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Impaired mitochondrial complex IV respiration in peripheral blood mononuclear cells discriminates acute-on-chronic liver failure from acute decompensation

  • Author Footnotes
    # The authors have contributed equally to this study.
    Mona-May Langer
    Footnotes
    # The authors have contributed equally to this study.
    Affiliations
    Department for Gastroenterology and Hepatology, University Hospital Essen, University Duisburg-Essen, Germany

    Department of Internal Medicine II, LMU University Hospital Munich, Germany
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  • Author Footnotes
    # The authors have contributed equally to this study.
    Chantal Eickelmann
    Footnotes
    # The authors have contributed equally to this study.
    Affiliations
    Institute for Pathophysiology, West German Heart and Vascular Centre, University of Essen Medical School, University Duisburg-Essen, Germany
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  • Author Footnotes
    # The authors have contributed equally to this study.
    Petra Kleinbongard
    Footnotes
    # The authors have contributed equally to this study.
    Affiliations
    Institute for Pathophysiology, West German Heart and Vascular Centre, University of Essen Medical School, University Duisburg-Essen, Germany
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  • Author Footnotes
    # The authors have contributed equally to this study.
    Christian M. Lange
    Correspondence
    Corresponding author. Address: Department of Internal Medicine II, LMU University Hospital Munich, Marchioninistr. 15, D-81337 München, Germany. Tel.: 0049 89 4400 75292, fax: 0049 89 4400 78887.
    Footnotes
    # The authors have contributed equally to this study.
    Affiliations
    Department for Gastroenterology and Hepatology, University Hospital Essen, University Duisburg-Essen, Germany

    Department of Internal Medicine II, LMU University Hospital Munich, Germany
    Search for articles by this author
  • Author Footnotes
    # The authors have contributed equally to this study.
      To the Editor:
      Systemic inflammation is discussed as a new paradigm in the development of acute decompensation (AD) and acute-on-chronic liver failure (ACLF).
      • D'Amico G.
      • Bernardi M.
      • Angeli P.
      Towards a new definition of decompensated cirrhosis.
      • Moreau R.
      • Jalan R.
      • Gines P.
      • Pavesi M.
      • Angeli P.
      • Cordoba J.
      • et al.
      Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis.
      • Trebicka J.
      • Fernandez J.
      • Papp M.
      • Caraceni P.
      • Laleman W.
      • Gambino C.
      • et al.
      The PREDICT study uncovers three clinical courses of acutely decompensated cirrhosis that have distinct pathophysiology.
      • Arroyo V.
      • Angeli P.
      • Moreau R.
      • Jalan R.
      • Claria J.
      • Trebicka J.
      • et al.
      The systemic inflammation hypothesis: towards a new paradigm of acute decompensation and multiorgan failure in cirrhosis.
      Systemic inflammation can result in dysregulation of mitochondrial bioenergetics in leukocytes,
      • Calvano S.E.
      • Xiao W.
      • Richards D.R.
      • Felciano R.M.
      • Baker H.V.
      • Cho R.J.
      • et al.
      A network-based analysis of systemic inflammation in humans.
      and, reciprocally, impaired mitochondrial function contributes causally to dysfunctional immunity.
      • Weinberg S.E.
      • Sena L.A.
      • Chandel N.S.
      Mitochondria in the regulation of innate and adaptive immunity.
      Two elegant studies by Moreau et al. and Zhang et al., published in the Journal of Hepatology, have revealed that AD and in particular ACLF are associated with mitochondrial dysfunction in organs and immune cells.
      • Moreau R.
      • Claria J.
      • Aguilar F.
      • Fenaille F.
      • Lozano J.J.
      • Junot C.
      • et al.
      Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF.
      ,
      • Zhang I.W.
      • Curto A.
      • Lopez-Vicario C.
      • Casulleras M.
      • Duran-Guell M.
      • Flores-Costa R.
      • et al.
      Mitochondrial dysfunction governs immunometabolism in leukocytes of patients with acute-on-chronic liver failure.
      In detail, an untargeted serum metabolome analysis by Moreau et al. revealed a metabolite fingerprint of ACLF indicating a rise in the blood levels of fatty acylcarnitines due to depressed mitochondrial ß-oxidation in peripheral organs, which seems to be paralleled by increased extra-mitochondrial glucose and amino acid metabolism.
      • Moreau R.
      • Claria J.
      • Aguilar F.
      • Fenaille F.
      • Lozano J.J.
      • Junot C.
      • et al.
      Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF.
      Zhang et al. performed an in-depth analysis of mitochondrial metabolism and ultrastructure in peripheral blood mononuclear cells (PBMCs) of patients with AD or ACLF, which identified cristae rarefication and mitochondrial swelling, as well as an impaired tricarboxylic acid cycle associated with alternative energy production from carbon and nitrogen sources.
      • Zhang I.W.
      • Curto A.
      • Lopez-Vicario C.
      • Casulleras M.
      • Duran-Guell M.
      • Flores-Costa R.
      • et al.
      Mitochondrial dysfunction governs immunometabolism in leukocytes of patients with acute-on-chronic liver failure.
      However, these studies left open whether the described changes are accompanied by a reduction in mitochondrial respiration itself. We therefore analyzed in detail the function of mitochondrial respiration complexes in PBMCs of patients with AD or ACLF. Detailed methods are described in the supplementary methods.
      Patient characteristics are shown in Table S1. One can note a typical clinical profile of patients with AD or ACLF, with the exception of high albumin levels in patients with ACLF. This is explained by the fact that some patients with ACLF had received albumin supplementation during hospitalization before progression to ACLF. Baseline respiration was slightly decreased in PBMCs of patients with AD compared to healthy controls, and significantly decreased in PBMCs of patients with ACLF (Fig. 1A). By administration of exogenous ADP complex I, respiration in these patients could be largely restored (Fig. 1B). Uncoupled respiration was not different in PBMCs of patients with AD or ACLF, compared to healthy controls (Fig. 1C). Complex II respiration, measured after stimulation of complex II by the administration of succinate and after inhibition of complex I by rotenone, was neither affected in PBMCs of patients with AD or ACLF, compared to healthy controls (Fig. 1D, E). In contrast, a profound reduction of the residual respiration in PBMCs of patients with ACLF was observed after inhibition of complex III with antimycin A, whereas PBMCs of patients with AD had similar levels of residual respiration like healthy controls (Fig. 1F). Furthermore, complex IV respiration could be only partially rescued by stimulation with ascorbate and N,N,N′,N′-tetra-methyl-p-phenylene diamine (TMPD), indicating significant impairment of complex IV in ACLF (Fig. 1G). Quantification of ATP in PBMCs revealed that the ATP content in PBMCs of patients with AD and ACLF is significantly decreased compared to PBMCs of healthy controls, indicating impaired energy supply and/or high energy consumption in the PBMCs of these patients (Fig. 1H).
      Figure thumbnail gr1
      Fig. 1Impaired mitochondrial respiration and ATP content in PBMCs of patients with ACLF.
      (A-G) Mitochondrial respiration of PBMCs in patients with AD or ACLF, compared to healthy controls was measured by O2k (Oroboros, Innsbruck, AT), as described in detail in the . Graphs show individual values with mean (bars) ± SEM. One-way ANOVA or Kruskal-Wallis test were used as appropriate after Normality Test. ∗p ≤0.05, ∗∗p ≤0.01, ∗∗∗p ≤0.001, ∗∗∗∗p ≤0.0001. (A) Baseline respiration. (B) ADP-stimulated complex I respiration. (C) Uncoupled respiration. (D) complex I+II, and (E) complex II respiration. (F) Residual respiration. (G) Complex IV respiration. (H) ATP content of PBMCs. (I) Correlation matrix of selected respiration values and laboratory values of patients suffering from AD and ACLF showing spearman r values and levels of significance. (J) Principal component analysis of the components of mitochondrial respiration shown in A-H of patients with AD (light blue) and ACLF (dark blue). ACLF, acute-on-chronic liver failure; AD, acute decompensation; CRP, C-reactive protein; CLIF OF, CLIF organ failure score; MELD, model for end-stage liver disease; PBMCs, peripheral blood mononuclear cells. (This figure appears in color on the web.)
      Collectively, baseline, residual and complex IV respiration are reduced in PBMCs of patients with ACLF, whereas complex I, uncoupled and complex II respiration are just slightly reduced in PBMCs of patients with AD and ACLF compared to healthy controls. Remarkably, impaired complex IV respiration discriminates ACLF from AD, while deterioration of other respiration complexes shows rather a continuum with progressive disease severity. Since a high proportion of patients with ACLF had alcohol-related cirrhosis, we compared residual and complex IV respiration in patients with vs. without alcohol-related cirrhosis. No significant differences between both patient groups were observed, suggesting that our findings are specific for ACLF and not for alcohol-related liver disease (residual respiration in alcohol-related vs. other cirrhosis: 2.45 +/- 2.37 vs. 4.20 +/- 2.59; p = 0.2; complex IV respiration in alcohol-related vs. other cirrhosis: 58.75 +/- 26.59 vs. 68.15 +/- 33.74; p = 0.9). In line with this notion, Fig. 1I-J show that residual and complex IV respiration correlate inversely with the CLIF organ failure score, but not with the model for end-stage liver disease score and albumin, and that mitochondrial respiration in ACLF is clearly distinct from AD. In this regard, a specific role for mitochondrial complex IV dysfunction was linked to immunosuppression and fate of effector T cells,
      • Tarasenko T.N.
      • Pacheco S.E.
      • Koenig M.K.
      • Gomez-Rodriguez J.
      • Kapnick S.M.
      • Diaz F.
      • et al.
      Cytochrome c oxidase activity is a metabolic checkpoint that regulates cell fate decisions during T cell activation and differentiation.
      a typical feature of ACLF.
      • Rueschenbaum S.
      • Ciesek S.
      • Queck A.
      • Widera M.
      • Schwarzkopf K.
      • Brune B.
      • et al.
      Dysregulated adaptive immunity is an early event in liver cirrhosis preceding acute-on-chronic liver failure.
      Overall, our findings underline and extend those of Zhang et al., who described that there is not a general “shutdown” of mitochondrial function in PBMCs from patients with ACLF. However, in line with the findings of Moreau et al. and Zhang et al., our finding of decreased ATP content in PBMCs from patients with AD or ACLF shows that immune cells from patients with AD and ACLF suffer from reduced energy production and underlines that inflammation is energetically expensive.

      Financial support

      This study was supported by the Deutsche Forschungsgemeinschaft (INST 20876/390-1 to CML).

      Authors’ contributions

      MML, CE, PK, CML: study design, sample collection, data acquisition, statistical analysis, interpretation of data, manuscript writing.

      Conflicts of interest

      The authors have no conflicts of interest to disclose.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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