Uncovering immune dysfunction in ACLF: cellular mechanisms, molecular pathways, and therapeutic frontiers

Abstract

Acute-on-chronic liver failure (ACLF) is a life-threatening condition characterized by acute hepatic decompensation, multi-organ failure, and high short-term mortality in patients with liver cirrhosis. A hallmark of ACLF is profound deterioration of the immune system, which contributes to organ-specific excessive inflammation and immune dysfunction, predisposing patients to infection and multi-organ failure. This review aims to elucidate the cellular and molecular mechanisms underlying systemic immune dysfunction in ACLF, highlighting key pathophysiological pathways and their clinical significance.

We provide an overview of ACLF including its global prevalence and clinical significance, against the background of the underlying immune dysfunction in its pathogenesis. The discussion focuses on innate immune alterations, such as impaired neutrophil and monocyte phagocytosis, excessive neutrophil extracellular trap (NET) formation, and monocyte/macrophage dysfunction contributing to immuneparesis and exaggerated inflammation, respectively, which evolve in an organ-specific manner. Dysregulation of natural killer (NK) cell cytotoxicity and adaptive immune dysfunction, including changes in T cell subpopulations and B cell antibody production in ACLF, are discussed.

We further dissect the emerging evidence of molecular pathways driving dysfunction of immune cells and their impaired ability to control infections in ACLF, emphasizing the roles of pathogen- and damage-associated molecular patterns (PAMPs/DAMPs), toll-like receptor (TLR) signaling, oxidative stress, mitochondrial dysfunction, epigenetic/metabolic reprogramming and immune checkpoint molecules. The review expands on immune cell communication within the immune system (innate and adaptive), with other non-parenchymal and parenchymal cells and at the inter-organ level, detailing interactions between immune cells of key organs and compartments affected during ACLF, including the liver, circulation, brain, gut and kidney.

Finally, we summarize the latest preclinical and clinical findings exploring biomarkers of immune dysfunction and immunomodulatory therapeutic strategies aimed at restoring immune homeostasis in patients with ACLF.

1. Introduction

Acute-on-chronic liver failure, ACLF, is a recently defined stage in patients with underlying chronic liver disease (with or without cirrhosis) characterized by high short-term mortality, multi-organ failure and immune dysfunction that develops after an acute decompensation event[1–4]. ACLF can arise in patients with cirrhosis being more prevalent during the decompensated stage[1, 5]. Bacterial infection, alcohol misuse and gastrointestinal bleeding are the most frequent factors precipitating ACLF[6]. According to recent meta-analysis[7], the global prevalence of ACLF among patients admitted with acutely decompensated cirrhosis is 35% (95% CI [33% to 38%]), with a 90-day mortality of 58% (95% CI [51% to 64%]) and the highest prevalence in South Asia. As defined by the CLIF organ failure score system, commonly affected organs in ACLF besides the liver are the kidneys, coagulation system, brain, circulation, and lungs, with the number of organ failure correlating with mortality rates[8].

Immune dysfunction is a central player in ACLF characterized by excessive inflammation, cell exhaustion and suppressed pathogen-fighting functions (immuneparesis)[9]. ACLF occurs in the setting of an increase in both pro-inflammatory (interleukin (IL) 6, tumor necrosis factor (TNF) α and IL1β) and anti-inflammatory cytokines (IL10). These mediators contribute to the development of fibrosis/cirrhosis, induce microvascular dysfunction and cellular damage that will further induce immune cell infiltration into damaged tissues. The pro-inflammatory milieu also modulates the vascular tone, hemodynamics and triggers metabolic reprogramming to meet high energy demand. In addition, amplified cell death signaling and damage-associated molecular patterns (DAMPs) release trigger an even more aggressive inflammatory response in a self-sustaining cycle of liver and extrahepatic organ damage[9, 10]. Immune dysfunction in both the innate and adaptive compartments lead to impaired host defense in phagocytosis, antigen presentation, cytokine secretion, migration and differentiation states[11] (Figure 1). Both inflammation and immune dysfunction have been shown to correlate with disease severity and mortality in ACLF[8].

Figure 1. Immune cell dysfunction in ACLF.

Diagram summarizing the molecular and cellular changes occurring in the innate (neutrophils, MO/Mφ and NK cells) and adaptive (T and B cells) cells leading to immune dysfunction in ACLF. MO: monocytes, Mφ: macrophages, NK: natural killer, ROS: reactive oxygen species, NETs: neutrophil extracellular traps, IL: interleukin, TNF: tumor necrosis factor, CXCL: Chemokine (C-X-C motif) Ligand, FPR1: Formyl peptide receptor 1, LCN2: lipocalin 2, NE: neutrophil elastase, MPO: myeloperoxidase, VCAM: vascular cell adhesion molecule, ICAM: intercellular cell adhesion molecule, IFN: interferon, CXC3CR1: C-X3-C motif chemokine receptor 1, CCR: C-C Motif Chemokine Receptor, TLR: toll-like receptor, CEACAM-1: Carcinoembryonic antigen-related cell adhesion molecule 1, BTLA: B and T Lymphocyte Attenuator, CTLA-4: cytotoxic T-lymphocyte-associated protein 4, PD-L2: Programmed Death-Ligand 2. Created with BioRender.com.

The clinical consequences of progressive immune dysfunction in ACLF include increased risk of infection due to the inability of circulating and tissue resident immune cells to control pathogens, organ damage and sustained inflammation leading to single or multi-organ failure. Triggers of the massive inflammatory responses in ACLF largely include DAMPs from damaged cells and pathogen-associated molecular patterns (PAMPs) from microbial translocation in which microorganisms (bacteria, fungi or virus) and/or their products translocate from the gut into the bloodstream[12].

Immune dysfunction exists in both the fibrotic/cirrhotic and ACLF stages, arising on a background of persistent low-grade inflammation, impaired gut barrier function, and continuous exposure to PAMPs/DAMPs[13–15]. As an acute response, this can lead to an exaggerated inflammatory response known as systemic inflammatory response syndrome (SIRS). SIRS is not exclusive to ACLF or cirrhosis, as it can be driven by PAMPs or by DAMPs triggered by a variety of insults[16]. In later phases if insult persists, repeated and prolonged exposure to gut-derived lipopolysaccharide (LPS) and other PAMPs trigger the activation of mechanisms related to immune cell exhaustion and blunted cytokine responses, leading to a compensatory anti-inflammatory response syndrome (CARS)[15, 17], predisposing ACLF patients to secondary infections and multi-organ failure. In the acute phase of ACLF, and depending of the precipitating event, SIRS symptoms can have clinical manifestations and are key determinants of early sepsis, organ failure, and survival[1].

Despite the efforts for identifying and stratifying ACLF patients at the bedside, currently available therapies are limited to supportive intensive care and liver transplantation[18–21]. Extracorporeal liver support systems are in clinical trials[22] with no clear impact on survival. This highlights unmet needs for advancing ACLF research to uncover the molecular mechanisms underlying its progression and pathophysiology for the development of novel therapies.

This review summarizes recent knowledge on the cellular and molecular mechanisms driving inflammation and immuneparesis as central forces driving ACLF pathophysiology. We also expand on immune cell control of infections and organ-specific regulation, interactions between immune cells in key organs in ACLF. Finally, we summarize novel targets, biomarkers and therapeutic candidates for ACLF patients.

2. Immune cell dysfunction and molecular mechanisms in ACLF

2.1. Innate Immune Dysfunction in ACLF

2.1.1. Neutrophils

Neutrophils are the primary effector cells of the innate immune system, playing a crucial role in host defense and responding to tissue injury. Neutrophils exert their antimicrobial functions through four different mechanisms: 1- degranulation (release of cytotoxic proteins); 2- neutrophil extracellular traps (NETs) formation (spill of decondensed chromatin decorated with histones and antimicrobial proteins); 3- oxidative burst (nicotinamide adenine dinucleotide phosphate oxidase activation generates superoxide, HOCl, hydrogen peroxide and other reactive oxygen species (ROS) with potent antimicrobial activity); and 4- phagocytosis (receptor-mediated internalization of large particles for degradation) [23]. All of these can be dysfunctional in ACLF.

Increased number of circulating neutrophils is a landmark of ACLF[24, 25] influencing the clinical prognosis in ACLF patients [26]. Overexpression of neutrophil-specific markers, including CD177 and the granular proteins neutrophil elastase (NE) and myeloperoxidase (MPO) correlated with increased mortality in ACLF patients[26]. Despite an increase in circulating neutrophil frequency, impairment in neutrophil functions leads to loss of antimicrobial control. Expression of chemokines and adhesion molecules such as carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1 indicate increased chemotactic activity in ACLF as well as infiltration in injured tissues[27]. Decreased expression of CD16 involved in phagocytosis and increased CD66b, regulating transendothelial migration, were found in ACLF[28]. ACLF neutrophils exhibit increased degranulation and formation of NETs both in vivo and in response to in vitro stimulation with phorbol 12-myristate 13-acetate, E. coli or N-formyl-methionyl-leucyl-phenylalanine[25, 27]. Importantly, a recent study showed that circulating NET components (cell-free DNA, MPO-DNA and citrulinated histone 3) can predict 90-day mortality in ACLF patients[29]. Oxidative burst activity is decreased in neutrophils from ACLF patients at baseline[30]. Moreover, these neutrophils fail to further increase ROS production upon stimulation (e.g. with E. coli), also showing diminished phagocytosis and bactericidal capacity, suggesting functional exhaustion due to chronic exposure to inflammatory factors such as cytokines or PAMPs[27, 31]. Neutrophil heterogeneity has been described across immune diseases during their maturation, activation and tissue location driving changes in neutrophil functionality, and balance between their protective vs pathologic roles[32]. While less in known about neutrophil heterogeneity in ACLF, emergency granulopoiesis (EG) has recently been shown to contribute to neutrophil dysfunction in patients with ACLF. Maladaptive EG is driven by the IL6-STAT3-CEBPB axis and leads to increased neutrophils with transcriptomic signatures associated with low-density (exhausted) neutrophils and granulocytic myeloid-derived suppressor cells known to have defective antimicrobial activity and suppress lymphocyte functions [33]. EG has also been described in the setting of infection[34, 35], frequently developed in ACLF patients, EG might be a central mechanism regulating neutrophil dynamics in ACLF. Still, the regulation of neutrophil heterogeneity in response to ACLF stages or etiological factors remains poorly understood.

In hepatitis B virus (HBV)-induced ACLF, neutrophils showed increased expression of the pathogen recognition receptors toll-like receptor (TLR)1 and TLR4 and CEACAM1[27]. On the other hand, neutrophils exhibited decreased expression of the chemokine receptor for bacterial formyl peptides. Formyl peptide receptor 1, compared to compensated cirrhotic non-ACLF or healthy controls[27, 36]. Transcriptomic analysis of these cohorts revealed upregulation of gene signatures related to degranulation, antibacterial immunity, extracellular matrix remodeling but a downregulation of genes related to communication with adaptive immunity, chemokine production and type 1 interferon (IFN) production[37].

The plasma environment in ACLF is central to neutrophil reprogramming. Circulating factors such as pro-inflammatory cytokines and LPS are persistently increased and activate neutrophils leading to their exhaustion[28]. The energy demand of increased inflammation in ACLF reshapes cellular metabolism leading to distinctive patterns of immunometabolism. Indeed, immune cells from patients with ACLF exhibit altered mitochondrial morphology and function, and adopt alternative carbon and nitrogen sources, such as adenosine and L-glutamine, to meet increased energy demands. The state of bioenergetic failure has profound effects on the efficacy of immune surveillance and response, exacerbating disease progression and organ failure[38].

Therefore, the interplay between elevated neutrophil counts, profound antimicrobial dysfunction, increased ROS and NETs and underlying bioenergetic compromise significantly contributes to the immunopathology of ACLF (Figure 2).

Figure 2. Neutrophil phenotypical and functional deregulation in ACLF.

In the ACLF environment, despite the need for effective defense against infections and microbial products, neutrophils exhibit multifaceted dysfunction. Key impairments include altered cytokine and chemokine release, impaired reactive oxygen species (ROS) production, defective phagocytosis, increased adhesion, transmigration, and tissue infiltration, as well as impaired degranulation and exacerbated neutrophil extracellular trap (NET) formation. Together, these defects compromise microbial clearance and contribute to disease progression. ROS: reactive oxygen species, NETs: neutrophil extracellular traps, IL: interleukin, TNF: tumor necrosis factor, CXCL: Chemokine (C-X-C motif) Ligand, LCN2: lipocalin 2, NE: neutrophil elastase, MPO: myeloperoxidase, TLR: toll-like receptor, CEACAM-1: Carcinoembryonic antigen-related cell adhesion molecule 1, PAMPs: pathogen-associated molecular patterns, DAMPs: damage-associated molecular patterns. Created with BioRender.com.

2.2.2. Monocytes and macrophages

Mononuclear phagocytes (monocytes, macrophages, dendritic cells) are key initiators of innate immunity, performing phagocytosis, antigen presentation, cytokine production, and immune cell activation. They regulate antimicrobial defense, inflammation, tissue injury, and fibrosis, important mechanisms failing during immuneparesis in cirrhosis[39]. Additionally to inflammatory cytokines, distinct vascular and intracellular cell adhesion molecules (VCAM1, ICAM1) as well as vascular endothelial growth factor A, link monocyte and macrophage chemotaxis and migration[40]. Systemic inflammation is amplified by sterile inflammatory mediators[41] such as lysophosphatidic acid (LPA), whose dysregulated downstream pathway suppressed MER proto-oncogene tyrosine kinase (MERTK)/CD163 expression and increased pro-inflammatory cytokine production of monocytes maintaining the inflammatory cascade in ACLF[42]. Dysfunctions across multiple compartments weaken host defence mechanisms and the immune response shifts from SIRS to CARS[14, 43–48, 43, 49]. Wasmuth et al. first described a sepsis-like immune failure in ACLF, with reduced human leukocyte antigen – DR isotype (HLA-DR) expression and impaired cytokine production of circulating monocytes[43]. Recently, these cells have been identified as CD14⁺HLA-DR_low monocytic myeloid-derived suppressor cells (M-MDSC) suppressing T cell responses, innate inflammatory cytokine production and phagocytosis of pathogens, hereby increasing infection susceptibility in ACLF[44, 45]. Studies using single cell approaches revealed a major heterogeneity in the mononuclear phagocytic population. A distinct subset of MERTK-expressing monocytes/macrophages expanded in diverse compartments in ACLF, demonstrating supressed inflammatory cytokine responsiveness to microbial stimuli and correlating with disease severity[46]. Furthermore, in ACLF[47, 48], and sepsis[50], increased programmed cell death protein (PD)1/PD ligand1 (PDL1) expression on monocytes/macrophages is associated with impaired cytokine production, bacterial clearance and increased infection risk. These tolerance-like properties are explained by the phenomenon of endotoxin tolerance, a model of innate immune memory induced by persistent microbial exposure[51]. Mechanistically, upregulation of microRNA (miR)-221/222 targeting the chromatin remodeler brahma-related gene 1 (SMARCA4) was described to induce epigenetic silencing of inflammatory genes, providing a potential link between chronic stimulation and immuneparesis in ACLF[52]. Similarly known for its tolerogenic characteristics in homeostasis is T cell immunoglobulin (TIM) 3, sparsely expressed on monocytes from patients with decompensated cirrhosis without overt infection, correlating with endotoxemia, impaired phagocytosis and diminished HLA-DR expression[53]. Upon HBV-ACLF, a circulating versican+CD14+ monocyte subset revealed activated IFN-stimulated genes and enhanced inflammatory properties, followed by an induction of anti-inflammatory CD163+ monocytes by apoptotic hepatocytes[54]. Novel findings revealed elevated phospholipase C in acutely decompensated (AD)/ACLF patients cleaving CD52 from monocytes, increasing soluble CD52 and CD52_low monocytes with impaired phagocytosis and cytokine production, hence inducing immuneparesis[55].

Functional heterogeneity of monocytes/macrophages is governed by coordinated transcriptional and metabolic programs in a compartment-specific manner (Figure 3). Within the distinct milieu in ACLF, these cells undergo profound metabolic rewiring by adopting distinct pathways. Bioenergetic failure significantly impair monocyte function, wherein circulating monocytes display severely compromised phagocytosis, oxidative burst, oxidative phosphorylation and glycolysis, collectively reflecting metabolic exhaustion[56]. Peripheral blood mononuclear cells (PBMC) from patients with ACLF exhibit profound mitochondrial structural damage, including cristae rarefication, swelling, reduced organelle size, and a compensatory increase in mitochondrial number. Despite this, mitochondrial adenosine triphosphate (ATP) generation was markedly impaired[38]. In HBV-ACLF, PBMC exhibit pronounced immune dysregulation associated with progressively worsening lipid and fatty acid metabolic disruption[57]. In monocytes, dysregulation of the glutamine metabolism causes suppressed antibacterial and inflammatory responses [58]. Intrahepatic macrophages are similarly reprogrammed; Triggering receptor expressed on myeloid cells (TREM)2⁺ lipid-associated macrophages enriched in HBV-ACLF linked to fatty acid metabolism and anti-inflammatory signalling[59].

Figure 3. Overview of monocyte and macrophage plasticity across compartments in the pathophysiology of acute-on-chronic liver failure (ACLF).

The schematic summarizes the current understanding of how monocyte and macrophage subsets adapt to the complex, multi-organ processes underlying ACLF, including progressive liver fibrosis, parenchymal injury, portal hypertension, gut dysbiosis, and pathological bacterial translocation, as well as the resulting activation of local and systemic inflammatory cascades. A broad range of cellular and non-cellular mediators involved in these interlinked mechanisms is illustrated. Distinct immune-cell compositions are depicted within the gut, liver, circulation, peritoneum, and lymph nodes, highlighting the multi-compartmental nature of the syndrome. Under homeostatic conditions, monocyte and macrophage subsets display stable, compartment-specific phenotypes and functions. In ACLF, sustained hepatic and systemic inflammation, excessive DAMP and PAMP exposure from hepatocyte injury and gut barrier failure, and profound alterations in lipid and metabolite profiles collectively reprogram their differentiation and activation, promoting dysfunctional, immunosuppressive phenotypes. Adapted from Geng et al., Frontiers in Network Physiology, 2022. ATX/LPA, autotaxin–lysophosphatidic acid axis; PM, peritoneal macrophage; PMN- and M-MDSC, polymorphonuclear and monocytic myeloid-derived suppressor cell; HSC, hepatic stellate cell; DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern. Created with BioRender.com.

In summary, monocyte and macrophage states in ACLF are characterized by immunesuppressive functions including impaired phagocytosis, antigen presentation, and antimicrobial effector functions, in part due to metabolic reprogramming.

2.1.3. Natural killer cells

Natural killer (NK) cells which are cytotoxic lymphocytes that mediate the major histocompatibility complex (MHC)-independent recognition and spontaneous killing of stressed cells, are significantly reduced in both frequency and cytotoxic function in the circulation and livers of ACLF patients[60, 61]. This correlates with worse outcomes and impaired pathogen defense[62, 63]. Phenotypically, NK cells in ACLF display increased expression of activating receptors (such as NKp30, NKp44, NKp46, and NKG2D) and certain inhibitory receptors, yet paradoxically, their cytotoxic capabilities—reflected in decreased killing capacity and attenuated production of critical effector molecules like TNFα and IFNγ—are markedly impaired[17, 61].

The cytokine environment in ACLF is characterized by the presence of C-X-C motif chemokine ligand (CXCL)10, which induces NK cell apoptosis and diminishes their cytotoxic population[62]. These changes reduce NK cell-mediated immune surveillance and disrupt their ability to control viral infections. In HBV-induced ACLF, accumulation of intrahepatic NK cells expressing cytotoxic markers (CD57+, CD3−, and NKG2D) leads to hepatocyte death via degranulation and activation of death receptor pathways including TNF-related apoptosis-inducing ligand, exacerbating liver injury[64]. Overall, the functional exhaustion of NK cells impairs their killing potential, resulting in decreased clearance of infected cells and poor regeneration of liver tissue[17, 65].

Recent transcriptomic analyses validated NK cell signatures indicative of heightened migration, but low proliferation and immune response in ACLF[17]. Moreover, single cell analysis of NK cells identified four different clusters in ACLF (adaptive, mature, inflamed and CD56 bright) with ACLF patients having lower frequency of mature over the inflamed cluster[61]. The identification of Cemip2, which encodes for a cell migration-inducing hyaluronidase, was specifically identified in the inflamed NK subset showing promising potential as a biomarker for progression and prognosis of ACLF[17].

2.2. Adaptive Immune Dysfunction in ACLF

2.2.1. T cells

T cell dysfunction in ACLF is characterized by lymphocytopenia, reduced numbers of resting memory CD4 and CD8 T cells, while the proportions of plasma cells, naïve-, activated memory-, and CD4 regulatory T cells (Tregs) are not affected[24, 66]. Multiple studies demonstrated progressive T cell exhaustion and senescence, driven by an upregulation of immune checkpoint molecules[54, 67–69]. Increased B and T lymphocyte attenuator (BTLA) expression on circulating and intrahepatic CD4 T induce cell exhaustion in HBV-ACLF involving reduced activation, proliferation, and cytokine production through Phosphatidylinositol 3-kinase (PI3K)- Protein Kinase B (AKT) signalling in correlation with disease severity and infection[68]. Similarly, an upregulation of both inhibitory- (cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), BTLA) and co-stimulatory immune checkpoint molecules (HLA-DR, TIM1) on CD4 and CD8 T cells ex vivo in early cirrhosis were related to the development of ACLF, while production of pro-inflammatory cytokines was diminished[69]. The expansion of immunesuppressive CD4⁺HLA-G⁺ T cells further aggravated adaptive immunesuppression mediated through HLA-G and CTLA-4 pathways impairing T cell responses and downregulating Th17-related cytokines[67]. Exhaustion of cytotoxic T cells in HBV-ACLF developed under the influence of immunosuppressive C-X-C motif chemokine receptor (CXCR)2+ neutrophils in association with disease progression[54]. With regards to mitochondrial dysfunction in T cells from ACLF patients, it has been shown that circulating extracellular vesicles (EV)-derived from ACLF plasma impair T cell viability by inducing mitochondrial depolarization, increased apoptosis, and metabolic dysfunction, thereby altering the composition of T cell populations and linking EV-mediated mitochondrial injury to adaptive immune dysregulation[70]. The imbalance between pro-inflammatory Th17 and Treg responses in relation to the immunopathogenesis of ACLF was demonstrated in HBV-ACLF. Th17 cell expansion was predominant over Tregs, and Treg functions were impaired[71, 72] leading to a hyperinflammatory state inducing hepatocyte injury, organ failure, and death[9]. Increased frequencies of circulating and intrahepatic Th17 cells with upregulated IL23R expression was observed in a mixed population of alcohol- and HBV-induced ACLF, correlating with biochemical markers of hepatic injury, Child–Pugh/Model for End-Stage Liver Disease (MELD) scores, and poor survival[73]. Tissue-resident memory (TRM) CD8 T are known to contribute to local immunesurveillance in the liver [74]. In AD patients with spontaneous bacterial peritonitis, macrophage-derived IL15 imprinted TRM-like CD8 T cells (CD103⁺CD69⁺CD49a⁺) enriched in ascitic fluid compared to blood. IL15 exposure induced transcriptional signatures of activation and exhaustion in TRM-like CD8 T cells, yet these cells retained cytotoxic effector functions even in an environment rich in soluble immune checkpoints (e.g., PD1/PDL1), and checkpoint blockade did not impair their cytotoxicity[75]. In addition, enhanced peripheral γδ T cell cytotoxic potential has been described in HBV-ACLF, characterized by increased granzyme B and CD107a expression and elevated TNFα and IL17 production. Despite reduced circulating γδ T cell frequencies, suggesting tissue recruitment or activation-induced cell death, their increased cytotoxic and pro-inflammatory activity correlated with liver injury, indicating a potential contribution to disease progression[76].

2.2.2. B cells

B cells are increasingly recognised in ACLF pathogenesis, although their precise role remains insufficiently defined. Profound alterations occur in both circulating and intrahepatic B cell compartments[24, 77]. In peripheral blood, memory B cells seem to be depleted[24]. In HBV-ACLF, single-cell ribonucleic acid (RNA) sequencing identified six major B cell subsets on peripheral blood mononuclear cells in ACLF, with a significant increase in naïve B cells displaying enrichment of inflammatory-response pathways, suggesting functional reprogramming toward a pro-inflammatory phenotype[78].

Intrahepatic B cells were characterized in depletion of naïve B cells concomitant with an expansion of activated memory B cells, atypical- and immunoglobulin (Ig)M+ memory B cells, and plasma cells[77]. Intrahepatic plasma cells were characterized by increased PD-L2 expression and higher secreted levels of granzyme B and IL10[77].

In the gut mucosa, most B cells produced secretory IgA, which shapes the microbiota and neutralises bacterial antigens, and whose retrograde passage into the circulation with increased gut permeability. IgA has recently been shown to be a sensitive marker of gut barrier dysfunction in ACLF, indicating microbial translocation, systemic inflammation and predicting disease severity and short-term mortality[79].

In summary, ACLF involves profound derangements of adaptive immunity, combining T cell exhaustion, an imbalance of Th17 cells/Tregs, and dysfunctional B cell responses increasing the risk of infections, multi-organ failure, and death.

2.3. Soluble mediators

ACLF pathophysiology does not exclusively involve immune cell dysfunction but is linked to a complex network of non-cellular mediators such as cytokines, chemokines, PAMPs, DAMPs, bioactive lipids, metabolic intermediates, and complement factors, playing pivotal roles in propagating immune dysregulation.

Eicosanoids, specialized pro-resolving lipid mediators (SPM) and lysophospholipids are important regulators of immune homeostasis, and their dysregulation contributes to the ACLF pathogenesis[80]. Aberrant eicosanoid signatures such as leukotriene E4 and 12-hydroxyheptadecatrienoic acid are associated with inflammation, disease severity and prognosis, and were markedly elevated in ACLF but not AD patients or healthy controls[81]. Untargeted lipidomic analyses revealed global lipid suppression in cirrhosis, particularly ACLF, with distinct sphingomyelin, cholesterylester, and lysophosphatidylcholine signatures distinguishing disease stages, liver dysfunction and infections[82]. Similarly, decreased phospholipids, sphingosine-1-phosphate and increased gut microbe-derived metabolites independently predict ACLF and mortality, highlighting the gut–liver–lipid interplay[83, 84]. Moreover, the loss of extracellular vesicle–associated CD5L, an anti-inflammatory glycoprotein that promotes SPM biosynthesis, weakened the resolution capacity of monocytes/macrophages and contributes to multi-organ failure including circulatory, brain, and respiratory dysfunction[85].

Beyond lipids, reduced hepatic synthesis of acute-phase proteins and complement factors (C)3/C3a impair opsonization and bacterial clearance[86, 87]. Reduced levels of albumin structural alterations are reflected by its defective antioxidant, scavenging, immune-modulating, and endothelial-protective properties, thereby amplifying oxidative stress, immuneparesis, and microvascular dysfunction[88].

The recognition of these mediators include the signaling pathways characterized by pattern recognition receptors including cell surface or endosome TLRs, or cytosolic receptors including NOD-like receptors leading to inflammasome activation, RIG-I-like receptors recognizing viral RNA, C-type lectins that detect fungal carbohydrate structures or absent in melanoma 2 (AIM2)-like receptors as sensor for cytoplasmic deoxyribonucleic acid (DNA), which have been extensively described in[89]. In the specific context of ACLF, both clinical[90] and preclinical alcohol-induced ACLF[25] studies have demonstrated the activation of TLRs[91] and the canonical (through caspase 1) and non-canonical (through caspase 4 and 5) activation of the inflammasome complex leads to pyroptosis and immunosupression. Interestingly deletion of gasdermin D in a murine model of alcohol-induced ACLF improved liver dysfunction but exacerbated extrahepatic damage in brain and kidney[92].

3. Organ-specific regulation in ACLF

3.1. Immune cell compartments and susceptibility to infection

Cirrhosis, and particularly ACLF as a systemic inflammatory condition, does not restrict to changes in the hepatic immunity but implies modulation of the cellular and humoral immune system across the body, most importantly in the liver, systemic circulation, peritoneum, mesenteric lymph nodes, brain and kidneys. In physiological conditions, the liver is a tolerogenic organ[93] while most other compartments remain immunogenic and combat evasion of pathogens. In cirrhosis, compartments other than the liver may turn tolerogenic, leading to susceptibility to infection.

The liver immune compartment in ACLF remains scarcely described given that liver biopsy is rarely performed (if not explanted). In general, it is characterized by diminished parenchyma, destructed sinusoidal endothelia and accumulation of scar tissue[94, 95]. Single cell transcriptomic data from liver immune cells of patients with cirrhosis, however at a stage prior to ACLF, revealed the occurrence of TREM2⁺CD9⁺ scar-associated macrophages, along with Atypical Chemokine Receptor/Plasmalemma Vesicle-Associated Protein (ACKR1⁺ PLVAP⁺) endothelia facilitating leucocyte migration[96]. Also, in explanted tissue from patients with ACLF compared to cirrhosis, CD14⁺MERTK⁺ macrophages, polymorphonuclear (PMN)- and M-MDSC accumulated in the liver and mesenteric lymph nodes[44–46].

Soluble and cellular immune components are dramatically changed in the systemic circulation, leading to an immunosuppressive circulatory compartment with significant reduction in antimicrobial defense. As detailed above, functional circulating monocytes are replaced by substantial proportions of immunemodulatory (CD14⁺MERTK⁺, 36%) and immunosuppressive (M-MDSC, 55%) mononuclear cells, characterized by dampened responses to pathogens that may occur in the blood stream or tissues[44–46]. Besides migration of immune cells across endothelia and compartments, the immunomodulatory plasma is shaping dynamic immune cell differentiation. Important components are the presence of microbial products (e.g. bacterial DNA)[97], the elevation of pro- and anti-inflammatory cytokines concomitantly[98, 99], sphingolipids, eicosanoids and other metabolic factors[42, 100], and indicators of immune cell activation such as adhesion molecules, cleaved immune cell receptors and enzymes active in essential immune signaling pathways[46, 55, 99]. Moreover, immune cell function has been shown to be disturbed by defective mitochondrial tricarboxylic acid cycle metabolism[58, 38].

Little is known about the immunity of the human peritoneum in health as the tissue is seldom available, peritoneal macrophages (PM) are important blood-derived phagocytes constantly exchanged in order to maintain homeostasis[101]. In the presence of ascites, distinct PM subsets have been identified: highly phagocytic CRIg^highMERTK+TIMD4+ PM with enhanced antimicrobial effector activity associated with reduced morbidity and mortality[102]. This population probably overlaps with large PMs (LPM), characterized by CD206⁺CD163⁺ expression, higher granularity, and increased levels of maturation markers such as CRIg, CD16, CD163 and MERTK, susceptible to tolerance induction. In contrast, small PMs are typically CD206⁻. TLR stimulation cleaved CD206 surface expression and released soluble CD206 in the early phase of spontaneous bacterial peritonitis, leading to temporal loss of LPM[103]. Also, adaptive immune cell enrichment in association with disease severity has been described, i.e. the distinct CXCR6+CD69+ clusters of late effector memory CD8+ T cells. These cells were hyperreactive to innate cytokines which was reversible by the Janus kinase (JAK)-inhibitor tofacitinib ex vivo[104].

Another compartment is the spleen. Metabolic dysfunction of splenic B cells has been described in cirrhosis[105]. Also, the hepatic immune cell compartment changed after splenectomy in patients with decompensated cirrhosis[106]. Yet, these circumstances need to be studied in ACLF.

While some immunosuppressive cells may at the stage of ACLF be distributed throughout the entire body (e.g. M-MDSC, CD14⁺MERTK⁺[44, 46]), others expand or dissolve in compartment specific manners. Examples represent CD14⁺AXL⁺[49, 107–110], and CD14+CD52+ phagocytes[55], implicating importance for future immunomodulatory strategies.

3.2. Gut permeability and pathological microbial translocation

Gut dysbiosis and pathological translocation of microbial components are one of the primary drivers leading to systemic inflammation, immune dysfunction and potential triggers of infectious complications in patients with cirrhosis eliciting ACLF[111–113].

Impaired intestinal barrier allows the translocation of pathogens, PAMPs and virulence factors (proteases, exotoxins, phospholipases, etc.) to reach the liver through the portal circulation[6]. These signals directly damage the host tissue and perpetually activate the host immune response. This effect is aggravated by DAMPs release from the injured host cells perpetuating the inflammatory state[41]. Specific signaling and receptors for hepatocyte-derived DAMPs including high mobility group box 1 (HMGB1), mitochondrial DNA (mDNA), histones and IL33 have been extensively reviewed in[114].

Reduced microbial diversity, an overgrowth of certain bacteria phyla such as Firmicutes and Proteobacteria and a reduction in beneficial fila including Bacteroidetes[115], has been described in cirrhosis and particularly in ACLF with significant overlap between oral and gut microbiome[116]. The presence of Proteobacteria (Escherichia coli, Klebsiella pneumonia or Pseudomonas aeruginosa) as opposed to Firmicutes is highly associated with poorer outcomes in patients with ACLF[115, 117]. Interestingly, predominance of specific microbial families has been correlated with organ-specific failures, poor cognition or opportunistic infections during cirrhosis and ACLF[118, 119].

Smets et al. revealed bacterial translocation requires dysfunction of gut-vascular macrophages besides gut epithelial cell death and vascular barrier damage. Specifically, macrophages with enhanced chemokine expression and reduced bacterial clearance interacted poorly with blood vessels, and depletion of vascular-lining macrophages resulted in bacterial translocation in mice and similarly in patients[120].

When the gut-liver axis is disrupted, bacterial toxins such as LPS activate TLR4 on intestinal epithelial cells and mucosal immune cells including monocytes, macrophages and dendritic cells (DCs), which in turn triggers a cytokine cascade leading to systemic inflammation[111–113]. Moreover, TLR-induced inflammation can affect epithelial survival tight junction permeability, and mucosal immunity. Intestinal DCs stimulate naive T cell and B cell differentiation through antigen presentation, conforming the adaptive mucosal immune system[12]. Differentiated CD4⁺ and CD8⁺ T are recruited to the lamina propria producing cytokines such as IFNγ that contribute to mucosal immunity[121]. Pathogens are also confined to the gastrointestinal lumen by IgA production from activated B cells[79, 122]. Immune dysfunction during ACLF with decreased antigen presentation by DC, impaired phagocytosis by monocytes/macrophages and shifted phenotypic changes in T cell maturation prevents the proper control of gut-derived pathogens within the mucosal and intestinal microenvironment. Moreover, persistent stimulation with microbial products leads to immune cell desensitization and reprogramming, partially contributing to immune dysfunction[113].

In the context of alcohol misuse and alcohol-related ACLF, gut dysbiosis is even more relevant since alcohol and its metabolites directly damage the intestinal epithelium and further deregulate microbial diversity. Bacterial fermentation products such as butyrate and propionate are short chain fatty acids (SCFA) are essential for maintaining gut barrier, as nutrients for the colonic epithelium as well as colonic regulators of pH[123]. Dysbiosis derived from alcohol misuse is characterized by a reduction of SCFA-producing bacteria, further contributing to barrier leakiness.

In ACLF, three different interventions have been tested targeting gut dysbiosis. Fecal microbiota transplant showed improved microbial diversity, cognitive function and reduced hospitalization time[124]. Recurrent hepatic encephalopathy (HE) is usually treated with rifaximin or other non-absorbable antibiotics which show reduced risk of decompensation and improved survival in patients with alcohol-related decompensated cirrhosis[125]. Finally, probiotics showed a reduction of infection rates and ammonia levels without improving survival in cirrhotic patients with HE[126, 127].

3.3. Immune cell – parenchymal and non-parenchymal cell interactions

Organ-specific interactions between immune cells and parenchymal and non-parenchymal cells is central to understand the immune contribution in ACLF pathophysiology (Figure 4).

Figure 4. Immune-derived organ-specific impairment in ACLF.

The pathophysiology of ACLF extends beyond liver failure, with immune dysregulation orchestrating organ-specific responses and inter-organ communication across extrahepatic sites, particularly the gut, brain, and kidney. NETs: neutrophil extracellular traps, LSEC: liver sinusoidal endothelial cells, HSC: hepatic stellate cells, NPC: non-parenchymal cell, BA: bile acid, PAMPs: pathogen-associated molecular patterns, DAMPs: damage-associated molecular patterns, HE: hepatic encephalopathy, RTEC: real tubular epithelial cell, LPS: lipopolysaccharide, DNA: deoxyribonucleic acid, ATP: adenosine triphosphate, mDNA: mitochondrial DNA, HMGB1: High mobility group box protein 1. Created with BioRender.com.

3.3.1. Immune-liver crosstalk and liver regeneration

The interaction between immune and parenchymal cells regulates liver homeostasis, injury and repair[128]. In ACLF, a wide variety of immune cells including monocytes, neutrophils and dendritic cells are recruited into the liver in response to PAMPs such as LPS or bacterial DNA from the leaky gut or DAMPs released during cell death including ATP, genomic or mDNA and HMGB1 among others [24, 129, 130]. Innate immune cell infiltration triggers a cascade of inflammatory cytokines and chemokines in the injured liver which orchestrate host defense and regulate hepatocyte and other non-parenchymal cell survival and regenerative response[129]. IFNγ and TNFα released by NK cells and macrophages can induce hepatocyte death and stimulate hepatocyte proliferation and survival through downstream pathways such as JAK-STAT and hepatocyte growth factor signaling. IL6 and IL8 are major regulators of liver regeneration, with IL6 especially implicated in activating regeneration pathways and IL8 contributing to immune cell recruitment for tissue repair[131].

Immune cell infiltration, release of cytotoxic and bioactive mediators from NK and T cells or NETs directly induce hepatocyte injury and death pathways [18,50]. In a mouse model of steatohepatitis, NETs were shown to modulate the phenotype of various cells within the liver sinusoids. NETs can directly induce hepatic stellate cell (HSC) activation indicated by increased expression of fibrogenic markers including α-smooth muscle actin (SMA) and IL1β release. NETs also promoted the activation of monocytes and release of pro-inflammatory TNFα, monocyte chemoattractant protein (MCP)1 and IL1β. These effects were abrogated by disrupting the NET structure with DNAse1 or by inhibiting Nod-like receptor family pyrin domain containing 3 (NLRP3) signaling highlighting NLRP3 inflammasome as a key driver in NET response. In vitro, components of NETs together but not individually activated HSC providing insights into NET-mediated fibrosis[132]. In ACLF, alcohol- or bile acid-induced NETs co-cultured with hepatocytes increased their release of Receptor-Interacting Protein Kinase (RIPK)3, IL1β and lactate dehydrogenase to the supernatant, indicating a direct role of NETs in inducing hepatocellular death[25]. Also, liver sinusoidal endothelial cells (LSEC) increase expression of dedifferentiation markers such as von Willebrand Factor, CD34, Fibrillin1 or adhesion molecules[25]. NETs can also induce pyroptosis and contribute to microthrombus formation and portal hypertension[133]. (Figure 5).

Figure 5. Effect of NETs in the main liver cells during ACLF.

Excessive NET formation is a key trait of neutrophils during ACLF, contributing to liver damage. NET components including both chromatin and bioactive molecules induce hepatocyte inflammation and death, LSEC damage, inflammation and formation of microthrombi, HSC activation and induction of a pro-inflammatory state in MO/Mφ. Both HSC activation and MO shift to a pro-inflammatory phenotype have been shown to be partially dependent on NLRP3. MO: monocytes, Mφ: macrophages, NETs: neutrophil extracellular traps, LSEC: liver sinusoidal endothelial cells, HSC: hepatic stellate cells, IL: interleukin, MCP-1: monocyte chemoattractant protein 1, LDH: lactate dehydrogenase, RIPK3: receptor-interacting serine/threonine kinase 3, NLRP3: NLR family pyrin domain containing 3, VCAM: vascular cell adhesion molecule, ICAM: intercellular cell adhesion molecule, α-SMA: alpha smooth muscle actin, LCN2: lipocalin 2, NE: neutrophil elastase, MPO: myeloperoxidase, NFκB: Nuclear factor kappa B, vWF: von Willebrand factor, Fbn1: fibrillin 1, citH3: citrulinated histone 3. Created with BioRender.com.

In healthy and regenerative states, macrophages induce differentiation of hepatocytes and support repair[134]. This subset of macrophages exert hepatoprotective functions by inhibiting necroptosis and pyroptosis of hepatocytes[135, 136]. In ACLF, despite recruitment of these macrophage populations, the hepatoprotective effect is lost influenced by high levels of circulating cytokines such as TNFα, IL6, IFNγ and granulocyte colony stimulating factor (G-CSF) [50]. In fact, preclinical murine models of ACLF have shown reduction of regeneration markers such as transforming growth factor-beta (Tgfb) or Snail family transcriptional repressor 1 (Snai1) but an induction of cell death predominantly through necroptosis and pyroptosis in the liver[25, 92]. Indeed, RIPK3, a central mediator in the necroptosis pathway, has been associated with clinical outcomes in HBV-related ACLF patients[137]. Moreover, RIPK3 inhibition in preclinical models of ACLF improved survival, inflammation, neutrophil activation and extrahepatic features of ACLF including neuroinflammation and kidney function[25]. In ALF, signaling through IL6/IL22-STAT3, that normally promotes hepatocyte survival and proliferation, does not induce cell proliferation, likely due to inhibitory mechanisms and persistent inflammation[138], similar mechanism could be happening in ACLF. Importantly, the excessive presence of IL22 binding protein, a natural antagonist of IL22, blocks hepatoprotective and pro-regenerative signaling, and correlates with poor outcomes in ACLF[139, 140]. IL22Fc therapy (recombinant fusion protein that maintains IL22 in circulation) improved proliferation of hepatocytes and ameliorated ACLF pathophysiology in a CCl₄ + sepsis mouse model of ACLF by restoring liver regeneration, upregulating pro-regenerative (pSTAT3, STAT3, cyclin D1) and downregulating anti-regenerative proteins such as p21[140].

Massive immune activation in ACLF shifts the pro-regenerative environment to one dominated by oxidative stress, cell death, and fibrosis[17]. As such, insufficient regeneration—despite pathway activation—is a hallmark of ACLF, driven by overwhelmed by inflammatory and anti-regenerative cues. New therapeutics targeting liver regeneration, including recombinant IL22Fc, G-CSF or the TLR4 inhibitor TAK-242, are under investigation[140–144], aiming to reestablish the beneficial immune-hepatocyte crosstalk.

3.3.2. Immune-brain communication and hepatic encephalopathy

Hepatic encephalopathy, a major complication of ACLF, is characterized by accumulation of circulating ammonia promoting cognitive impairment[145]. Ammonia buildup is multifactorial from reduced detoxification capacity of the liver, increased intestinal production of ammonia, and impaired renal clearance[146]. Moreover, the intense systemic inflammation and neurotoxic substances (such as LPS, bilirubin or SCFA cross the blood-brain barrier due to increased permeability[147, 148]. This triggers activation of microglia and astrocytes, and induces neuroinflammation and microglia death. Intestinal microbiome has been shown to influence not only gut barrier integrity but also the blood–brain barrier contributing to neuroinflammation [149, 150].

In the neuroinflammatory response in HE and in neurodegenerative diseases [151, 152] microglia activation correlates with upregulation of pro-inflammatory cytokines (IL1β, TNFα, IL6) and chemokines like MCP1 in the cortex, hippocampus, and cerebellum[153]. This neuroinflammation is perpetuated by cytokine release from microglia, which activates astrocytes and alters neurotransmitter balance, notably impairing cognitive and motor functions[154].

Peripheral immune activation—from increased neutrophil, monocyte, or T cell cytokines and ROS production in the circulation— and haemodynamics in ACLF contribute to brain inflammation[155]. Indeed, systemic inflammatory markers like C-reactive protein correlate with the degree of brain inflammation and HE severity [9, 156].

The communication between immune cells and the brain in ACLF involves bidirectional signaling through circulating mediators, neuroinflammation driven by microglial and astrocyte activation, and impaired clearance of toxins. These mechanisms underlie the pathogenesis of HE and point to potential therapeutic targets in modulating brain–immune system interactions in ACLF.

3.3.3. Immune-kidney crosstalk and renal failure

Acute kidney injury (AKI) is a severe and most common complication in ACLF[157]. Immune cell dysregulation plays a significant role and contributes to renal pathophysiology during ACLF.

First, buildup of circulating bilirubin and bile acids in ACLF have toxic effects on renal tubular epithelial cells and promote the formation of bile casts leading to tubulinterstitial inflammation and cell death[158–160]. Second, ACLF is associated with severe cardiovascular dysfunction and a hyperdynamic circulatory state, which can lead to poor renal perfusion, microthrombosis and ischemia[161]. Third, inflammatory mediators and recruitment of monocytes and neutrophils promote endothelial dysfunction and interstitial damage in the kidney contributing to microvascular dysfunction, renal inflammation and oxidative stress-induced damage[158, 162]. Importantly, the contribution of these mechanisms may vary depending on the precipitating factor. A recent study using mice models of ischemia reperfusion injury or cecal ligation and puncture reported a major contributing role of neutrophils in the setting of hypoperfusion-induced AKI whereas this was not the case in sepsis-related AKI which was characterized by pro-inflammatory cytokines, mitochondrial dysfunction and energetic failure[163].

In a preclinical model of alcohol-induced ACLF kidney dysfunction correlated with increased creatinine, blood urea nitrogen production and altered histology characterized by NETs indicating neutrophil infiltration in renal epithelial damage[25]. Neutrophil Gelatinase-Associated Lipocalin (NGAL or lipocalin 2) has been recently described as a marker of renal tubular epithelial injury in cirrhosis and ACLF. NGAL is also produced by damaged renal epithelial cells, so elevated levels indicate kidney damage, especially within the tubules, and are associated with poor clinical outcomes in patients with liver disease[164–166].

Kidney injury can be worsened by infection and sepsis, common precipitating factors of ACLF[6, 9]. In sepsis, hypoperfusion, mitochondrial functions such as oxidative phosphorylation and ATP production decrease while oxidative stress and apoptosis increase, leading to DAMP and mitochondrial DNA release that can further exacerbate inflammatory responses in the renal and systemic milieu[167].

4. Therapeutic implications, biomarkers and future directions

4.1. Immunomodulatory therapies in ACLF

Immune reconstitution is believed to prevent bacterial infections as precipitators of decompensation and organ failure and hereby alleviate the morbidity and improve survival in patients with ACLF in the future, although previously studied strategies failed.

Amongst, plasma exchange (PLEX) has not been shown beneficial in ACLF according to a recent systematic review[168], although earlier studies had described beneficial effects for the subgroup of HBV-ACLF patients as well as a metanalysis including mainly Asian studies with predominant percentage of HBV-ACLF patients[169]. A haemofiltration system including exchange of albumin and adsorption of DAMPs and other molecules, the DIALIVE device, has been shown beneficial in ACLF patients of diverse aetiologies in a first small randomized controlled trial (RCT)[170], larger studies are ongoing. Cytosorb^® filter added to haemofiltration absorbing circulating cytokines has also been studied in smaller cohorts of ACLF patients, but significantly improved prognosis has not been proven[171–173]. Elevated prostaglandin E2 (PGE₂) levels had been shown to suppress bacterial killing and cytokine responses in patients with AD, and albumin administration lowered circulating PGE₂ levels and restored TNFα production by macrophages in vitro[100], however not in an in vivo study in patients with decompensated cirrhosis[174].

The well-studied G-CSF administration promoting the release of CD34+ haematopoietic stem cells from the bone marrow which may differentiate into monocytes and replace dysfunctional subsets. G-CSF in this indication has been administered alone or in combination with erythropoietin or haematopoietic stem cells. While initial studies indicated beneficial effects on patients’ survival[175, 176], others did not including a recent multicentre randomised phase II study and cannot be recommended at this stage[177, 178]. In a mouse model of alcohol-associated liver disease, G-CSF administration proved to be a double edge sword, where G-CSF worsened liver damage in combination with LPS [179]. Potential interest remains in the TLR4 inhibitor TAK-242 with and without G-CSF combination, which inhibited inflammation, promoted regeneration and improved survival in preclinical in vivo studies[141, 180].

The accumulation of M-MDSC in the circulation and other tissues in ACLF aggravates immuneparesis[45]. Recent attempts ex vivo in monocytes from ACLF patients and in vivo in mice hinted at the potential of TLR3 agonist pI:pC to reduce M-MDSC and augment immune responses[46]. Immune checkpoint inhibition dramatically enhancing anti-tumor immunity may also improve responses against bacterial infection in ACLF. PD1 axis activation reduced bactericidal functions of macrophages in vitro and deserves further investigation[48, 181]. Also, high IL22/low IL22BP/IL22 ratios were associated with ACLF mortality and excessive IL22BP secretion neutralized IL22 in vitro [139]. In an CCl4/bacterial infection mouse model recapitulating ACLF, IL22Fc therapy enhanced regeneration and attenuated bacterial infection[140]. RIPK3, is one of the effector molecules in the necroptosis pathway together with RIPK1 and mixed lineage kinase domain-like protein. Recent preclinical[25] and clinical[182] studies highlight the relevance of RIPK3 as a biomarker and therapeutic strategy for ACLF. Pharmacological inhibition of RIPK3 with GSK047 reduced inflammation, neutrophil activation and improved extrahepatic organ function in a preclinical model of alcohol-induced ACLF[25].

There are a number of additional strategies that have been outlined above showing proof-of-concept to augment immune responses in ACLF in vitro, ex vivo and in vivo in animal models and may merit clinical evaluation summarized in Table 1. TLR 7/8 agonists (e.g. R848) stimulate NADPH oxidase 2 synthesis via mTOR- and AKT, MAPK, p47phox signaling pathways. In an in vivo model of cirrhosis, R848 decreased bacterial infection and improved survival by potentiating ROS production and bacterial killing by neutrophils[183]. Also, TAM receptors (Tyro-3, AXL, MERTK) converge on immunomodulatory pathways inhibiting TLR-signaling and enhancing efferocytosis[184] and MERTK expression is upregulated on monocytes and diverse tissue macrophages in ACLF. MERTK inhibitors have been clinically developed, and ex vivo proof of principle data has shown, that inhibition of MERTK may enhance immune responses of monocytes from ACLF patients[46]. AXL by contrast is overexpressed in cirrhosis patients without ACLF and in a very compartment-specific manner, suggesting therapeutic TAM receptor inhibition needs to be highly targeted and specific[49, 107]. Moreover up-regulation of autotaxin in ACLF patients promoted LPA production from lysophosphatidylcholines. High LPA levels suppressed monocyte MERTK/CD163 expression and increased their pro-inflammatory cytokine production and represents another targetable axis[42]. Immune cell metabolism has been shown indispensable for their physiologic function, and dysregulation causes immuneparesis also in the context of cirrhosis and ACLF[185]. Methionine sulfoximine, an inhibitor of glutamine synthase, restored the tricarboxylic acid cycle hereby augmenting phagocytic and inflammatory capacities of monocytes in vitro[58]. - Recently, the phospholipase C (PLC)/CD52 pathway has been implicated with regulation of monocyte function in patients with cirrhosis and ACLF; in ACLF high levels of PLC cleave CD52 expression on the cells hereby breaking down monocyte phagocytosis, cytokine production and migratory capabilities, suggesting inhibition of PLC and stabilization of CD52 in ACLF patients may be of therapeutic value and merit clinical investigation[55]. An integrated extracorporeal circuit (UTOpia) combining human induced pluripotent stem cell-derived hepatocyte-like cell (iHLC) with granulocyte and monocyte apheresis (GMA) was tested in a preclinical model of ACLF induced by bile duct ligation-induced fibrosis and LPS injection. ACLF mice under UTOpia treatment showed improved survival, liver biochemistry, reduced hyperammonemia, hyperbilirubinemia and systemic inflammation compared to HepG2, iHLC or GMA cell-based devices[186]

Table 1. Novel therapeutic strategies for ACLF based on preclinical models and clinical research including ongoing clinical trials.

Table 1 summarizes immunomodulatory targets for potential future immunotherapy for patients with ACLF.

Target	Agent	Mechanism	Phase	Reference
DAMPs	plasma exchange (PLEX)	By high- or standard volume PLEX, DAMPs released following tissue injury, PAMPs in case of infection and cytokines are cleared from the circulation in order to prevent secondary immune cell activation and tissue injury	Phase III (diverse clinical studies including radomised controlled trials (RCT))	[168, 169]
PAMPs	DIALIVE	Haemofiltration with additional filters for the exchange of albumin and adsorption of PAMPs (endotoxins).	Phase III (RCT)	[170]
circulating cytokines	CytoSorb®	Absortion of cytokines, bilirubin, bile acids in addition to haemofiltration in order to reduce auto-intoxication and hyperinflammation.	Phase III (1 RCT, 2 retrospective studies)	[171–173]
PGE2	Albumin	Albumin administration in patients with AD lowered circulating PGE2 levels and restored TNF-α production by plasma-conditioned MoMF in vitro and in an animal model, however not in vivo in patients with decompensated cirrhosis	Phase III	[100, 174]
CD34+ hematopoietic stem cells	G-CSF	G-CSF promotes release of CD34+ hematopoietic stem cells from the bone marrow which may differentiate into monocytes and replace dysfunctional subsets	Phase II	[175–178]
TLR4/NLRP3	TAK-242/RLX	Inhibition of macrophage TLR4/NLRP3 by TAK-242 or relaxin (RLX) reduces liver injury and inflammation in rodent models	Pre-clinical (animal model)	[180]
	G-CSF + TAK-242	Combining release of haematopoietic stem cells and inhibition of macrophage TLR4	Pre-clinical (animal model)	[141]
IL22	IL22Fc	in a CCL4/bacterial infection mouse model recapitulating ACLF, IL22Fc therapy enhanced regeneration and attenuated bacterial infection	Pre-clinical (animal model)	[140]
RIPK3	RIPK3 Inhibitor (GSK047)	Necroptosis inhibition improves inflammation, neutrophil activation and extrahepatic damage (kidney dysfunction and neuroinflammation).	Pre-clinical (animal model, ex vivo)	[25]
M-MDSC	pI:pC	Decreases M-MDSCs, augments phagocytic capacity	Preclinical (animal model, ex vivo,)	[44]
PD1	PD1/PDL1 inhibitors	immune-suppressive PDL1+ monocytes correlate with disease severity/infection risk in ACLF; PD1 axis activation on macrophages reduces bactericidal functions, alters cytokine production	Pre-clinical (ex vivo)	[48, 181]
TLR7/8	CL097, R848	decreased bacterial infection and improved survival by potentiating ROS production and bacterial killing by neutrophils	Pre-clinical (ex vivo)	[183]
MERTK	UNC569	MERTK inhibition restored LPS-induced pro-inflammatory cytokine response in monocytes from patients with ACLF ex vivo	Pre-clinical (ex vivo)	[46]
Autotaxin/LPA	LPA	autotaxin in ACLF patients promoted LPA production from lysophosphatidylcholines; LPA suppressed monocyte MERTK/CD163 expression and increased pro-inflammatory cytokine production	Pre-clinical (ex vivo)	[42]
Glutamine synthetase	Glutamine synthetase inhibitor (MSO)	Improves monocyte phagocytosis, increases pro- and reduces anti-inflammatory cytokine production	Pre-clinical (ex vivo)	[58]
CD52	PLC-inhibition	in ACLF high levels of PLC cleave CD52 expression on monocytes breaking down phagocytosis, cytokine production and migratory capabilities	Pre-clinical (ex vivo)	[55]
p21 and HNF4α	Integrated extracorporeal circuit (UTOpia) combining iHLC & GMA	iHLC-secreted α-fetoprotein suppressed p21 and enhanced regeneration. UTOpiA also restored HNF4α activity and decreased IL6 and TNFα.	Pre-clinical (ex vivo)	[186]

In addition, in relation to individualised future immunotherapy it will be very important to establish immunological biomarkers in order to identify patients at risk who may benefit from the respective treatment. Novel potential biomarkers to assess immune dysfunction, prognosis and survival are highlighted in Table 2.

Table 2. Immunological biomarkers for ACLF based on preclinical models and clinical research.

The table summarizes recent studies identifying potential biomarkers related to immune cells in ACLF to predict mortality, risk of infection/sepsis, monocyte function or organ damage/failure.

Biomarker	Method	Indicating	advantage	limitation	reference / source
CLIF-ACLFscore	Composite score including leucocyte count	mortality	Multidimensional, online tool	for AD/ACLD patients only	[187])
CLIF-SIG (systemic inflammation gene) score	RNA sequencing, composite of 28 immune cell related DE genes	systemic inflammation, transition to ACLF, mortality	28 validated genes from whole blood, validated in patients w/o cirrhosis	high costs, time delay of results	[188])
MLR / NLR	differential blood count	mortality, sepsis	low cost, fast, easy to calculate, comparable to CLIF-SOFA	leucocyte quantification method	[189][190])
sIgA	ELISA	mortality, gut-barrier dysfunction	surrogate for pathological bacterial translocation	small derivation and validation cohorts	[79]
monocyte HLA-DR / M-MDSC	flow cytometry	mortality, monocyte function	routine technique, validated in sepsis patients	not ACLF specific	[43][44]
PDL1+ monocytes	flow cytometry	mortality, SI, infection risk	routine technique, ev therapeutic monitoring	preliminary data	[47, 191])
CD52+monocyte, sCD52/PLC	flow cytometry, ELISA	mortality, monocyte function	routine technique, ev therapeutic monitoring	preliminary data	[55]
MERTK	flow cytometry, western blot	mortality, monocyte function	routine technique, ev therapeutic monitoring	preliminary data	[46]
PRO-C6	ELISA	mortality, extrahepatic organ failure, extrahepatic matrix modulation	ev extrahepatic OF marker	Preliminary, retrospective, uncertain pathophysiology	[37]

4.2. Challenges and future research directions in restoring immune homeostasis in ACLF

Immune dysfunction is a major component of ACLF pathology; therefore, it is expected that therapies that restore immune balance will improve ACLF outcomes. While the number of clinical trials has increased in recent years, challenges remain. First, ACLF is an acute clinical syndrome with rapid progression, poor outcomes in a vulnerable patient population that make clinical trials challenging. Second, standardization of enrollment criteria and study end-points is needed to allow comparison of different clinical trials and therapeutic candidates. Third, while preclinical studies identified new therapeutic targets, animal models have limitations in representing the complexity of human disease. Fourth, therapeutic strategy with a single agent may not be sufficient to ameliorate all aspects of the complex defects of hepatocyte dysfunction, inflammation and immune dysfunction. Therefore, ACLF research would benefit from large-scale translational studies and clinical data sharing to empower AI based tools for analysis of clinical outcomes, biomarker discovery and prediction models. The field is in need of prognosticating markers of response to therapy and new interventions. Finally, the underlying disease etiology, whether viral hepatitis or steatohepatitis, likely has disease-specific impacts on immune defects in ACLF. Future collaborative clinical trials should address these unsolved urgent questions in ACLF immunology.

Key points.

1. Immune dysfunction in ALCF involves common and cell-specific molecular and functional changes in both innate and adaptive immune cells.

2. Non-cellular signaling mediators importantly contribute to the deregulation of immune cells in a compartment and stage specific manner.

3. Immune cells concomitantly respond and contribute to the pathophysiology of multi-organ failure involving the liver, and most frequently also the brain, kidneys and gut.

4. Distinct immunomodulatory strategies may improve ACLF pathophysiology and reduce patient’s susceptibility to infection. Promising candidates for biomarkers assessing immune function and therapeutic intervention have been proposed.

5. To date, major challenges for assessing the indication and efficacy of novel ACLF immunotherapies include the compartment- and stage-specific adaptations of the immune milieu, lack of markers revealing the dynamics of immune function, patient vulnerability, clinical trial consensus and diverse disease aetiologies.

Abbreviations

AIM2

Absent in melanoma 2

AKI

Acute kidney injury

AD

Acutely decompensated

ACLF

Acute-on-chronic liver failure

ATP

Adenosine triphosphate

ACKR1+ PLVAP+

Atypical Chemokine Receptor/Plasmalemma Vesicle-Associated Protein

BTLA

B and T lymphocyte attenuator

CEACAM

Carcinoembryonic antigen-related cell adhesion molecule

CARS

Compensatory anti-inflammatory response syndrome

C

Complement factors

CI

Confidence interval

CXCL

C-X-C motif chemokine ligand

CXCR

C-X-C motif chemokine receptor

DCs

Dendritic cells

DNA

Deoxyribonucleic acid

EG

Emergency granulopoiesis

EV

Extracellular vesicles

GMA

Granulocyte and monocyte apheresis

HE

Hepatic encephalopathy

HSC

Hepatic stellate cell

HBV

Hepatitis B virus

HMGB1

High mobility group box 1

HLA-DR

Human leukocyte antigen – DR isotype

Ig

Immunoglobulin

iHLC

Induced pluripotent stem cell-derived hepatocyte-like cell

UTOpia

Integrated extracorporeal circuit

IFN

Interferon

IL

Interleukin

JAK

Janus kinase

LPM

Large PMs

LPS

Lipopolysaccharide

LSEC

Liver sinusoidal endothelial cells

LPA

Lysophosphatidic acid

MHC

Major histocompatibility complex

MERTK

MER proto-oncogene tyrosine kinase

miR

MicroRNA

mDNA

Mitochondrial DNA

MELD

Model for End-Stage Liver Disease

MCP

Monocyte chemoattractant protein

M-MDSC

Monocytic myeloid-derived suppressor cells

MPO

Myeloperoxidase

NK

Natural killer

NE

Neutrophil elastase

NET

Neutrophil extracellular trap

NGAL

Neutrophil Gelatinase-Associated Lipocalin

NLRP3

Nod-like receptor family pyrin domain containing 3

PAMPs/DAMPs

Pathogen- and damage-associated molecular patterns

PBMC

Peripheral blood mononuclear cells

PM

Peritoneal macrophages

PI3K

Phosphatidylinositol 3-kinase

PLC

Phospholipase C

PLEX

Plasma exchange

PMN

Polymorphonuclear

PD

Programmed cell death protein

SPM

Pro-resolving lipid mediators

PGE2

Prostaglandin E2

AKT

Protein Kinase B

RCT

Randomized controlled trial

ROS

Reactive oxygen species

RIPK

Receptor-Interacting Protein Kinase

Tregs

Regulatory T cells

RNA

Ribonucleic acid

SCFA

Short chain fatty acids

SIRS

Systemic inflammatory response syndrome

TIM

T cell immunoglobulin

TRM

Tissue-resident memory

TLR

Toll-like receptor

TREM

Triggering receptor expressed on myeloid cells

TNF

Tumor necrosis factor

VCAM1, ICAM1

Vascular and intracellular cell adhesion molecules