 24.2–63.4] vs. 288 U/L [range 46-807]), extended areas of cell death (TUNEL: 0.23% [range 0.17-0.29] vs. 2.6% [range 1.3-5.2]), and RIPK3 positivity (1% [range 0.75-1.1] vs. 3.2% [range 1.6-4.4]), a marker for necroptotic cell death. In contrast to the inflammatory LPS model, treatment with G-CSF alone reduced ALT levels (101 U/L [range 51-284], p <0.05), liver cell death (TUNEL 1.2% [range 0.4-1.9], p <0.001) and RIPK3 expression (1.9% [range 0.9-2.5] p <0.001). TAK-242 alone reduced ALT levels (to 96.5 U/L [range 45-229], p <0.05), RIPK3 expression (1.7% [range 0.9-2.1], p <0.001) and liver cell death (TUNEL 1.4% [range 0.4-3.3], p <0.001], however, without reaching statistical significance. The combination of G-CSF and TAK-242 was superior to both single treatments and improved ALT levels (to 74.5 U/L [range 44-297]), liver cell death (TUNEL 0.45% [range 0.11-0.95], p <0.001 compared with TAK-242 and p <0.01 compared with G-CSF) and RIPK3 expression (1.23% [range 1-1.78], p <0.001 compared to CCl4+GalN) (Fig. 6B). Furthermore, treatment with TAK-242 + G-CSF was also associated with activation of the STAT3 pathway and a trend to increased anti-apoptotic BCL2 expression as assessed in pooled liver samples by Western Blot (Fig. 6C).
      We also evaluated whether hepatocyte proliferation was modulated by treatment with G-CSF ± TAK-242 in this model. CCl4-treated animals injected with GalN showed high levels of proliferating hepatocytes (Ki67: CCl4 0.1% ± 0.1 vs. CCl4 + GalN 2% ± 1.7; Cyclin A2: CCl4 0.1% ± 0.2 vs. CCl4 + GalN 1.9% ± 1.6) and hepatocytes in cell cycle arrest (p21: CCl4 0.5% ± 0.2 vs. CCl4 + GalN 6.4% ± 4.7). Treatment with G-CSF, with or without TAK-242, reduced the degree of proliferating and senescent hepatocytes, whereas TAK-242 alone did not alter the response to injury (Ki67: CCl4+GalN+TAK-242 3% ± 2.1 vs. CCl4+GalN+TAK-242+G-CSF 1.2% ± 1.4 [p <0.01]; Cyclin A2: CCl4+GalN+TAK-242 1.9% ± 1.6 vs. CCl4+GalN+TAK-242+G-CSF 0.3% ± 0.2 [p <0.001]; p21: CCl4+GalN+TAK-242 7.1% ± 4.5 vs. CCl4+GalN+TAK-242+G-CSF 1.7% ± 2.3 [p <0.001]) (Fig. 6D).
      Taken together these data suggest that in this model with low-grade inflammation, the combination of G-CSF and TAK-242 impacts positively on liver injury, regeneration and reduces markers of senescence. It also confirms that the regenerative effect of G-CSF is preserved in an environment with reduced inflammation.

      Relationship between cell death and liver regeneration and the effect of G-CSF and TAK-242

      To explore the role of cell death as a modulator and inducer of regeneration, we used RIPA56, a selective RIPK1 inhibitor, to prevent GalN-induced necroptotic cell death in the different groups of animals with CCl4-induced chronic liver injury. RIPA56 treatment effectively prevented GalN-driven cell death (total cell death [TUNEL p <0.001] and necroptosis [RIPK3 p <0.001], both compared to CCl4+GalN) and was associated with a decrease in hepatocyte proliferation compared to CCl4+GalN (Ki67 2% ± 1.7 vs. 0.2% ± 0.2, p <0.001; Cyclin A2 1.9% ± 1.6 vs. 0.03% ± 0.1, p <0.001) (Fig. S7), which suggests that in this environment with low-grade inflammation, the regenerative response correlates directly with the severity of liver injury, as previously observed.
      • Bird T.G.
      • Muller M.
      • Boulter L.
      • Vincent D.F.
      • Ridgway R.A.
      • Lopez-Guadamillas E.
      • et al.
      TGFbeta inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.
      We then calculated the ratio between expression of Cyclin A2, as a marker of cell cycle progression, and the severity of total liver cell death (TUNEL) to delineate the pro-regenerative capacity in relation to liver injury of both drugs in all models studied; an increase in the ratio of Cyclin A2/TUNEL would represent enhanced regenerative activity with less cell death and vice versa (Fig. S8). In the 24-hour CCl4-LPS model, LPS completely inhibited regenerative responses (CyclinA2/TUNEL ratio: CCl4 0.42; CCl4+LPS 0.02; CCl4+LPS+G-CSF 0.04), with or without G-CSF. In contrast, in the CCl4-GalN model the regenerative response was preserved (CyclinA2/TUNEL ratio: CCl4+GalN 0.54). In both “short-term” models, administration of TAK-242 was associated with enhanced liver cell regenerative response (CyclinA2/TUNEL ratio: CCl4+LPS+TAK-242 1.4; CCl4+GalN+TAK-242 0.9) supporting the hypothesis that creating an ‘inflammation-free’ environment is important to restore regenerative capacities, as also described recently in a model of CCl4 and Klebsiella pneumonia-induced ACLF.
      • Xiang X.
      • Feng D.
      • Hwang S.
      • Ren T.
      • Wang X.
      • Trojnar E.
      • et al.
      Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming of impaired regeneration pathways in mice.
      Moreover, the anti-inflammatory environment (in the presence of TAK-242) allowed G-CSF to exert its positive effect on liver injury, notably in the CCl4-LPS model, and to enhance regeneration after 5 days of therapy (CyclinA2/TUNEL ratio: CCl4+ LPS+TAK-242+G-CSF 1.7); thus, supporting the pro-regenerative capacity of G-CSF especially after long-term treatment.

      Discussion

      The results of this study provide novel insights into the mechanisms underlying the molecular pathogenesis of ACLF, focusing on the relationship between inflammation and regeneration. The data provide the rationale for combining G-CSF, a recombinant protein that mobilises stem cells from the bone marrow, and a TLR4 inhibitor, TAK-242, that has been shown to reduce hepatic inflammation and mortality in animal models of ACLF. Herein, we show for the first time that in models of LPS- or GalN-induced ACLF, G-CSF and TAK-242 act in combination to improve the severity of liver injury by reducing inflammation and cellular senescence and improving regeneration.
      ACLF has a dismal prognosis,
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      Validation of CLIF-C ACLF score to define a threshold for futility of intensive care support for patients with acute-on-chronic liver failure.
      and its treatment is an urgent unmet need. In ACLF, TLR4-driven inflammation and lack of hepatic regeneration determines the disease course.
      • Engelmann C.
      • Sheikh M.
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      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      ,
      • Dubuquoy L.
      • Louvet A.
      • Lassailly G.
      • Truant S.
      • Boleslawski E.
      • Artru F.
      • et al.
      Progenitor cell expansion and impaired hepatocyte regeneration in explanted livers from alcoholic hepatitis.
      ,
      • Xiang X.
      • Feng D.
      • Hwang S.
      • Ren T.
      • Wang X.
      • Trojnar E.
      • et al.
      Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming of impaired regeneration pathways in mice.
      ,
      • Arroyo V.
      • Moreau R.
      • Jalan R.
      • Gines P.
      • Study E.-C.C.C.
      Acute-on-chronic liver failure: a new syndrome that will re-classify cirrhosis.
      Additionally, DAMPs,
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      The lipopolysaccharide-sensing caspase(s)-4/11 are activated in cirrhosis and are causally associated with progression to multi-organ injury.
      released from non-apoptotic, immunogenic forms of cell death (such as necroptosis), and PAMPs,
      • Engelmann C.
      • Adebayo D.
      • Oria M.
      • De Chiara F.
      • Novelli S.
      • Habtesion A.
      • et al.
      Recombinant alkaline phosphatase prevents acute on chronic liver failure.
      ,
      • Engelmann C.
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      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      derived from infection and/or intestinal bacterial translocation, both initiate a cascade of cytokine-driven inflammatory responses that underlie the pathogenesis of the syndrome in which the TLR4 pathway plays an important role.
      • Engelmann C.
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      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      ,
      • Macdonald S.
      • Andreola F.
      • Bachtiger P.
      • Amoros A.
      • Pavesi M.
      • Mookerjee R.
      • et al.
      Cell death markers in patients with cirrhosis and acute decompensation.
      ,
      • Engelmann C.
      • Claria J.
      • Szabo G.
      • Bosch J.
      • Bernardi M.
      Pathophysiology of decompensated cirrhosis: portal hypertension, circulatory dysfunction, inflammation, metabolism and mitochondrial dysfunction.
      This systemic inflammation is also associated with the release of endogenous G-CSF, which can induce further tissue injury.
      • Claria J.
      • Stauber R.E.
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      • Moreau R.
      • Jalan R.
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      • et al.
      Systemic inflammation in decompensated cirrhosis: characterization and role in acute-on-chronic liver failure.
      ,
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      • Gines P.
      • Study E.-C.C.C.
      Acute-on-chronic liver failure: a new syndrome that will re-classify cirrhosis.
      Paradoxically, G-CSF has been used to treat patients with ACLF, with a view to mobilising the bone marrow stem cell with variable results.
      • Garg V.
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      Granulocyte colony-stimulating factor mobilizes CD34(+) cells and improves survival of patients with acute-on-chronic liver failure.
      ,
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      Although preclinical studies suggested that G-CSF may help in tissue repair,
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      and some clinical trials in patients with ACLF showed promising results,
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      the large multicentre randomised clinical trial of G-CSF failed to show a beneficial effect. In fact, in the subgroup of patients with the most severe inflammation, there were some suggestions that G-CSF may even be deleterious.
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      In a rat model of septic liver injury, 4-day pre-treatment with G-CSF resulted in increased liver expression of LPS-binding protein and TLR4. Administration of LPS to these animals led to enhanced liver cell death and mortality showing a negative sensitising effect of G-CSF.
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      Granulocyte colony stimulating factor induces lipopolysaccharide (LPS) sensitization via upregulation of LPS binding protein in rat.
      However, in human trials, administration of G-CSF to treat sepsis showed no evidence of efficacy but G-CSF did not induce any excess mortality. It is important to note that these patients had no underlying liver disease.
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      Filgrastim in patients with pneumonia and severe sepsis or septic shock.
      When rats were treated with G-CSF in a rodent model of partial (70%) hepatectomy, administration of LPS was associated with severe liver injury and excess mortality compared with partial hepatectomy and LPS injection alone.
      • Liu A.
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      G-CSF pretreatment aggravates LPS-associated microcirculatory dysfunction and acute liver injury after partial hepatectomy in rats.
      These results indicate that G-CSF may sensitise the liver to LPS through TLR4, which may be deleterious in patients with ACLF, as these patients present with high levels of circulating DAMPs and PAMPs, potent ligands for this receptor.
      • Macdonald S.
      • Andreola F.
      • Bachtiger P.
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      • et al.
      Cell death markers in patients with cirrhosis and acute decompensation.
      ,
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      • Affo S.
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      • Sancho-Bru P.
      • et al.
      Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis.
      To better understand the mechanisms underlying the potential deleterious effect of G-CSF, we used murine models of ACLF that mimic the human phenotype. Administration of LPS to mice with advanced fibrosis led to ACLF,
      • Engelmann C.
      • Sheikh M.
      • Sharma S.
      • Kondo T.
      • Loeffler-Wirth H.
      • Zheng Y.B.
      • et al.
      Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.
      ,
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      A model of acute kidney injury in mice with cirrhosis and infection.
      which was associated with severe inflammation, liver injury, hepatocellular senescence, and high mortality rate. G-CSF exaggerated the inflammatory response and further induced cell death by decreasing the BCL2/BAX ratio and hence favouring cell death. Inhibiting TLR4 receptor signalling with TAK-242, which was associated with marked reduction in the severity of systemic and hepatic inflammation, prevented the increased mortality with G-CSF. Furthermore, TAK-242 allowed G-CSF to act through activation of the STAT3 pathway to prevent further cell death by releasing BCL2 and inducing hepatocyte proliferation. These observations may indicate that the combination may indeed be synergistic in their action rather than simply additive. It may be argued that true recovery needs to be proven at multiple levels, which may be true especially for hepatotoxic injuries in acute liver injury. However, ACLF comprises a complex pathogenesis including inflammation, cell death and inadequate regeneration, with predominance of one or the other in different disease stages. We observed that the combinatorial therapy addresses all aspects and preserves tissue integrity exhibited by low ALT levels and 100% survival. G-CSF may also target side effects of TAK-242, which may impair response to pathogens such as phagocytosis increasing the risk of secondary infections.
      • Lv J.
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      • et al.
      TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages.
      However, TAK-242 did not increase the risk of infections in human trials and was shown to be safe in sepsis.
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      A randomized, double-blind, placebo-controlled trial of TAK-242 for the treatment of severe sepsis.
      Nevertheless, any future trial will need to monitor closely for infections when patients are treated with a TLR4 antagonist.
      ACLF is characterised by a lack of regeneration and LPS-driven inflammation is potentially of pathogenic importance.
      • Xiang X.
      • Feng D.
      • Hwang S.
      • Ren T.
      • Wang X.
      • Trojnar E.
      • et al.
      Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming of impaired regeneration pathways in mice.
      In the LPS-precipitated model of ACLF, the number of Ki67+ hepatocytes were markedly reduced despite activation of Cyclin A2. This may be related to the inhibitory effect of hepatocellular senescence on hepatic regeneration, indicated by increased hepatocellular p21 expression. In human liver tissues of patients with ACLF, the presence of infiltrating CD68+ macrophages was associated with low numbers of proliferating hepatocytes
      • Shubham S.
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      Cellular and functional loss of liver endothelial cells correlates with poor hepatocyte regeneration in acute-on-chronic liver failure.
      and in a ACLF mouse model, infection was an important determinant of inhibition of regeneration.
      • Xiang X.
      • Feng D.
      • Hwang S.
      • Ren T.
      • Wang X.
      • Trojnar E.
      • et al.
      Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming of impaired regeneration pathways in mice.
      Taken together, the data support the hypothesis that targeting inflammation may improve regeneration.
      The relationship between TLR4 signalling and cell proliferation is contradictory; previous in vitro work on tumour cell lines indicated a pro-proliferative effect on malignant parenchymal cells,
      • Jiang N.
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      Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells through the Toll-like receptor 4/nuclear factor-kappaB pathway.
      • Zu Y.
      • Ping W.
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      Lipopolysaccharide-induced toll-like receptor 4 signaling in esophageal squamous cell carcinoma promotes tumor proliferation and regulates inflammatory cytokines expression.
      • Yue Y.
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      • Zhang Z.
      • Li L.
      • Liu L.
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      High mobility group box 1/toll-like receptor 4/myeloid differentiation factor 88 signaling promotes progression of gastric cancer.
      whereas TLR4 stimulation by LPS was proposed by others to halt pluripotent progenitor cells’ ability to divide.
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      ,
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      In cirrhotic animals, administration of LPS inhibited regeneration, as evidenced by activation of the IFNy-STAT1 pathway with increased expression of cyclin-dependent kinase complex inhibitor while reducing the expression of Cyclin D1. Additionally, administration of G-CSF to LPS-induced ACLF animals led to markedly increased expression of p21 in the hepatocytes, which was associated with enhanced expression of other senescence mediators such as Serpin1E, p16 and TGFb1. TLR4 signalling also leads to TGFb1 release, which is a classical mediator of the senescence-associated secretory phenotype, activating p21 independently of p53.
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      TGFbeta inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.
      ,
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      Taken together, senescence is an important consequence of LPS-induced ACLF, which is aggravated by G-CSF. These data allow us to hypothesise that senescence and the concomitant inflammatory environment may be involved in the inhibition of hepatic regeneration by senescence in ACLF. The fact that inhibiting the TLR4 pathway by TAK-242 in G-CSF treated animals reduces inflammation, prevents senscence and fosters regeneration further supports this hypothesis. Evidence for the co-existence of pathogen/endotoxin-driven inflammation and lack of regeneration is robust and alcoholic hepatitis, one of the leading precipitating events for ACLF, seems to be one of the potential indications for treatment with this combinatorial therapy. Dubuquoy et al. indicated that livers from patients with alcoholic hepatitis lack sufficient hepatocyte proliferation whilst showing increased proliferation of hepatic progenitor cells and ductular reaction.
      • Dubuquoy L.
      • Louvet A.
      • Lassailly G.
      • Truant S.
      • Boleslawski E.
      • Artru F.
      • et al.
      Progenitor cell expansion and impaired hepatocyte regeneration in explanted livers from alcoholic hepatitis.
      The same disease is also characterised by high levels of circulating LPS and inflammation.
      • Michelena J.
      • Altamirano J.
      • Abraldes J.G.
      • Affo S.
      • Morales-Ibanez O.
      • Sancho-Bru P.
      • et al.
      Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis.
      To further validate our observations and determine whether this pro-regenerative effect could be reproduced in an environment of non-LPS-driven, sterile injury, we created a new model of ACLF using GalN on the background of advanced fibrosis. We confirmed similar effects of the combination therapy in this model. Although G-CSF alone reduced tissue injury, its effect on tissue repair, when combining it with TAK-242, was superior to the individual treatments. This observation shows that TAK-242 overcomes the effect of injury-related release of DAMPs and TLR4 activation, confirming the beneficial effect of the combination therapy even when the superimposed injury is non-LPS related.
      This study is limited by the fact that ACLF is a heterogeneous entity and animal models might mimic only part of the disease spectrum. However, using an inflammatory and a sterile model of ACLF, mimicking extrahepatic and hepatic insults, allowed us to explore the major pathogenic factors involved in disease development and progression. Although our study provides important insight into the inflammation-senescence-regeneration sequence and provides a potential novel therapeutic strategy, further research is needed to understand the involvement of endothelial cells and other subsets of immune cells (such as macrophages), which might modulate regenerative responses; their involvement is likely as indicated in this study by changes in the markers of vascular regeneration, such as angiopoietin, proliferin and platelet-derived growth factor, possibly also acting through modulation of TGFb1 expression.
      • Hu J.
      • Srivastava K.
      • Wieland M.
      • Runge A.
      • Mogler C.
      • Besemfelder E.
      • et al.
      Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat.
      ,
      • Starlinger P.
      • Assinger A.
      Importance of platelet-derived growth factors in liver regeneration.
      It may also be necessary to apply the combinatorial therapy in models where infection induces ACLF
      • Xiang X.
      • Feng D.
      • Hwang S.
      • Ren T.
      • Wang X.
      • Trojnar E.
      • et al.
      Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming of impaired regeneration pathways in mice.
      and models of alcoholic hepatitis to test the TAK-242/G-CSF combination in models mimicking the most frequent precipitating events. The models applied here target mechanistic aspects of pathogenetic changes, which occur throughout the whole spectrum of ACLF, providing the basis for translation into humans and for future mechanistic studies.
      Although the clinical course of ACLF is not confounded by sex, mechanistic preclinical studies should ideally include both male and female animals. Herein, we chose to use male animals only because the model applied in this study was previously developed in male animals.
      In conclusion, the results of this study provide new evidence for the importance of the LPS-TLR4 pathway in modulating the systemic and hepatic inflammation that is associated with ACLF, as well in inducing hepatocyte senescence and inhibiting regeneration. We report the novel observation that the combinatorial therapy of TLR4 inhibition using TAK-242 and G-CSF reduces liver injury and improves hepatocyte proliferation through reduction in inflammation and senescence. This overcomes the inhibition of hepatic regeneration, a characteristic feature of ACLF. As both drugs are known to be safe, they can be repurposed and evaluated in combination for patients with ACLF.

      Abbreviations

      ACLF, acute-on-chronic liver failure; ALT, alanine aminotransferase; BAX, BCL2 associated X protein; BCL2, B-cell lymphoma 2 protein; CCl4, carbon tetrachloride; CK19, cytokeratin 19; DAMP, damage-associated molecular pattern; GalN, galactosamine; G-CSF, granulocyte-colony stimulating factor; HMGB1, high mobility group box-1; LPS, lipopolysaccharide; OD, optical density; PAMP, pathogen-associated molecular pattern; PMA, phorbol 12-myristate-13-acetate; SASP, senescence-associated secretory phenotype; TLR4, Toll-like receptor 4; TGFb1, transforming growth factor beta-1.

      Financial support

      The German Research Foundation (DFG) ( EN 1100/2-1 ) and Berlin Institute of Health (BIH) funded Cornelius Engelmann. This study was also received funding by a grant from the European Union Horizon 2020 research and innovation programme under grant agreement number 954096.

      Authors’ contributions

      RJ, TB, FA, CE, FA, ND, SJF - contributed to the conception and design of the study. RJ, ND, FA - provided administrative, study supervision, obtained funding, material support. CE, MF, SFG, FA, AK, SN, LH, MH, AF - performed experiments and substantially contributed to the acquisition of data and its analysis. All authors were involved in the interpretation of data. CE drafted the manuscript. All authors revised the manuscript critically for important intellectual content.

      Data availability statement

      Data are available on request from the corresponding author.

      Conflict of interest

      Rajiv Jalan has research collaborations with Yaqrit. Rajiv Jalan is the founder of Yaqrit Limited, which is developing UCL inventions for treatment of patients with cirrhosis. Rajiv Jalan is an inventor of ornithine phenylacetate, which was licensed by UCL to Mallinckrodt. He is also a Founder of Hepyx Ltd. and Cyberliver Ltd. Cornelius Engelmann has received advisory fees from Novartis and CSL Behring. He is shareholder of Hepyx Ltd. Fausto Andreola is shareholder of Hepyx Ltd. Rajiv Jalan, Cornelius Engelmann, Fausto Andreola and Thomas Berg are the named inventors on the patents surrounding the use of G-TAK in ACLF, which have been filed as a priority application. This patent has been licensed to Hepyx Ltd.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgement

      We would like to thank Alexander Hettich for creating the graphical abstract.

      Supplementary data

      The following are the supplementary data to this article:

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