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Innate immune cells in cirrhosis

  • Christine Bernsmeier
    Correspondence
    University Centre for Gastrointestinal and Liver Diseases, Petersgraben 4, 4031 Basel, Switzerland
    Affiliations
    Department of Biomedicine, University of Basel, Switzerland

    University Centre for Gastrointestinal and Liver Diseases, Basel, Switzerland
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  • Schalk van der Merwe
    Correspondence
    Department of Gastroenterology and Hepatology, University Hospital Gasthuisberg, 49 Here street, 3000 Leuven, Belgium
    Affiliations
    Laboratory of Hepatology, Department of Chronic Diseases, Metabolism and aging (CHROMETA), University of Leuven, Leuven, Belgium

    Department of Gastroenterology and Hepatology, University Hospital Gasthuisberg, Leuven, Belgium
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  • Axel Périanin
    Correspondence
    Corresponding authors. Addresses: INSERM1149, Centre de Recherche sur l'Inflammation, Faculté Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. Tel.: +33 157277473
    Affiliations
    INSERM U1149, Centre de Recherche sur l'Inflammation, Paris, France

    UMRS1149, Université Paris Diderot-Paris 7, Paris, France

    Centre National de la Recherche Scientifique (CNRS), Paris, France
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Published:March 30, 2020DOI:https://doi.org/10.1016/j.jhep.2020.03.027

      Summary

      Cirrhosis is a multisystemic disease wherein inflammatory responses originating from advanced liver disease and its sequelae affect distant compartments. Patients with cirrhosis are susceptible to bacterial infections, which may precipitate acute decompensation and acute-on-chronic liver failure, both of which are associated with high short-term mortality. Innate immune cells are an essential first line of defence against pathogens. Activation of liver macrophages (Kupffer cells) and resident mastocytes generate proinflammatory and vaso-permeating mediators that induce accumulation of neutrophils, lymphocytes, eosinophils and monocytes in the liver, and promote tissue damage. During cirrhosis progression, damage- and pathogen-associated molecular patterns activate immune cells and promote development of systemic inflammatory responses which may involve different tissues and compartments. The antibacterial function of circulating neutrophils and monocytes is gradually and severely impaired as cirrhosis worsens, contributing to disease progression. The mechanisms underlying impaired antimicrobial responses are complex and incompletely understood. This review focuses on the continuous and distinct perturbations arising in innate immune cells during cirrhosis, including their impact on disease progression, as well as reviewing potential therapeutic targets.

      Keywords

      Linked Article

      • Macrophage markers and innate immunity in cirrhosis
        Journal of HepatologyVol. 73Issue 6
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          We read with great interest the review article “Innate immune cells in cirrhosis” by Bernsmeier et al.1 We strongly agree on the important role of innate immune cells, especially monocytes and macrophages viz. Kupffer cells, in the development and progression of liver cirrhosis and complications such as acute decompensation (AD) and acute-on-chronic liver failure (ACLF). Even though the review is very comprehensive, we think it is missing a section on macrophage biomarkers to further support the role of macrophages in cirrhosis development and progression including complications.
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      Introduction

      Cirrhosis is a multisystem disease that develops as a result of chronic liver injury when healthy parenchyma is progressively replaced by fibrotic tissue. The consequences of cirrhosis stem not only from hepatocyte loss or portal hypertension but also from continuous local and systemic inflammation that further perpetuates liver injury and leads to increased susceptibility to infection and risk of organ failure.
      From a clinical perspective, analysis of natural history studies showed that prognosis in cirrhosis correlated with clinically recognisable stages, as patients progressed from compensated to decompensated cirrhosis. In compensated cirrhosis, in the absence of ascites or bleeding oesophageal varices, liver-related mortality remained low, but it dramatically increased as ascites, variceal bleeding and bacterial infections developed.
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      (Fig. 1A).
      Figure thumbnail gr1
      Fig. 1Current concept of diverse innate immune cell actions in various tissues and compartments in the context of cirrhosis-associated immune dysfunction.
      (A) Evolution of clinical stages of cirrhosis and the associated prognosis. Clinical stages of cirrhosis defined by the Child-Pugh Score (upper arrow), the clinical classification suggested by D'Amico et al. (middle arrow,
      • D'Amico G.
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      Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 118 studies.
      ) and the onset of AD and ACLF (lower arrow,
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      Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis.
      ) which can evolve at any stage following a precipitating event and directly increase disease severity. Although AD and ACLF carry a high mortality, there is a possibility of re-compensation. (B) Conceptual evolution of the pathophysiology of cirrhosis-associated innate immune dysfunction in relation to the complex pathophysiology of cirrhosis progression and its clinical stages. During progression of fibrosis, portal hypertension and its associated clinical manifestations, the risk for episodes of infection increases and relates to the development of cirrhosis-associated immune dysfunction. This involves numerous immune cells and non-cellular components (see symbol legend) in various compartments. Briefly, chronic inflammation, parenchymal damage and scaring of the liver activate local tissue inflammation and contribute to a systemic inflammatory response mediated by cell debris, DAMPs, cytokines and reverse migration of immunoregulatory innate immune cells. Concurrently, endothelial- and vascular barrier dysfunction of the gut relates to pathological bacterial translocation and endotoxemia. Microbial products and PAMPs further enhance the hepatic inflammatory response and differentiation of immune cells in the gut, the liver, the circulation and other organs such as the peritoneum. Differentiation of diverse innate immune cells is specific not only for the underlying aetiology but also to the stages of cirrhosis severity and their compartment. The pathophysiological processes are highly complex and the figure relates to the most relevant findings only (see text for details). Adapted from.
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      ACLF, acute-on-chronic liver failure; AD, acute decompensation; CLD, chronic liver disease; DAMPs, damage-associated molecular patterns; HVPG, hepatic venous pressure gradient; PAMPs, pathogen-associated molecular patterns; PHT, portal hypertension.
      A new concept that has evolved over the last decades centres around the view of cirrhosis as a multisystem disease, with immune dysfunction central to its evolution.
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      According to this concept, immune dysfunction in cirrhosis can be viewed as a continuum that starts with the onset of chronic hepatic inflammation, worsens with the development of cirrhosis and portal hypertension, is further aggravated by bacterial translocation and may finally culminate in complete immune exhaustion in acute-on-chronic liver failure (ACLF
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      Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis.
      ) (Fig. 1B).
      Immune dysfunction in cirrhosis mainly involves components of the innate immune system. This system is widely distributed throughout human tissue compartments and represents the first line of defence against pathogens. Innate immunity is critical to maintain homeostasis by preventing microbe invasion, by regulating appropriate responses to sterile injury, and by shaping adaptive immunity through antigen presentation. The innate immune system is composed of physical and chemical barriers, involving humoral and cell-mediated components. In cirrhosis, knowledge has been accumulating regarding the involvement of subsets of innate immune cells in immune dysfunction. Evidence now suggests that specifically neutrophils, monocytes, macrophages and dendritic cells and to a lesser extent eosinophils, basophils, mastocytes, innate lymphoid cells, and mucosal-associated invariant T (MAIT) cells actively contribute to inflammation and susceptibility to infection.
      In this review, we aim to elaborate on the importance of innate immune cells in cirrhosis and the associated multisystem inflammatory state. We introduce the concept that the natural history of cirrhosis and the development of its complications can be explained by evolving immune dysfunction from the onset of chronic liver disease to end-stage decompensated cirrhosis; we propose that a better understanding of immune dysfunction at the molecular level may help to develop novel therapeutic approaches.
      Immune activation in chronic liver disease evolves from chronic liver injury of any aetiology, long before the onset of fibrosis or cirrhosis. In parallel to inflammation restorative immune cell populations infiltrate the liver. However, if resolution of inflammation does not occur, cirrhosis evolves characterised by hepatic and systemic inflammation.

      Neutrophils

      Neutrophil host defence activities

      Neutrophils represent the largest family of circulating leucocytes and are the first cells mobilised during tissue injury. They play a key role in inflammation and host defence against pathogens (bacteria, yeasts, fungi). Their anti-infectious function requires various rapid and temporally coordinated activities which are triggered upon activation of specific receptors, regulating neutrophil migration into tissues, pathogen sensing, recognition and killing.
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      This step is essential for diapedesis, a short process (<10 min) regulated by various chemoattractants (C5a, Paf, LTB4, n-formylated peptides, IL8, IL6, SDF1α), which control neutrophil migration speed (chemokinesis), polarity and direction (chemotaxis).
      Once in tissues, neutrophils capture pathogens either by direct interaction with them or indirectly via Fc receptors, FcγRIIA (CD32) and FcγRIIIB (CD16), and complement receptors, CR1 (CD35) and CR3, and CD11b/CD18 integrin. Signalling events triggered by these opsonin receptors induce a series of coordinated antibacterial responses such as the engulfment of pathogens into phagosomes (phagocytosis) and the release of antibacterial agents stored in azurophilic, specific and tertiary granules (degranulation or exocytosis) including proteases (elastase, cathepsin G, lysozyme), myeloperoxidase (MPO), and defensins. Simultaneously, NADPH oxidase 2 (NOX2) complex is activated at the plasma membrane and massively generates superoxide anion (O2.-) respiratory burst) which is essential for bacterial killing.
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      ,
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      • Winterbourn C.C.
      • Nauseef W.M.
      Myeloperoxidase: a front-line defender against phagocytosed microorganisms.
      Superoxide is immediately converted into more toxic reactive oxygen species (ROS), i.e. OH-, H2O2, 1O2 (singlet oxygen), hypochloric acid (HOCl)/bleach water, (NaOCl) and chloramine (R-NHCL). H2O2 is used by MPO to halogenate proteins, which enhances bactericidal activity.
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      • Winterbourn C.C.
      • Nauseef W.M.
      Myeloperoxidase: a front-line defender against phagocytosed microorganisms.
      Finally, large strands of decondensed DNA are extruded from dying neutrophils, carrying with them mainly cationic proteins of cytosolic and granular origin (elastase, cathepsin G, lactoferrin, MPO). These structures called neutrophil extracellular traps (NETs) capture and destroy pathogens.
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      In healthy individuals, the defence activities of neutrophils are harmoniously coordinated and regulated, leading to rapid bacterial eradication, resolution of inflammation and tissue preservation. During cirrhosis development, neutrophil responses are modified over time, leading to compromised bacterial elimination and disease progression.

      Neutrophils move from an aggressive to a paralysed state during cirrhosis development

      During cirrhosis development (Child-Pugh A, B and C), systemic inflammation persists with high serum levels of proinflammatory cytokines (TNFα, IL-1β, IL-6, IL-17, IL-18, interferon-gamma [IFNγ]), soluble receptors (sTNFRI, IL1sRI, IL1Ra, sCD14, Fas-R), endothelial activation markers (ICAM-1, VCAM-1, vascular endothelial growth factor [VEGF], nitrates/nitrites) and neutrophils overexpressing CD11b and CD62L (LSEL) but decreased L-selectins.
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      degranulation,
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      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      ,
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ROS production,
      • Rolas L.
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      • Djerdjouri B.
      • Elkrief L.
      • et al.
      Inhibition of mammalian target of rapamycin aggravates the respiratory burst defect of neutrophils from decompensated patients with cirrhosis.
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      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
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      bacterial killing
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      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      ,
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
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      • Francés R.
      • Shah N.
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      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
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      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
      and tend to worsen during disease progression from Child-Pugh A to C, as shown for phagocytosis
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      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      ,
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      • Abeles R.D.
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      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
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      Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis.
      This is consistent with weak antibacterial activities (ROS production, MPO release, bacterial killing) induced by formylpeptide in neutrophils from patients with acute decompensation (AD) (Child-Pugh C) and high levels of white blood cells (WBCs 7.6–9.6×103 cell/μl).
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      • Makhezer N.
      • Hadjoudj S.
      • El-Benna J.
      • Djerdjouri B.
      • Elkrief L.
      • et al.
      Inhibition of mammalian target of rapamycin aggravates the respiratory burst defect of neutrophils from decompensated patients with cirrhosis.
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      Patients with AD show more marked exhaustion of neutrophil bacterial capacity compared to patients with cirrhosis and alcoholic hepatitis (Child-Pugh C; WBC count 14.1×103 cells/μl),
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ,
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      but not to those with ACLF (Child-Pugh C, WBC count 9.2×103cells/μl
      • Taylor N.J.
      • Manakkat Vijay G.K.
      • Abeles R.D.
      • Auzinger G.
      • Bernal W.
      • Ma Y.
      • et al.
      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
      ). These neutrophil modifications were not related to the aetiology of cirrhosis (hepatitis B/C, alcohol-related liver disease [ALD]) nor gastrointestinal bleeding and serum levels of glutamic-pyruvate transaminase (GPT) or alkaline phosphatase.
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      ,
      • Garfia C.
      • García-Ruiz I.
      • Solís-Herruzo J.A.
      Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis.
      ,
      • Khanam A.
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      Paradoxically, basal neutrophil ROS production is increased in some decompensated patients
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      • Taylor N.J.
      • Manakkat Vijay G.K.
      • Abeles R.D.
      • Auzinger G.
      • Bernal W.
      • Ma Y.
      • et al.
      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
      who had the highest risk of developing multiple organ failure and complications (Child-Pugh B/C, WBC count 8–14×103cells/μl).
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      • Taylor N.J.
      • Manakkat Vijay G.K.
      • Abeles R.D.
      • Auzinger G.
      • Bernal W.
      • Ma Y.
      • et al.
      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
      This “primed” basal activity indicates a pre-activation state of neutrophils, and was suggested to deplete neutrophil energy.
      • Sipeki N.
      • Antal-Szalmas P.
      • Lakatos P.L.
      • Papp M.
      Immune dysfunction in cirrhosis.
      However, its intensity remained relatively weak and raised questions about its relevance to bacterial killing, which requires massive ROS production. The increased basal ROS production during disease progression and defective phagocytic activity of neutrophils in decompensated patients were associated with a greater risk of infection, organ failure and short-term mortality.
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      ,
      • Taylor N.J.
      • Manakkat Vijay G.K.
      • Abeles R.D.
      • Auzinger G.
      • Bernal W.
      • Ma Y.
      • et al.
      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
      In the damaged neutrophils of patients with decompensated cirrhosis (Child-Pugh C), major signalling defects were identified, affecting phospholipase C activity,
      • Garfia C.
      • García-Ruiz I.
      • Solís-Herruzo J.A.
      Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis.
      the activation of AKT, MAP kinases (ERK1/2 and p38-MAPK)
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      and a major NOX2 component, phospho-p47phox(S345).
      • Rolas L.
      • Makhezer N.
      • Hadjoudj S.
      • El-Benna J.
      • Djerdjouri B.
      • Elkrief L.
      • et al.
      Inhibition of mammalian target of rapamycin aggravates the respiratory burst defect of neutrophils from decompensated patients with cirrhosis.
      NOX2 expression (gp91phox+p22phox) is also strongly reduced due to the external degradation of gp91phox by high elastase levels in patient serum
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      ,
      • Stanley A.J.
      • MacGregor I.R.
      • Dillon J.F.
      • Bouchier I.A.
      • Hayes P.C.
      Neutrophil activation in chronic liver disease.
      and by impairment of a mammalian target of rapamycin (mTOR)-dependent NOX2 synthesis process
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      and mTOR expression.
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      This deficiency may raise concerns about mTOR metabolic activities and about mTOR antagonist (rapamycin)-based therapy of immunocompromised patients. A defective IL33/ST2 signalling pathway associated with overexpression of G protein-coupled receptor kinase 2 (GRK2) was involved in the defective IL8-mediated migration of neutrophils from patients with decompensated cirrhosis (Child-Pugh B/C, WBC count 6–11×103cells/μl).
      • Artru F.
      • Bou Saleh M.
      • Maggiotto F.
      • Lassailly G.
      • Ningarhari M.
      • Demaret J.
      • et al.
      IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.
      The impairment of neutrophil's defence function comprises both intracellular and extracellular alterations. The intrinsic neutrophil impairments reported above (Table 1) can be reproduced with pathological concentrations of various agents present in patient serum such as bilirubin,
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      hyperammonemia and hyponatremia,
      • Shawcross D.L.
      • Wright G.A.K.
      • Stadlbauer V.
      • Hodges S.J.
      • Davies N.A.
      • Wheeler-Jones C.
      • et al.
      Ammonia impairs neutrophil phagocytic function in liver disease.
      elastase,
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      ,
      • Stanley A.J.
      • MacGregor I.R.
      • Dillon J.F.
      • Bouchier I.A.
      • Hayes P.C.
      Neutrophil activation in chronic liver disease.
      prostaglandin E2 (PGE2
      • O'Brien A.J.
      • Fullerton J.N.
      • Massey K.A.
      • Auld G.
      • Sewell G.
      • James S.
      • et al.
      Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2.
      ), LPS
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      ,
      • Macnaughtan J.
      • Mookerjee R.P.
      • van der Merwe S.
      • Jalan R.
      Lipopolysaccharide-induced neutrophil dysfunction following Transjugular Intrahepatic Portosystemic Stent Shunt (TIPSS) Insertion is associated with organ failure and mortality.
      or anti-inflammatory cytokines (IL-4, IL-10, transforming growth factor-beta [TGFβ]). These deficiencies are likely to be aggravated in ALD
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      since ethanol by itself causes transphosphatidylation, a phospholipase D-catalysed reaction which generates a biologically inactive metabolite, phosphatidylethanol, instead of phosphatic acid.
      • Exton J.H.
      Regulation of phospholipase D.
      Transphosphatidylation inhibits most of the antibacterial activities of neutrophils.
      • Exton J.H.
      Regulation of phospholipase D.
      Of note, most neutrophil deficiencies are also observed during sepsis, in patients without cirrhosis,
      • Alves-Filho J.C.
      • Spiller F.
      • Cunha F.Q.
      Neutrophil paralysis in sepsis.
      consistent with deleterious effects of bacterial endotoxins on neutrophil function.
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      ,
      • Markwick L.J.L.
      • Riva A.
      • Ryan J.M.
      • Cooksley H.
      • Palma E.
      • Tranah T.H.
      • et al.
      Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis.
      Extrinsic alterations were observed for phagocytosis due to a lack of bacterial opsonization
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      or a low level of serum complement.
      • Potter B.J.
      • Trueman A.M.
      • Jones E.A.
      Serum complement in chronic liver disease.
      Impaired neutrophil migration was related to defective chemoattractant activity
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ,
      • Van Epps D.E.
      • Strickland R.G.
      • Williams R.C.
      Inhibitors of leukocyte chemotaxis in alcoholic liver disease.
      or the presence of a serum inhibitor.
      • DeMeo A.N.
      • Andersen B.R.
      Defective chemotaxis associated with a serum inhibitor in cirrhotic patients.
      Increased expression of adhesion molecules (ICAM/VCAM) by endothelial cells
      • Albillos A.
      • Lario M.
      • Alvarez-Mon M.
      Cirrhosis-associated immune dysfunction: distinctive features and clinical relevance.
      and neutrophils (CD62L (LSEL) or CD11b) may explain the neutrophil hyper adhesion and subsequent defective transmigration,
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      and increased patient susceptibility to infection.
      Table 1Intrinsic alterations of neutrophil antibacterial activities in decompensated cirrhosis.
      Functional/signalling activitiesPathophysiological consequencesCirrhosis stageReferences
      Migration
       Rolling: ↓L-Selectins↑ Neutrophilia“Organ failure”Rosenbloom et al., JAMA 1995
      • Rosenbloom A.J.
      • Pinsky M.R.
      • Bryant J.L.
      • Shin A.
      • Tran T.
      • Whiteside T.
      Leukocyte activation in the peripheral blood of patients with cirrhosis of the liver and SIRS. Correlation with serum interleukin-6 levels and organ dysfunction.
       ↓ Adhesion:Neutrophil marginationStable

      Decompensated
      Tritto et al., J Hepatol 2011
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      ;

      DeMeo et al., NEJM 1972
      • DeMeo A.N.
      • Andersen B.R.
      Defective chemotaxis associated with a serum inhibitor in cirrhotic patients.
       ↑ CD11b

       ↑ CD11b, ↓ CD62L (LSEL)
      “Organ failure”

      Advanced
      Rosenbloom et al., JAMA 1995,
      • Rosenbloom A.J.
      • Pinsky M.R.
      • Bryant J.L.
      • Shin A.
      • Tran T.
      • Whiteside T.
      Leukocyte activation in the peripheral blood of patients with cirrhosis of the liver and SIRS. Correlation with serum interleukin-6 levels and organ dysfunction.


      Fiuza et al., Clin Diagn Lab Immunol 2002
      • Fiuza C.
      • Salcedo M.
      • Clemente G.
      • Tellado J.M.
      Granulocyte colony-stimulating factor improves deficient in vitro neutrophil transendothelial migration in patients with advanced liver disease.
       ↓ CD11bDecompensatedMarkwick et al., Gastro 2015
      • Markwick L.J.L.
      • Riva A.
      • Ryan J.M.
      • Cooksley H.
      • Palma E.
      • Tranah T.H.
      • et al.
      Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis.
       ↓ TransmigrationAdvancedFiuza et al., J Inf Dis 2000
      • Fiuza C.
      • Salcedo M.
      • Clemente G.
      • Tellado J.M.
      In vivo neutrophil dysfunction in cirrhotic patients with advanced liver disease.
       ↓ Chemotaxis/chemokinesis

       ↓ CXCR2
      ↑ Bacterial infectionAdvanced

      Decompensated

      Decompensated
      Fiuza et al., Clin Diagn Lab Immunol 2002,

      Artru F, et al. J Hepatol, 2020
      • Artru F.
      • Bou Saleh M.
      • Maggiotto F.
      • Lassailly G.
      • Ningarhari M.
      • Demaret J.
      • et al.
      IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.


      Artru F, et al. J Hepatol, 2020
      • Artru F.
      • Bou Saleh M.
      • Maggiotto F.
      • Lassailly G.
      • Ningarhari M.
      • Demaret J.
      • et al.
      IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.
      Degranulation↑Bacterial infectionND

      AD
      Rajkovic & Williams, Hepatol 1986
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ;

      Boussif et al., J Hepatol 2016
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      ROS production
       ↑ Basal Production↑ Energy depletionSevere

      Stable

      Western ACLF
      Mookerjee et al., Hepatol 2007
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      ;

      Tritto et al., J Hepatol 2011
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      ;

      Taylor et al., AP&T 2014
      • Taylor N.J.
      • Manakkat Vijay G.K.
      • Abeles R.D.
      • Auzinger G.
      • Bernal W.
      • Ma Y.
      • et al.
      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
       ↓ gp91phox/p22phox level

       ↓P-p47phox
      ↑ Bacterial infectionND

      Decompensated

      AD

      AD
      Rajkovic & Williams, Hepatol 1986
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ;

      Garfia et al., J Hepatol 2004
      • Garfia C.
      • García-Ruiz I.
      • Solís-Herruzo J.A.
      Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis.
      ;

      Rolas et al., Gut 2018
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.


      Rolas et al., Hepatol 2013
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • et al.
      Neutrophil extracellular traps kill bacteria.
      Phagocytosis↑ Bacterial infection
       ↓ Engulfment, ↓CD16Advanced

      Stable

      Stable advanced

      Stable, Western ACLF
      Rajkovic & Williams, Hepatol 1986
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ;

      Fiuza et al., J Inf Dis 2000,
      • Fiuza C.
      • Salcedo M.
      • Clemente G.
      • Tellado J.M.
      In vivo neutrophil dysfunction in cirrhotic patients with advanced liver disease.


      Tritto et al., J Hepatol 2011
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Francés R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      ;

      Markwick et al., Gastro 2015
      • Markwick L.J.L.
      • Riva A.
      • Ryan J.M.
      • Cooksley H.
      • Palma E.
      • Tranah T.H.
      • et al.
      Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis.


      Taylor et al., AP&T 2014
      • Stanley A.J.
      • MacGregor I.R.
      • Dillon J.F.
      • Bouchier I.A.
      • Hayes P.C.
      Neutrophil activation in chronic liver disease.
       ↓ Bacterial killingRajkovic & Williams, Hepatol 1986
      • Rajkovic I.A.
      • Williams R.
      Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.
      ;

      Boussif et al., J Hepatol 2016
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      NET↑ Bacterial infectionDecompensatedAgraz-Cibrian et al., J Inf Dev Count 2016
      • Agraz-Cibrian J.M.
      • Segura-Ortega J.E.
      • Delgado-Rizo V.
      • Fafutis-Morris M.
      Alterations in neutrophil extracellular traps is associated with the degree of decompensation of liver cirrhosis.
      Signalling↑ Bacterial infection
       ↓ PLCDecompensatedGarfia et al., J Hepatol 2004
      • Garfia C.
      • García-Ruiz I.
      • Solís-Herruzo J.A.
      Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis.
       ↓P-AKT, ↓P-ERK1/2, ↓P-p38 MAPK

       ↓mTOR expression

       ↑ GRK2 expression
      AD

      AD

      Decompensated
      Boussif et al., J Hepatol 2016
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.


      Rolas et al., Gut 2018
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.


      Artru et al., J Hepatol, 2020
      • Taylor N.J.
      • Manakkat Vijay G.K.
      • Abeles R.D.
      • Auzinger G.
      • Bernal W.
      • Ma Y.
      • et al.
      The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality.
      During the development of cirrhosis, neutrophils are subjected to biochemical changes and impairment of their anti-infectious function which worsens according to the disease severity. In the severe stages of cirrhosis (acute decompensation), many cellular activities that support host defence function are highly deficient including the parameters regulating the diapedesis, bacterial recognition and various killing activities including bacterial engulfment/phagocytosis, degranulation (exocytosis), the massive and rapid production of reactive oxygen species (respiratory burst) and NETs. These functional deficiencies are associated with impaired intracellular signalling and protein expression, and partly explain the high susceptibility of patients to microbial infections. AD, acute decompensation; NET, neutrophil extracellular trap; PLC, phospholipase C; ERK, extracellular signal-regulated kinase 1/2; MAPK, mitogen-activated protein kinases; GRK2, G protein-coupled receptor kinase 2; ND, not determined.

      Therapeutic approaches to improve neutrophil antibacterial responses and reduce susceptibility to infection

      Recent ex vivo studies with potent TLR7/8 agonists (CL097, R848) show that the deficient degranulation, ROS production and bacterial killing by neutrophils from patients with cirrhosis can be rapidly corrected in vitro providing evidence that intrinsic deficiencies are reversible in decompensated alcohol-related cirrhosis (Child-Pugh C)
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      (Fig. 2). These TLR agonists stimulate NOX2 synthesis via an mTOR-dependent mechanism
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      and potentiate signalling pathways (AKT, MAPK, p47phox).
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      In an in vivo rat model of cirrhosis, R848 decreased bacterial infection and improved animal survival, leading to it being patented.
      • Périanin A.
      • Rolas L.
      Methods and Pharmaceutical Compositions for The Treatment of Bacterial Infections in Patients Suffering From Cirrhosis.
      Stimulation of granulopoiesis by granulocyte colony-stimulating factor (G-CSF) increased circulating neutrophil counts
      • Fiuza C.
      • Salcedo M.
      • Clemente G.
      • Tellado J.M.
      Granulocyte colony-stimulating factor improves deficient in vitro neutrophil transendothelial migration in patients with advanced liver disease.
      ,
      • Moreau R.
      • Rautou P.-E.
      G-CSF therapy for severe alcoholic hepatitis: targeting liver regeneration or neutrophil function?.
      and transendothelial migration.
      • Fiuza C.
      • Salcedo M.
      • Clemente G.
      • Tellado J.M.
      Granulocyte colony-stimulating factor improves deficient in vitro neutrophil transendothelial migration in patients with advanced liver disease.
      ,
      • Garg V.
      • Garg H.
      • Khan A.
      • Trehanpati N.
      • Kumar A.
      • Sharma B.C.
      • et al.
      Granulocyte colony-stimulating factor mobilizes CD34(+) cells and improves survival of patients with acute-on-chronic liver failure.
      Other indirect strategies demonstrating benefit were based on endotoxin removal from patient plasma,
      • Mookerjee R.P.
      • Stadlbauer V.
      • Lidder S.
      • Wright G.A.K.
      • Hodges S.J.
      • Davies N.A.
      • et al.
      Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.
      albumin supplementation,
      • Irvine K.M.
      • Ratnasekera I.
      • Powell E.E.
      • Hume D.A.
      Causes and consequences of innate immune dysfunction in cirrhosis.
      ,
      • O'Brien A.J.
      • Fullerton J.N.
      • Massey K.A.
      • Auld G.
      • Sewell G.
      • James S.
      • et al.
      Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2.
      and treatment with probiotics
      • Stadlbauer V.
      • Mookerjee R.P.
      • Hodges S.
      • Wright G.A.K.
      • Davies N.A.
      • Jalan R.
      Effect of probiotic treatment on deranged neutrophil function and cytokine responses in patients with compensated alcoholic cirrhosis.
      and branched-chain amino acids.
      • Nakamura I.
      Impairment of innate immune responses in cirrhotic patients and treatment by branched-chain amino acids.
      The neutrophil antibacterial function becomes more and more deficient as cirrhosis worsens and is associated with defective signalling and synthesis of major effector proteins (mTOR, NOX2).
      Figure thumbnail gr2
      Fig. 2Proposed models to reverse the deficient neutrophil antibacterial activity in cirrhosis.
      In severe cirrhosis, human neutrophil antibacterial activities are highly deficient due to impaired signalling events (phosphorylation of MAPK, AKT, p47phox) and protein expression such as mTOR and NOX2 (gp91phox/p22phox), which is responsible for ROS production.
      • Rolas L.
      • Makhezer N.
      • Hadjoudj S.
      • El-Benna J.
      • Djerdjouri B.
      • Elkrief L.
      • et al.
      Inhibition of mammalian target of rapamycin aggravates the respiratory burst defect of neutrophils from decompensated patients with cirrhosis.
      ,
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      Treatment of neutrophils of patients with cirrhosis with potent TLR7/8 agonists (R848/CL097) stimulates major signalling events, NOX2 synthesis and the bactericidal activity induced by the bacterial formyl-peptide fMLP.
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      ,
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      The R848 restorative property was confirmed in vivo on ROS production by blood granulocytes from a rat model of cirrhosis, with beneficial effects on bacterial infection and animal survival.
      • Périanin A.
      • Rolas L.
      Methods and Pharmaceutical Compositions for The Treatment of Bacterial Infections in Patients Suffering From Cirrhosis.
      ROS, reactive oxygen species.
      The pharmacological restoration of neutrophil's ROS-dependent antibacterial function
      • Boussif A.
      • Rolas L.
      • Weiss E.
      • Bouriche H.
      • Moreau R.
      • Périanin A.
      Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.
      ,
      • Rolas L.
      • Boussif A.
      • Weiss E.
      • Lettéron P.
      • Haddad O.
      • El-Benna J.
      • et al.
      NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
      ,
      • Markwick L.J.L.
      • Riva A.
      • Ryan J.M.
      • Cooksley H.
      • Palma E.
      • Tranah T.H.
      • et al.
      Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis.
      reveals a potential approach to treat or prevent infection in cirrhosis,
      • Périanin A.
      • Rolas L.
      Methods and Pharmaceutical Compositions for The Treatment of Bacterial Infections in Patients Suffering From Cirrhosis.
      which could have a particular role as adjunct therapy against antibiotic-resistant bacteria. Liver function could also benefit since ROS mediate macrophage M2 polarisation and orchestrate liver repair.
      • Yang W.
      • Tao Y.
      • Wu Y.
      • Zhao X.
      • Ye W.
      • Zhao D.
      • et al.
      Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair.
      Deciphering the molecular basis of cirrhosis-associated protein depletion in neutrophils (adhesion molecules, mTOR, NOX2) is important for the development of interventions that preserve liver function and reduce severely affected patients' susceptibility to infection.
      The deficient antimicrobial activities of neutrophils in decompensated cirrhosis can be reversed in vitro and in vivo with potent TLR7/8 agonists (R848) that stimulate signalling and protein synthesis (NOX2), and bacterial elimination.

      Monocytes

      Mononuclear phagocytes include monocytes, macrophages and dendritic cells and play a pivotal role in initiating innate immune responses. Their functions involve phagocytosis and killing of bacteria, antigen presentation, inflammatory cytokine production in response to bacterial challenge, as well as recruitment and activation of immune effector cells. Phagocytes regulate antimicrobial defence, perpetuation and cessation of inflammation and tissue injury, fibrogenesis and tumourigenesis, explaining their central role in the pathophysiology of immune dysfunction in patients with cirrhosis.

      Regulation of monocytes along different stages of cirrhosis progression

      Circulating monocytes

      The relationship between disease-specific differentiation/dysfunction of circulating monocytes and morbidity/mortality has been described in patients with AD and ACLF (compared to stable cirrhosis) and less frequently in those with chronic liver disease and compensated cirrhosis (compared to healthy individuals).
      The first report of monocyte dysfunction in patients with cirrhosis goes back to a letter from 1979,
      • Hassner A.
      • Kletter Y.
      • Jedvab M.
      • Aronson M.
      • Shibolet S.
      Impaired monocyte function in liver cirrhosis.
      delineating the impaired phagocytic capacity of yeast in a study involving patients with biopsy-proven cirrhosis. Wasmuth et al. were the first to discover reduced expression of a major histocompatibility complex (MHC) class II receptor (human leucocyte antigen [HLA]-DR) on circulating monocytes from patients with ACLF, also showing that this reduced expression was associated with adverse prognosis.
      • Wasmuth H.E.
      • Kunz D.
      • Yagmur E.
      • Timmer-Stranghöner A.
      • Vidacek D.
      • Siewert E.
      • et al.
      Patients with acute on chronic liver failure display ‘sepsis-like’ immune paralysis.
      A substantial number of studies have since corroborated these findings.
      • Lin C.-Y.
      • Tsai I.-F.
      • Ho Y.-P.
      • Huang C.-T.
      • Lin Y.-C.
      • Lin C.-J.
      • et al.
      Endotoxemia contributes to the immune paralysis in patients with cirrhosis.
      • Xing T.
      • Li L.
      • Cao H.
      • Huang J.
      Altered immune function of monocytes in different stages of patients with acute on chronic liver failure.
      • Berres M.-L.
      • Schnyder B.
      • Yagmur E.
      • Inglis B.
      • Stanzel S.
      • Tischendorf J.J.W.
      • et al.
      Longitudinal monocyte human leukocyte antigen-DR expression is a prognostic marker in critically ill patients with decompensated liver cirrhosis.
      • Zimmermann H.W.
      • Seidler S.
      • Nattermann J.
      • Gassler N.
      • Hellerbrand C.
      • Zernecke A.
      • et al.
      Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.
      • Berry P.
      • Antoniades C.
      • Carey I.
      • McPhail M.
      • Hussain M.
      • Davies E.
      • et al.
      Severity of the compensatory anti-inflammatory response determined by monocyte HLA-DR expression may assist outcome prediction in cirrhosis.
      • Peter J.
      • Frey O.
      • Stallmach A.
      • Bruns T.
      Attenuated antigen-specific T cell responses in cirrhosis are accompanied by elevated serum interleukin-10 levels and downregulation of HLA-DR on monocytes.
      • Gadd V.L.
      • Patel P.J.
      • Jose S.
      • Horsfall L.
      • Powell E.E.
      • Irvine K.M.
      Altered peripheral blood monocyte phenotype and function in chronic liver disease: Implications for hepatic recruitment and systemic inflammation.
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      • Satsangi S.
      • Duseja A.
      • Sachdeva M.
      • Tomer S.
      • Arora S.K.
      • Taneja S.
      • et al.
      Monocyte human leukocyte antigen - antigen D related, neutrophil oxidative burst and cytokine analysis in patients of decompensated cirrhosis with and without acute-on chronic liver failure.
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      Monocytes with reduced HLA-DR expression were shown to gradually decrease with severity of cirrhosis,
      • Lin C.-Y.
      • Tsai I.-F.
      • Ho Y.-P.
      • Huang C.-T.
      • Lin Y.-C.
      • Lin C.-J.
      • et al.
      Endotoxemia contributes to the immune paralysis in patients with cirrhosis.
      ,
      • Xing T.
      • Li L.
      • Cao H.
      • Huang J.
      Altered immune function of monocytes in different stages of patients with acute on chronic liver failure.
      ,
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      ,
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      and were functionally linked to reduced production of TNF-α and IL-6 in response to LPS.
      • Wasmuth H.E.
      • Kunz D.
      • Yagmur E.
      • Timmer-Stranghöner A.
      • Vidacek D.
      • Siewert E.
      • et al.
      Patients with acute on chronic liver failure display ‘sepsis-like’ immune paralysis.
      ,
      • Lin C.-Y.
      • Tsai I.-F.
      • Ho Y.-P.
      • Huang C.-T.
      • Lin Y.-C.
      • Lin C.-J.
      • et al.
      Endotoxemia contributes to the immune paralysis in patients with cirrhosis.
      ,
      • Berres M.-L.
      • Schnyder B.
      • Yagmur E.
      • Inglis B.
      • Stanzel S.
      • Tischendorf J.J.W.
      • et al.
      Longitudinal monocyte human leukocyte antigen-DR expression is a prognostic marker in critically ill patients with decompensated liver cirrhosis.
      ,
      • Berry P.
      • Antoniades C.
      • Carey I.
      • McPhail M.
      • Hussain M.
      • Davies E.
      • et al.
      Severity of the compensatory anti-inflammatory response determined by monocyte HLA-DR expression may assist outcome prediction in cirrhosis.
      ,
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      ,
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      Some authors postulated a role for the anti-inflammatory cytokine IL-10 in monocyte modulation towards this “endotoxin tolerant”
      • Pena O.M.
      • Pistolic J.
      • Raj D.
      • Fjell C.D.
      • Hancock R.E.W.
      Endotoxin tolerance represents a distinctive state of alternative polarization (M2) in human mononuclear cells.
      population,
      • Wasmuth H.E.
      • Kunz D.
      • Yagmur E.
      • Timmer-Stranghöner A.
      • Vidacek D.
      • Siewert E.
      • et al.
      Patients with acute on chronic liver failure display ‘sepsis-like’ immune paralysis.
      ,
      • Lin C.-Y.
      • Tsai I.-F.
      • Ho Y.-P.
      • Huang C.-T.
      • Lin Y.-C.
      • Lin C.-J.
      • et al.
      Endotoxemia contributes to the immune paralysis in patients with cirrhosis.
      ,
      • Berry P.
      • Antoniades C.
      • Carey I.
      • McPhail M.
      • Hussain M.
      • Davies E.
      • et al.
      Severity of the compensatory anti-inflammatory response determined by monocyte HLA-DR expression may assist outcome prediction in cirrhosis.
      ,
      • Peter J.
      • Frey O.
      • Stallmach A.
      • Bruns T.
      Attenuated antigen-specific T cell responses in cirrhosis are accompanied by elevated serum interleukin-10 levels and downregulation of HLA-DR on monocytes.
      ,
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      however, this mechanism likely involves multiple cytokines, endotoxins and cellular signals.
      • Berres M.-L.
      • Schnyder B.
      • Yagmur E.
      • Inglis B.
      • Stanzel S.
      • Tischendorf J.J.W.
      • et al.
      Longitudinal monocyte human leukocyte antigen-DR expression is a prognostic marker in critically ill patients with decompensated liver cirrhosis.
      ,
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      ,
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      ,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      In response to extracellular binding of pathogenic peptides, HLA-DR elicits T cell responses by activating the T cell receptor. HLA-DR expression is increased upon classical or alternative immune stimulation, illustrating its role as a monocyte activation marker. By contrast, HLA-DR downregulation occurs in states of acquired immunosuppressive monocyte differentiation induced by various stimuli.
      • Gordon S.
      • Taylor P.R.
      Monocyte and macrophage heterogeneity.
      Endotoxemia, shown to be present in advanced cirrhosis,
      • Zapater P.
      • Francés R.
      • González-Navajas J.M.
      • de la Hoz M.A.
      • Moreu R.
      • Pascual S.
      • et al.
      Serum and ascitic fluid bacterial DNA: a new independent prognostic factor in noninfected patients with cirrhosis.
      leads to immediate loss of circulating HLA-DR+ cells in the circulation, while replenishment with and differentiation of monocytes may occur within 24 h under homeostatic conditions.
      • Patel A.A.
      • Zhang Y.
      • Fullerton J.N.
      • Boelen L.
      • Rongvaux A.
      • Maini A.A.
      • et al.
      The fate and lifespan of human monocyte subsets in steady state and systemic inflammation.
      Reduced expression of HLA-DR on monocytic cells in advanced cirrhosis was recently attributed to the generation of monocytic myeloid-derived suppressor cells (M-MDSCs, CD14+CD15−CD11b+HLA-DR−)
      • Bronte V.
      • Brandau S.
      • Chen S.-H.
      • Colombo M.P.
      • Frey A.B.
      • Greten T.F.
      • et al.
      Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.
      in response to systemic inflammatory response syndrome (SIRS) and circulating pathogen-associated molecular patterns (PAMPs). Functionally M-MDSCs in cirrhosis were typified by distinct immunosuppressive properties, i.e. reduced T cell activation, TNF-α/IL-6 production in response to TLR stimulation and phagocytic capacity.
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      The number of circulating human monocytes was increased in relation to cirrhosis severity from Child-Pugh A to C and AD, respectively.
      • Zimmermann H.W.
      • Seidler S.
      • Nattermann J.
      • Gassler N.
      • Hellerbrand C.
      • Zernecke A.
      • et al.
      Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.
      ,
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      Monocytes have classically been subdivided into 3 subsets: CD14++CD16− (classical, migratory), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical, patrolling monocytes).
      • Jakubzick C.V.
      • Randolph G.J.
      • Henson P.M.
      Monocyte differentiation and antigen-presenting functions.
      A recent study revisiting monocyte taxonomy using single-cell RNA sequencing (scRNAseq) identified a substantial genetic heterogeneity of the CD14++CD16+ subset.
      • Villani A.-C.
      • Satija R.
      • Reynolds G.
      • Sarkizova S.
      • Shekhar K.
      • Fletcher J.
      • et al.
      Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors.
      Interestingly, an expansion of CD14++CD16+ monocytes has been observed in the circulation and the liver of patients with cirrhosis progression.
      • Zimmermann H.W.
      • Seidler S.
      • Nattermann J.
      • Gassler N.
      • Hellerbrand C.
      • Zernecke A.
      • et al.
      Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.
      ,
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      Recently, TAM receptors (protein tyrosine kinase 3 [TYRO-3], anexelekto [AXL], Mer receptor tyrosine kinase [MERTK]), which are important regulators of innate immune homeostasis that inhibit TLR signalling pathways and promote phagocytic removal of apoptotic cells,
      • Rothlin C.V.
      • Ghosh S.
      • Zuniga E.I.
      • Oldstone M.B.A.
      • Lemke G.
      TAM receptors are pleiotropic inhibitors of the innate immune response.
      have been implicated in innate immune dysfunction in cirrhosis. In parallel with advancing cirrhosis and portal hypertension prior to AD and ACLF, a circulating immunoregulatory population of distinct AXL-expressing (CD14+HLA-DR+AXL+) monocytes expanded. These monocytes were characterised by preserved phagocytic capacity but reduced T cell activation and TNF-α/IL-6 production in response to TLR stimulation.
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      In AD and ACLF, the emergence of a MERTK-expressing circulating monocyte population (CD14+HLA-DR+MERTK+) that dampened innate immune responses to microbial challenge was associated with disease severity and adverse outcomes,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      an important finding that has subsequently been confirmed.
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      ,
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      RNA-seq analysis has shown that monocytes in ACLF have a distinct transcriptional profile compared to those in decompensated cirrhosis. It was also shown recently that glutamine metabolism regulated monocyte functions, such as phagocytic capacity and cytokine production.
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      As outlined, the phagocytic capacity of monocytes seems to be maintained throughout disease progression to decompensated cirrhosis, but was reduced at the stage of AD and ACLF potentially due to the expansion of M-MDSCs.
      • Gadd V.L.
      • Patel P.J.
      • Jose S.
      • Horsfall L.
      • Powell E.E.
      • Irvine K.M.
      Altered peripheral blood monocyte phenotype and function in chronic liver disease: Implications for hepatic recruitment and systemic inflammation.
      ,
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      ,
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      ,
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      ,
      • Liaskou E.
      • Zimmermann H.W.
      • Li K.-K.
      • Oo Y.H.
      • Suresh S.
      • Stamataki Z.
      • et al.
      Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics.
      In contrast to neutrophils, bacterial killing and oxidative burst capacity have been differentially reported, but seemed to remain preserved throughout all stages of cirrhosis, except for monocytes from patients with alcohol-related liver disease and active drinking.
      • Gadd V.L.
      • Patel P.J.
      • Jose S.
      • Horsfall L.
      • Powell E.E.
      • Irvine K.M.
      Altered peripheral blood monocyte phenotype and function in chronic liver disease: Implications for hepatic recruitment and systemic inflammation.
      ,
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      ,
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      ,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      ,
      • Bruns T.
      • Peter J.
      • Hagel S.
      • Herrmann A.
      • Stallmach A.
      The augmented neutrophil respiratory burst in response to Escherichia coli is reduced in liver cirrhosis during infection.
      ,
      • Vergis N.
      • Khamri W.
      • Beale K.
      • Sadiq F.
      • Aletrari M.O.
      • Moore C.
      • et al.
      Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase.
      The differentiation of circulating monocytes in patients with cirrhosis is continually adapting to the current inflammatory milieu, which is characterised by continual PAMP and DAMP exposure that increases with disease severity, activation and death of other circulating immune cells and SIRS at the stage of infection and subsequent AD and ACLF. The modulation of the milieu over time seems to generate immunoregulatory monocytic populations that expand over the course of compensated to decompensated disease, and then immunosuppressive populations that expand with the onset of liver failure. The hypothetical aim of monocyte plasticity in cirrhosis is to restore systemic and compartmental immune homeostasis but this increases the risk of systemic or compartmental infectious complications (Fig. 3).
      In advanced cirrhosis and ACLF distinct immunoregulatory and immunosuppressive populations of circulating monocytic cells evolve in relation to disease severity, amongst CD14++CD16+, M-MDSC, and monocytes expressing AXL and MERTK, respectively.
      Figure thumbnail gr3
      Fig. 3Differentiation of circulating monocytic cells with cirrhosis progression.
      Under healthy homeostatic conditions monocytic cells in the blood are heterogenous. Classically they have been subdivided into CD14++CD16− (classical), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical) subsets,
      • Jakubzick C.V.
      • Randolph G.J.
      • Henson P.M.
      Monocyte differentiation and antigen-presenting functions.
      recently a classification by presence and absence of an array of cell surface markers was suggested.
      • Murray P.J.
      • Allen J.E.
      • Biswas S.K.
      • Fisher E.A.
      • Gilroy D.W.
      • Goerdt S.
      • et al.
      Macrophage activation and polarization: nomenclature and experimental guidelines.
      Paralleling progression of cirrhosis and portal hypertension distinct monocytic subsets accumulated and displaced functionally intact monocytic cells.
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      Subset differentiation appeared to be plastic and evolved context and stage specific. While monocytic cell differentiation in the compensated stage barely differed from healthy conditions, distinct immunoregulatory and immunosuppressive monocytic subsets emerged in decompensated stages and in AD/ACLF and may relate to increased infection susceptibility. Immunosuppressive M-MDSCs expanded during progression to decompensated stages (Child B/C) and represented half of all circulating CD14+ cells in AD/ACLF.
      • Wasmuth H.E.
      • Kunz D.
      • Yagmur E.
      • Timmer-Stranghöner A.
      • Vidacek D.
      • Siewert E.
      • et al.
      Patients with acute on chronic liver failure display ‘sepsis-like’ immune paralysis.
      ,
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      ,
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      During decompensation distinct stage-specific immunoregulatory subsets expanded such as CD14++CD16+,
      • Liaskou E.
      • Zimmermann H.W.
      • Li K.-K.
      • Oo Y.H.
      • Suresh S.
      • Stamataki Z.
      • et al.
      Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics.
      ,
      • Zimmermann H.W.
      • Bruns T.
      • Weston C.J.
      • Curbishley S.M.
      • Liaskou E.
      • Li K.-K.
      • et al.
      Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver.
      CD14+CD16highHLA-DRhighAXL+
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      in a predominantly inflammatory milieu, and CD14+CD16highHLA-DRhighCD163+MERTK+
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      ,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      in a predominantly tolerogenic milieu such as AD/ACLF. ACLF, acute-on-chronic liver failure; AD, acute decompensation; CARS, compensatory anti-inflammatory response syndrome; DAMPs, damage-associated molecular patterns; M-MDSCs, monocytic myeloid-derived suppressor cells; PAMPs, pathogen-associated molecular patterns; SIRS, systemic inflammatory response.

      Liver infiltrating monocytes

      Monocytes in the liver may represent circulating monocytes trafficking through the sinusoids (CD14+CD68−). Moreover, migrating monocytes either remain as monocytes within the tissue, acquiring antigen-presenting capability, or mature into macrophages.
      • Jakubzick C.V.
      • Randolph G.J.
      • Henson P.M.
      Monocyte differentiation and antigen-presenting functions.
      ,
      • Strauss O.
      • Dunbar P.R.
      • Bartlett A.
      • Phillips A.
      The immunophenotype of antigen presenting cells of the mononuclear phagocyte system in normal human liver--a systematic review.
      ,
      • Weston C.J.
      • Zimmermann H.W.
      • Adams D.H.
      The role of myeloid-derived cells in the progression of liver disease.
      In the cirrhotic liver, CD14++CD16+ monocytes were increased, accumulated at sites of inflammation/fibrosis and were characterised by enhanced phagocytosis, antigen presentation, T cell activation, and secretion of chemokines, growth factors, proinflammatory and profibrogenic mediators (that activated hepatic stellate cells [HSCs]). CD14++CD16+ cells hereby contributed to the perpetuation of hepatic inflammation and fibrogenesis.
      • Zimmermann H.W.
      • Seidler S.
      • Nattermann J.
      • Gassler N.
      • Hellerbrand C.
      • Zernecke A.
      • et al.
      Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.
      ,
      • Liaskou E.
      • Zimmermann H.W.
      • Li K.-K.
      • Oo Y.H.
      • Suresh S.
      • Stamataki Z.
      • et al.
      Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics.
      Their presence was due to increased CX3CR1 dependent migratory potential, and maturation from CD14++CD16− monocytes. It has been proposed that proinflammatory CD14++CD16+ cells may transmigrate back to the circulation and contribute to SIRS responses, whereas anti-inflammatory monocytes remain in the liver, suppress T cells and promote endotoxin tolerance.
      • Zimmermann H.W.
      • Seidler S.
      • Nattermann J.
      • Gassler N.
      • Hellerbrand C.
      • Zernecke A.
      • et al.
      Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.
      ,
      • Liaskou E.
      • Zimmermann H.W.
      • Li K.-K.
      • Oo Y.H.
      • Suresh S.
      • Stamataki Z.
      • et al.
      Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics.
      ,
      • Aspinall A.I.
      • Curbishley S.M.
      • Lalor P.F.
      • Weston C.J.
      • Blahova M.
      • Liaskou E.
      • et al.
      CX(3)CR1 and vascular adhesion protein-1-dependent recruitment of CD16(+) monocytes across human liver sinusoidal endothelium.
      ,
      • Zimmermann H.W.
      • Bruns T.
      • Weston C.J.
      • Curbishley S.M.
      • Liaskou E.
      • Li K.-K.
      • et al.
      Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver.
      Triggering receptor expressed on myeloid cells 1 (TREM-1) was highly upregulated on circulating monocytes, monocyte-derived macrophages and Kupffer cells (KCs) in patients with fibrosis and promoted hepatic inflammation and fibrosis in a mouse model of cirrhosis.
      • Nguyen-Lefebvre A.T.
      • Ajith A.
      • Portik-Dobos V.
      • Horuzsko D.D.
      • Arbab A.S.
      • Dzutsev A.
      • et al.
      The innate immune receptor TREM-1 promotes liver injury and fibrosis.
      TREM-2 expression was observed on monocytes, macrophages and also neutrophils in fibrous septa, sinusoids and areas of inflammation in human cirrhotic liver.
      • Perugorria M.J.
      • Esparza-Baquer A.
      • Oakley F.
      • Labiano I.
      • Korosec A.
      • Jais A.
      • et al.
      Non-parenchymal TREM-2 protects the liver from immune-mediated hepatocellular damage.
      Recently, Rachamandran et al. investigated monocyte and macrophage heterogeneity using scRNAseq in healthy and cirrhotic human livers and defined a novel scar-associated profibrogenic TREM2+CD9+ macrophage subpopulation, that differentiated from circulating monocytes.
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

      Potential immunotherapy by targeting monocytes

      Given the association of monocyte dysfunction with infectious complications that may accelerate morbidity and mortality from cirrhosis, several potential targets for immunotherapy have been defined. There are a number of proof of concept studies, but few treatment options are under clinical evaluation (Table 2). Concepts and substances with potential therapeutic use are inhibitors of TAM receptors (AXL/MERTK),
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      ,
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      immune stimulant poly(I:C)
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      and glutamine synthase.
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      Clinical studies have evaluated a potential role for albumin in restoring monocyte function.
      • O'Brien A.J.
      • Fullerton J.N.
      • Massey K.A.
      • Auld G.
      • Sewell G.
      • James S.
      • et al.
      Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2.
      ,
      • China L.
      • Maini A.
      • Skene S.S.
      • Shabir Z.
      • Sylvestre Y.
      • Colas R.A.
      • et al.
      Albumin counteracts immune-suppressive effects of lipid mediators in patients with advanced liver disease.
      Therapy with the glycoprotein G-CSF is a potentially interesting concept to improve liver regeneration and innate immune responses in cirrhosis by releasing bone marrow-derived CD34+ haematopoietic stem cells in order to replace dysfunctional circulating innate immune cells (granulocytes, monocytes, dendritic cells) with functional counterparts. Clinical investigations revealed differential results in respect to fibrosis resolution, regeneration, liver function, adverse events and survival.
      • Garg V.
      • Garg H.
      • Khan A.
      • Trehanpati N.
      • Kumar A.
      • Sharma B.C.
      • et al.
      Granulocyte colony-stimulating factor mobilizes CD34(+) cells and improves survival of patients with acute-on-chronic liver failure.
      ,
      • Kedarisetty C.K.
      • Anand L.
      • Bhardwaj A.
      • Bhadoria A.S.
      • Kumar G.
      • Vyas A.K.
      • et al.
      Combination of granulocyte colony-stimulating factor and erythropoietin improves outcomes of patients with decompensated cirrhosis.
      • Verma N.
      • Kaur A.
      • Sharma R.
      • Bhalla A.
      • Sharma N.
      • De A.
      • et al.
      Outcomes after multiple courses of granulocyte colony-stimulating factor and growth hormone in decompensated cirrhosis: a randomized trial.
      • Newsome P.N.
      • Fox R.
      • King A.L.
      • Barton D.
      • Than N.-N.
      • Moore J.
      • et al.
      Granulocyte colony-stimulating factor and autologous CD133-positive stem-cell therapy in liver cirrhosis (REALISTIC): an open-label, randomised, controlled phase 2 trial.
      In view of these results, selective patients with ACLF may benefit from G-CSF while compensated patients did not; subsequent studies are ongoing.
      Table 2Potential future immunotherapeutic strategies to modulate monocyte function in cirrhosis.
      TargetSubstanceTherapeutic conceptCirrhosis stageReferences
      TAM receptors (AXL, MERTK)UNC569, BGB324Inhibition of MERTK (UNC569) restored LPS-induced proinflammatory response in ACLF ex vivo; inhibition of AXL (BGB324) restored proinflammatory cytokine responses in cirrhosis ex vivoACLF (UNC569); Child B/C (BGB324)Bernsmeier et al., Gastro 2015
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      ; Brenig et al., Life Sci Alliance 2019
      • Brenig R.
      • Pop O.T.
      • Triantafyllou E.
      • Geng A.
      • Singanayagam A.
      • Perez-Shibayama C.
      • et al.
      Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.
      M-MDSCPolyI:CTLR-3 agonist poly(I:C) reduced proportions of immunosuppressive M-MDSC and augmented their antimicrobial function in vitroACLFBernsmeier et al., Gut 2018
      • Bernsmeier C.
      • Triantafyllou E.
      • Brenig R.
      • Lebosse F.J.
      • Singanayagam A.
      • Patel V.C.
      • et al.
      CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.
      Glutamine synthaseMethionine sulfoximineInhibition of glutamine synthase restored TNF-α production and phagocytosis in ACLF-plasma conditioned monocytes in vitroACLFKorf et al., Gut 2019
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      PGE2AlbuminProstaglandin E2 levels decreased in vivo and improved TNF-α production in conditioned MoMFADO'Brien Nature Med 2013
      • O'Brien A.J.
      • Fullerton J.N.
      • Massey K.A.
      • Auld G.
      • Sewell G.
      • James S.
      • et al.
      Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2.
      ; China et al., Clin Gastro Heptol 2019
      • China L.
      • Maini A.
      • Skene S.S.
      • Shabir Z.
      • Sylvestre Y.
      • Colas R.A.
      • et al.
      Albumin counteracts immune-suppressive effects of lipid mediators in patients with advanced liver disease.
      Bone marrow-derived stem cellsG-CSFMobilises CD34+ haematopoietic stem cells from the bone marrow, with the capacity to differentiate into multiple cell lineages and replace dysfunctional circulating innate immune cells (granulocytes, monocytes, dendritic cells)Eastern ACLF (49), decompensated (87, 88), compensated (89)Garg et al., Gastro 2012
      • Garg V.
      • Garg H.
      • Khan A.
      • Trehanpati N.
      • Kumar A.
      • Sharma B.C.
      • et al.
      Granulocyte colony-stimulating factor mobilizes CD34(+) cells and improves survival of patients with acute-on-chronic liver failure.
      ; Kedarisetty et al., Gastro 2015
      • Kedarisetty C.K.
      • Anand L.
      • Bhardwaj A.
      • Bhadoria A.S.
      • Kumar G.
      • Vyas A.K.
      • et al.
      Combination of granulocyte colony-stimulating factor and erythropoietin improves outcomes of patients with decompensated cirrhosis.
      ; Verma et al. Hepatology 2018
      • Verma N.
      • Kaur A.
      • Sharma R.
      • Bhalla A.
      • Sharma N.
      • De A.
      • et al.
      Outcomes after multiple courses of granulocyte colony-stimulating factor and growth hormone in decompensated cirrhosis: a randomized trial.
      ; Newsome et al., Lancet 2018
      • Newsome P.N.
      • Fox R.
      • King A.L.
      • Barton D.
      • Than N.-N.
      • Moore J.
      • et al.
      Granulocyte colony-stimulating factor and autologous CD133-positive stem-cell therapy in liver cirrhosis (REALISTIC): an open-label, randomised, controlled phase 2 trial.
      Studies revealing proof of concept for immunotherapeutic strategies: TAM receptor (AXL/MERTK) inhibition, immune stimulant poly(I:C) and glutamine synthase. Clinical studies evaluating a potential role for albumin and G-CSF. ACLF, acute-on-chronic liver failure; AXL, anexelekto; G-CSF; granulocyte colony-stimulating factor; MERTK, myeloid-epithelial-reproductive tyrosine kinase; M-MDSC, monocytic myeloid-derived suppressor cells; MoMF, monocyte-derived macrophage; NAFLD, non-alcoholic fatty liver disease; PGE2, prostaglandin E2; TLR-3, toll-like receptor 3; TNF-α, tumour necrosis factor α.

      Tissue macrophages

      Liver macrophages in cirrhosis

      As the key metabolic organ, the liver is strategically placed to drain the abdominal organs through the portal vein and receive systemic blood via the hepatic artery. The large sinusoidal network lined by highly permeable fenestrated endothelial cells exposes hepatocytes to the content of plasma that includes gut-derived bacterial products. To prevent immune activation in healthy KCs, LSECs and liver dendritic cells (DCs) actively promote tolerance by effectively removing bacterial products and secreting tolerogenic factors. KCs and liver DCs regulate this microenvironment by constitutively secreting IL-10.
      • Sutti S.
      • Bruzzì S.
      • Heymann F.
      • Liepelt A.
      • Krenkel O.
      • Toscani A.
      • et al.
      CX3CR1 mediates the development of monocyte-derived dendritic cells during hepatic inflammation.
      KCs also inhibit T cell expansion and induce Foxp3+CD25+ IL-10-producing regulatory T cells. They reprogram infiltrating monocytes to regulatory IL10+IL-12- DCs that further promote tolerance.
      • Heymann F.
      • Peusquens J.
      • Ludwig-Portugall I.
      • Kohlhepp M.
      • Ergen C.
      • Niemietz P.
      • et al.
      Liver inflammation abrogates immunological tolerance induced by Kupffer cells.
      ,
      • Heymann F.
      • Tacke F.
      Immunology in the liver--from homeostasis to disease.
      In addition, hepatocytes, HSCs and endothelial cells actively drive infiltrating monocytes to a more tolerogenic KC-like phenotype.
      • Bonnardel J.
      • T'Jonck W.
      • Gaublomme D.
      • Browaeys R.
      • Scott C.L.
      • Martens L.
      • et al.
      Stellate cells, hepatocytes, and endothelial cells imprint the Kupffer cell identity on monocytes colonizing the liver macrophage niche.
      ,
      • Blériot C.
      • Ginhoux F.
      Understanding the heterogeneity of resident liver macrophages.
      KCs phagocytose and scavenge DAMPs, released during hepatic tissue injury, through cell surface receptors. These include ATP, uric acid, DNA fragments, cholesterol crystals and high mobility group box 1. They also effectively recognise PAMPs and remove circulating bacteria, preventing systemic immune activation.
      • Kubes P.
      • Jenne C.
      Immune responses in the liver.
      In addition, LSECs clear cellular debris, antigens and immune complexes from the circulation.
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      During the development of chronic liver disease and cirrhosis, these mechanisms fail to further maintain tolerance, which has profound consequences for the liver and systemic inflammatory environment.
      • Wu J.
      • Meng Z.
      • Jiang M.
      • Zhang E.
      • Trippler M.
      • Broering R.
      • et al.
      Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific.
      Cirrhosis profoundly alters the local liver environment, increasing sinusoidal resistance, which leads to intrahepatic shunting through vascularised fibrotic septae when portal hypertension develops.
      • Laleman W.
      • Claria J.
      • Van der Merwe S.
      • Moreau R.
      • Trebicka J.
      Systemic inflammation and acute-on-chronic liver failure: too much, not enough.
      Hepatic innate immune cells continue to release cytokines and chemokines, attracting inflammatory cells from the circulation. Chronic injury and inflammasome activation alter the phenotype of LSECs. Changes in the local microenvironment including the composition of hyaluronan, increased adhesion molecules such as leucocyte CD44 and CD18, and chemokines allow extravasation of immune cells across the sinusoidal endothelial barrier.
      • Heymann F.
      • Peusquens J.
      • Ludwig-Portugall I.
      • Kohlhepp M.
      • Ergen C.
      • Niemietz P.
      • et al.
      Liver inflammation abrogates immunological tolerance induced by Kupffer cells.
      ,
      • Shetty S.
      • Lalor P.F.
      • Adams D.H.
      Liver sinusoidal endothelial cells - gatekeepers of hepatic immunity.
      ,
      • Støy S.
      • Sandahl T.D.
      • Hansen A.L.
      • Deleuran B.
      • Vorup-Jensen T.
      • Vilstrup H.
      • et al.
      Decreased monocyte shedding of the migration inhibitor soluble CD18 in alcoholic hepatitis.
      These mainly include neutrophils, CCR2+ monocytes and lymphocytes.
      • Zimmermann H.W.
      • Bruns T.
      • Weston C.J.
      • Curbishley S.M.
      • Liaskou E.
      • Li K.-K.
      • et al.
      Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver.
      ,
      • Laleman W.
      • Claria J.
      • Van der Merwe S.
      • Moreau R.
      • Trebicka J.
      Systemic inflammation and acute-on-chronic liver failure: too much, not enough.
      The cellular component undergoes profound changes during progression from inflammation through fibrosis to cirrhosis.
      • Laleman W.
      • Claria J.
      • Van der Merwe S.
      • Moreau R.
      • Trebicka J.
      Systemic inflammation and acute-on-chronic liver failure: too much, not enough.
      On the one hand, hepatocyte loss worsens and hepatocellular failure ensues, leading to decreased complement, acute-phase proteins and albumin, which results in defective bacterial opsonization and impaired clearance of bacterial products. On the other hand, KCs are continuously activated during the progression of chronic liver disease, initially by DAMPs released by dying hepatocytes. Activated TLR4/CRIg-expressing KCs lose their tolerogenic phenotype and continue to secrete proinflammatory cytokines and chemokines including MCP-1/CCL2
      • Weston C.J.
      • Zimmermann H.W.
      • Adams D.H.
      The role of myeloid-derived cells in the progression of liver disease.
      ,
      • Kolios G.
      • Valatas V.
      • Kouroumalis E.
      Role of Kupffer cells in the pathogenesis of liver disease.
      that amplify the immune response, recruiting bone marrow-derived monocytes and neutrophils to the liver. Monocyte-derived macrophages may in fact become the dominant phagocytic cell population in the diseased liver. As cirrhosis worsens, bacterial product translocation increases from the gut.
      • Weston C.J.
      • Zimmermann H.W.
      • Adams D.H.
      The role of myeloid-derived cells in the progression of liver disease.
      In the final stage, the development of portal hypertension, continuous hepatocyte loss and local and systemic immune cell activation set the stage for immune exhaustion, the development of bacterial infections, organ dysfunction and progressive hepatocyte failure.
      Recently, MERTK-expressing monocytes and macrophages were identified in the circulation but also in the liver and other tissue sites, such as the peritoneum and lymph nodes in patients with AD, and especially those with ACLF. HLA-DR+MERTK+ monocytes represent an immunomodulatory pro-restorative cell population, characterised by enhanced efferocytosis of apoptotic neutrophils. However, this population show blunted cytokine responses when exposed to LPS,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      ,
      • Triantafyllou E.
      • Pop O.T.
      • Possamai L.A.
      • Wilhelm A.
      • Liaskou E.
      • Singanayagam A.
      • et al.
      MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.
      and likely originate in the anti-inflammatory/tolerogenic environment prevailing in ACLF. MERTK-expressing macrophages may contribute to resolution of inflammation and restoration at the tissue level, which may be particularly relevant in the fibrotic/cirrhotic liver, but may aggravate systemic immunoparesis by reverse migration.
      • Korf H.
      • du Plessis J.
      • van Pelt J.
      • De Groote S.
      • Cassiman D.
      • Verbeke L.
      • et al.
      Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.
      ,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.

      Intestinal macrophages in cirrhosis

      The development of cirrhosis has immunological consequences extending far beyond the liver. The intestine is one of the most immunologically affected and clinically relevant sites in cirrhosis, as progressive intestinal barrier dysfunction in advanced cirrhosis enables translocation of bacterial products to the liver, peritoneum and systemic circulation that may clinically manifest as spontaneous bacterial peritonitis, worsening encephalopathy, bacteraemia and sepsis. Recently activated CD14+Trem1+iNOS+ intestinal macrophages were observed in the duodenum of patients with decompensated cirrhosis undergoing screening for varices. These cells likely represent monocyte-derived intestinal macrophages that are attracted to the gut in response to MCP-1 which is released by the intestinal epithelium in response to increased bacterial translocation.
      • Du Plessis J.
      • Vanheel H.
      • Janssen C.E.I.
      • Roos L.
      • Slavik T.
      • Stivaktas P.I.
      • et al.
      Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function.
      In murine models of cirrhosis, the mucus layer is reduced in thickness, with a loss of goblet cells and decreased MUC2 expression. This is associated with bacterial overgrowth in the inner most mucus layer. The closer proximity of bacteria to epithelial cells, a deficiency of Paneth cell defensins, as well as a defective vascular barrier likely all contribute to the passage of bacterial products in cirrhosis.
      • Sorribas M.
      • Jakob M.O.
      • Yilmaz B.
      • Li H.
      • Stutz D.
      • Noser Y.
      • et al.
      FXR-modulates the gut-vascular barrier by regulating the entry sites for bacterial translocation in experimental cirrhosis.
      ,
      • Teltschik Z.
      • Wiest R.
      • Beisner J.
      • Nuding S.
      • Hofmann C.
      • Schoelmerich J.
      • et al.
      Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.
      Circulating monocytes, monocyte-derived macrophages in liver and other tissues and tissue macrophages have site-specific differentiation: this implies that alongside immunosuppressive populations prevailing in the systemic circulation, activated proinflammatory monocytes may prevail in tissues such as the gut in the same patient.
      Gut-resident macrophages represent a heterogeneous mix of CX3CR1-expressing cells strategically positioned within the different layers of the intestine. In the lamina propria (LP), these macrophages actively contribute to host defence and barrier integrity coupled with high phagocytic activity. Cx3cr1−/− mice have reduced numbers of intestinal macrophages and show increased bacterial translocation.
      • Medina-Contreras O.
      • Geem D.
      • Laur O.
      • Williams I.R.
      • Lira S.A.
      • Nusrat A.
      • et al.
      CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice.
      In order to promote tolerance, LP macrophages and DCs constitutively secrete IL-10, are hyporesponsive to bacterial stimulation and do not produce proinflammatory cytokines when stimulated by LPS.
      • Scott N.A.
      • Mann E.R.
      Regulation of mononuclear phagocyte function by the microbiota at mucosal sites.
      It has been shown that CD103(+)-DCs were activated with heightened phagocytosis and migration in rats with experimental cirrhosis but no evidence of bacterial translocation, whereas these functions were paralysed in infected rats.
      • Muñoz L.
      • José Borrero M.
      • Ubeda M.
      • Lario M.
      • Díaz D.
      • Francés R.
      • et al.
      Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.
      Recent fate mapping and single-cell sequencing experiments in genetically modified mice showed that self-renewing LP macrophages reside in specialised niches that function to support the epithelial and vascular barrier.
      • De Schepper S.
      • Verheijden S.
      • Aguilera-Lizarraga J.
      • Viola M.F.
      • Boesmans W.
      • Stakenborg N.
      • et al.
      Self-maintaining gut macrophages are essential for intestinal homeostasis.
      Meanwhile, bone marrow-derived LP macrophages are involved in innate immune responses and phagocytosis.
      • De Schepper S.
      • Verheijden S.
      • Aguilera-Lizarraga J.
      • Viola M.F.
      • Boesmans W.
      • Stakenborg N.
      • et al.
      Self-maintaining gut macrophages are essential for intestinal homeostasis.
      It is tempting to speculate that selective depletion of these specialised LP macrophage niches in cirrhosis is a key factor in the loss of epithelial and vascular barrier integrity. However, it is also possible that constant immune activation with an influx of monocyte-derived macrophages into the LP may be detrimental to barrier integrity, as these cells may secrete factors such as nitric oxide, IL-6 and IL-8 that may contribute to increased permeability.
      • Du Plessis J.
      • Vanheel H.
      • Janssen C.E.I.
      • Roos L.
      • Slavik T.
      • Stivaktas P.I.
      • et al.
      Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function.

      Eosinophils and basophils

      Eosinophils are known for their role in inflammation, asthma and parasitic diseases. Novel contributions have emerged in autoimmune diseases and antibacterial host-defence.
      • Diny N.L.
      • Rose N.R.
      • Čiháková D.
      Eosinophils in autoimmune diseases.
      In patients with decompensated cirrhosis, eosinophil number increased in the blood (>300/μl)
      • Diny N.L.
      • Rose N.R.
      • Čiháková D.
      Eosinophils in autoimmune diseases.
      ,
      • Noirot C.
      • Leynadier F.
      • Luce H.
      • Abuaf N.
      • Bernard P.F.
      • Dry J.
      and liver in the early stages of biliary cirrhosis, which was associated with mastocyte infiltration and hepatic expression of IL-6, IFNγ, TGFβ, and IL-2 and the granulopoietic-stimulating factor IL-5. Infiltrating eosinophils degranulate major cationic proteins, eosinophil cationic protein; major basic protein and eosinophil-derived neurotoxin, consistent with a role in primary biliary cirrhosis.
      • Diny N.L.
      • Rose N.R.
      • Čiháková D.
      Eosinophils in autoimmune diseases.
      The role of basophils during cirrhosis progression has barely been studied.

      Dendritic cells

      DCs operate at the interface between the innate sensing of pathogens and the activation of adaptive immunity. The role of DCs in human cirrhosis remains underexplored. Circulating DCs that cross hepatic sinusoids to reach afferent lymphatics are educated by the hepatic tolerogenic microenvironment to adopt a regulatory phenotype.
      • Weston C.J.
      • Zimmermann H.W.
      • Adams D.H.
      The role of myeloid-derived cells in the progression of liver disease.
      ,
      • Thomson A.W.
      • Knolle P.A.
      Antigen-presenting cell function in the tolerogenic liver environment.
      Monocyte-derived DCs from patients with compensated cirrhosis retained similar capacity for maturation, activation and antigen presentation as those from healthy individuals.
      • Tanoue S.
      • Chang L.-Y.
      • Li Y.
      • Kaplan D.E.
      Monocyte-derived dendritic cells from cirrhotic patients retain similar capacity for maturation/activation and antigen presentation as those from healthy subjects.
      In a rat model of cirrhosis, activation and function of intestinal CD103(+)-DCs was attenuated by bacterial translocation.
      • Muñoz L.
      • José Borrero M.
      • Ubeda M.
      • Lario M.
      • Díaz D.
      • Francés R.
      • et al.
      Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.
      For liver DCs, studies in mice suggested a pro-resolution function in tissue repair and fibrosis regression through a matrix metallopeptidase 9-dependent mechanism.
      • Weston C.J.
      • Zimmermann H.W.
      • Adams D.H.
      The role of myeloid-derived cells in the progression of liver disease.
      ,
      • Jiao J.
      • Sastre D.
      • Fiel M.I.
      • Lee U.E.
      • Ghiassi-Nejad Z.
      • Ginhoux F.
      • et al.
      Dendritic cell regulation of carbon tetrachloride-induced murine liver fibrosis regression.
      DCs may thus deserve more detailed characterisation and evaluation of their potential as immunotherapeutic targets.
      • Muñoz L.
      • José Borrero M.
      • Ubeda M.
      • Lario M.
      • Díaz D.
      • Francés R.
      • et al.
      Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.
      ,
      • Jiao J.
      • Sastre D.
      • Fiel M.I.
      • Lee U.E.
      • Ghiassi-Nejad Z.
      • Ginhoux F.
      • et al.
      Dendritic cell regulation of carbon tetrachloride-induced murine liver fibrosis regression.
      ,
      • Connolly M.K.
      • Bedrosian A.S.
      • Mallen-St Clair J.
      • Mitchell A.P.
      • Ibrahim J.
      • Stroud A.
      • et al.
      In liver fibrosis, dendritic cells govern hepatic inflammation in mice via TNF-alpha.

      Mastocytes

      Mastocytes are haematopoietic sentinel cells that reside in connective tissues and participate in many pathophysiological processes through production of various mediators released in part from their numerous granules. In addition to their role in IgE-dependent allergy, they induce liver damage and HSC activation during progression to fibrosis/cirrhosis in mice
      • Hargrove L.
      • Kennedy L.
      • Demieville J.
      • Jones H.
      • Meng F.
      • DeMorrow S.
      • et al.
      Bile duct ligation-induced biliary hyperplasia, hepatic injury, and fibrosis are reduced in mast cell-deficient KitW-sh mice.
      due to their infiltration along with eosinophils and neutrophils.
      • Kennedy L.L.
      • Hargrove L.A.
      • Graf A.B.
      • Francis T.C.
      • Hodges K.M.
      • Nguyen Q.P.
      • et al.
      Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents.
      Mast cell markers (c-Kit, chymase, Trypase) increase and help the differentiation of connective tissue.
      • Jarido V.
      • Kennedy L.
      • Hargrove L.
      • Demieville J.
      • Thomson J.
      • Stephenson K.
      • et al.
      The emerging role of mast cells in liver disease.
      Chymase is produced in large quantities by mastocytes and has emerged as a downregulator of neutrophil ROS production.
      • Madjene L.C.
      • Danelli L.
      • Dahdah A.
      • Vibhushan S.
      • Bex-Coudrat J.
      • Pacreau E.
      • et al.
      Mast cell chymase protects against acute ischemic kidney injury by limiting neutrophil hyperactivation and recruitment.

      Innate lymphoid cells

      Innate lymphoid cells (ILCs) include ILC1, ILC2, ILC3 and natural killer (NK) cells,
      • Eberl G.
      • Colonna M.
      • Di Santo J.P.
      • McKenzie A.N.J.
      Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology.
      and are derived from the lymphoid lineage. They mirror T cells but do not express acquired antigen receptors or undergo clonal expansion. ILCs do not express myeloid- or DC markers but respond to signals from infected/injured tissues, initiate immune responses and regulate immune homeostasis
      • Klose C.S.N.
      • Artis D.
      Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis.
      and fibrogenesis.
      • Hams E.
      • Bermingham R.
      • Fallon P.G.
      Macrophage and innate lymphoid cell Interplay in the genesis of fibrosis.
      ,
      • Peters A.L.
      • Luo Z.
      • Li J.
      • Mourya R.
      • Wang Y.
      • Dexheimer P.
      • et al.
      Single cell RNA sequencing reveals regional heterogeneity of hepatobiliary innate lymphoid cells in a tissue-enriched fashion.
      A detailed characterisation of their phenotype, tissue-residency and functionality in human liver revealed that ILC3s account for the majority of intrahepatic ILCs. The frequency of ILC2s, sparsely present in non-fibrotic livers, increased in parallel with fibrosis severity where they secreted the pro-fibrotic cytokine IL-13 and amphiregulin.
      • Forkel M.
      • Berglin L.
      • Kekäläinen E.
      • Carlsson A.
      • Svedin E.
      • Michaëlsson J.
      • et al.
      Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.
      • Jeffery H.C.
      • McDowell P.
      • Lutz P.
      • Wawman R.E.
      • Roberts S.
      • Bagnall C.
      • et al.
      Human intrahepatic ILC2 are IL-13positive amphiregulinpositive and their frequency correlates with model of end stage liver disease score.
      • Gonzalez-Polo V.
      • Pucci-Molineris M.
      • Cervera V.
      • Gambaro S.
      • Yantorno S.E.
      • Descalzi V.
      • et al.
      Group 2 innate lymphoid cells exhibit progressively higher levels of activation during worsening of liver fibrosis.
      ILC2 activation involved stimulation with IL-33 and thymic stromal lymphopoetin produced by hepatocytes, HSCs, and KCs.
      • Forkel M.
      • Berglin L.
      • Kekäläinen E.
      • Carlsson A.
      • Svedin E.
      • Michaëlsson J.
      • et al.
      Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.
      ,
      • McHedlidze T.
      • Waldner M.
      • Zopf S.
      • Walker J.
      • Rankin A.L.
      • Schuchmann M.
      • et al.
      Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis.
      The frequency of ILC3 in peripheral blood was increased in patients with advanced cirrhosis and ILC3 induced fibrogenesis in in vitro and in vivo models.
      • Wang S.
      • Li J.
      • Wu S.
      • Cheng L.
      • Shen Y.
      • Ma W.
      • et al.
      Type 3 innate lymphoid cell: a new player in liver fibrosis progression.
      NK cells exhibited antifibrotic properties as they regulate activated HSCs.
      • Radaeva S.
      • Sun R.
      • Jaruga B.
      • Nguyen V.T.
      • Tian Z.
      • Gao B.
      Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners.
      • Muhanna N.
      • Abu Tair L.
      • Doron S.
      • Amer J.
      • Azzeh M.
      • Mahamid M.
      • et al.
      Amelioration of hepatic fibrosis by NK cell activation.
      • Gur C.
      • Doron S.
      • Kfir-Erenfeld S.
      • Horwitz E.
      • Abu-Tair L.
      • Safadi R.
      • et al.
      NKp46-mediated killing of human and mouse hepatic stellate cells attenuates liver fibrosis.
      • Fasbender F.
      • Widera A.
      • Hengstler J.G.
      • Watzl C.
      Natural killer cells and liver fibrosis.
      ILCs have thus been proposed to represent potential targets for antifibrotic therapy.
      • Zhang Y.
      • Tang J.
      • Tian Z.
      • van Velkinburgh J.C.
      • Song J.
      • Wu Y.
      • et al.
      Innate lymphoid cells: a promising new regulator in fibrotic diseases.

      Mucosal-associated invariant T cells

      Mucosal-associated invariant T (MAIT) cells are a subset of non-conventional T cells abundant in human blood, gut, and liver that display innate, effector-like qualities. MAIT cells are activated by bacterial metabolites (riboflavin) via the MHC class I-related molecule (MR1). They modulate host defences and inflammation by producing inflammatory cytokines and via granzyme B-mediated killing of infected cells.
      • Kurioka A.
      • Walker L.J.
      • Klenerman P.
      • Willberg C.B.
      MAIT cells: new guardians of the liver.
      ,
      • Toubal A.
      • Nel I.
      • Lotersztajn S.
      • Lehuen A.
      Mucosal-associated invariant T cells and disease.
      Circulating and hepatic MAIT cells were reduced in patients with alcoholic or non-alcoholic fatty liver disease-related cirrhosis, while they accumulated in liver fibrotic septae. Functionally, they were activated and profibrogenic in their interactions with HSCs. Prophylactic antibiotic therapy in decompensated cirrhosis was associated with higher MAIT cell frequency, and decreased activation.
      • Hegde P.
      • Weiss E.
      • Paradis V.
      • Wan J.
      • Mabire M.
      • Sukriti S.
      • et al.
      Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver.
      In alcohol-related cirrhosis, MAIT cells were concomitantly activated and exhausted.
      • Riva A.
      • Patel V.
      • Kurioka A.
      • Jeffery H.C.
      • Wright G.
      • Tarff S.
      • et al.
      Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease.
      Depletion of circulating, functionally exhausted MAIT cells and their accumulation in fibrotic septae were further reported in patients with autoimmune liver disease.
      • Böttcher K.
      • Rombouts K.
      • Saffioti F.
      • Roccarina D.
      • Rosselli M.
      • Hall A.
      • et al.
      MAIT cells are chronically activated in patients with autoimmune liver disease and promote profibrogenic hepatic stellate cell activation.
      MAIT cells thus represent a potential future antifibrogenic target.
      Mediators generated by injured liver and resident cells promote the recruitment of phagocytes which aggravate liver damage, and alter distant tissue homeostasis which in turn impact on liver function/regeneration via molecular and cellular mediators.

      Interconnections between innate immune cells and compartments

      The innate and adaptive immune systems interact to produce proinflammatory and antibacterial agents that protect tissues against pathogens. In health, the liver is central to the elimination of bacteria/bacterial products from the intestines (such as LPS, peptides, nucleic acid fragments) and endogenous factors released following tissue injury. Liver dysfunction associated with hepatocellular failure and constant immune activation alters immune function in distant organs (Fig. 1), which in turn fuels systemic inflammation, impacting the liver, and ultimately fuelling a vicious cycle. Some examples of pro-/anti-inflammatory crosstalk between innate immune cells, tissues, and mediators are summarised in Table 3. Major crosstalk occurs when pathological recruitment of circulating neutrophils, eosinophils and monocytes follows the onset of chronic inflammation in the liver. This process is mediated by activated KCs, LSECs, HSCs, resident mastocytes, and adipocytes which produce vaso-permeating agents and chemoattractants, facilitating tissue infiltration of recruited phagocytes and inducing tissue damage.
      • Khanam A.
      • Trehanpati N.
      • Riese P.
      • Rastogi A.
      • Guzman C.A.
      • Sarin S.K.
      Blockade of neutrophil's chemokine receptors CXCR1/2 Abrogate liver damage in acute-on-chronic liver failure.
      ,
      • Kennedy L.L.
      • Hargrove L.A.
      • Graf A.B.
      • Francis T.C.
      • Hodges K.M.
      • Nguyen Q.P.
      • et al.
      Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents.
      ,
      • Jarido V.
      • Kennedy L.
      • Hargrove L.
      • Demieville J.
      • Thomson J.
      • Stephenson K.
      • et al.
      The emerging role of mast cells in liver disease.
      As cirrhosis develops, disruption of the intestinal barrier and failure to clear bacterial products by intestinal macrophages further activate liver immune cells. By the time portal hypertension develops, bacterial products are also shunted into the systemic circulation, maintaining inflammation.
      • Seki E.
      • Schnabl B.
      Role of innate immunity and the microbiota in liver fibrosis: crosstalk between the liver and gut.
      Inversely, spleen-derived immune cells and cytokines moving via the portal vein to the injured liver can contribute to portal hypertension.
      • Li L.
      • Duan M.
      • Chen W.
      • Jiang A.
      • Li X.
      • Yang J.
      • et al.
      The spleen in liver cirrhosis: revisiting an old enemy with novel targets.
      In an attempt to suppress inflammation, to repair tissue damage and to clear apoptotic neutrophils, the expansion of a MERTK+HLA-DRhigh monocyte and macrophage subsets occurs,
      • Bernsmeier C.
      • Pop O.T.
      • Singanayagam A.
      • Triantafyllou E.
      • Patel V.C.
      • Weston C.J.
      • et al.
      Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.
      which suppress neutrophil activation and NET formation, induce neutrophil apoptosis and enhance their clearance.
      • Triantafyllou E.
      • Pop O.T.
      • Possamai L.A.
      • Wilhelm A.
      • Liaskou E.
      • Singanayagam A.
      • et al.
      MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.
      Future immunotherapy has been proposed in order to restore innate immune function in cirrhosis hereby preventing infectious complications, decompensation, and potentially the need for transplantation, and death. Potential targets include TLR7/8, TLR3, TAM receptors and glutamine synthase.
      Table 3Interconnections between innate immune cells, and compartments in liver diseases.
      Partner 1Partner 2Mediators – pathophysiological consequencesPhenotypeReferences
      Cirrhosis
       Kupffer cellsMonocytesCCL2 released by activated KC promotes monocyte infiltration into liver and damage in cirrhosisProinflammatoryKhanam et al., 2017
      • Khanam A.
      • Trehanpati N.
      • Riese P.
      • Rastogi A.
      • Guzman C.A.
      • Sarin S.K.
      Blockade of neutrophil's chemokine receptors CXCR1/2 Abrogate liver damage in acute-on-chronic liver failure.
       Liver mast cellsNeutrophils

      Eosinophils

      Monocytes
      Histamine released by resident MC increases vascular permeability and leucocyte infiltrationProinflammatoryKennedy et al., 2014
      • Kennedy L.L.
      • Hargrove L.A.
      • Graf A.B.
      • Francis T.C.
      • Hodges K.M.
      • Nguyen Q.P.
      • et al.
      Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents.


      Jarido et al., 2017
      • Jarido V.
      • Kennedy L.
      • Hargrove L.
      • Demieville J.
      • Thomson J.
      • St