From hepatitis B virus infection to acute‐on‐chronic liver failure: The dynamic role of hepatic macrophages

Acute‐on‐chronic liver failure (ACLF) is a progressive disease that is associated with rapid worsening of clinical symptoms and high mortality. A multicentre prospective study from China demonstrated that patients with hepatitis B virus‐related ACLF (HBV‐ACLF) exhibited worse clinical characteristics and higher mortality rates compared to non‐HBV‐ACLF patients. Immune dysregulation is closely linked to the potential mechanisms of initiation and progression of ACLF. Innate immune response, which is represented by monocytes/macrophages, is up‐regulated across ACLF development. This suggests that monocytes/macrophages play an essential role in maintaining the immune homeostasis of ACLF. Information that has been published in recent years shows that the immune status and function of monocytes/macrophages vary in ACLF precipitated by different chronic liver diseases. Monocytes/macrophages have an immune activation effect in hepatitis B‐precipitated‐ACLF, but they exhibit an immune suppression in cirrhosis‐precipitated‐ACLF. Therefore, this review aims to explain whether this difference affects the clinical outcome in HBV‐ACLF patients as well as the mechanisms involved. We summarize the novel findings that highlight the dynamic polarization phenotype and functional status of hepatic macrophages from the stage of HBV infection to ACLF development. Moreover, we discuss how different HBV‐related liver disease tissue microenvironments affect the phenotype and function of hepatic macrophages. In summary, increasing developments in understanding the differences in immune phenotype and functional status of hepatic macrophages in ACLF patients will provide new perspectives towards the effective restoration of ACLF immune homeostasis.

mechanisms of ACLF and developing specific strategies for ACLF should be put on the agenda.
The majority of ACLF cases in Asia Pacific and Africa are attributed to hepatitis B virus (HBV) infection. 4The multicentre prospective study of the Chinese Group on the Study of Severe Hepatitis B (COSSH) demonstrated that the clinical characteristics of patients with HBV-related ACLF (HBV-ACLF) were significantly worse than the ones observed in those with alcohol-associated ACLF. 5 The shortterm (28/90 days) mortality of HBV-ACLF patients was significantly higher compared to that of the non-HBV-ACLF ones. 5Immune dysregulation is the most prominent change and disorder that is triggered by HBV reactivation. 6This is especially true with regard to innate immune response, represented by monocytes/macrophages, which is up-regulated as ACLF progresses that highlights the importance of the innate immune cells in the pathogenesis of ACLF. 6acrophages, the first cellular protective line of the innate immune defence in the liver, play a central role in maintaining homeostasis in the liver and in the initiation and progression of liver diseases, and have emerged as a critical player and therapeutic target in many chronic liver diseases. 7acrophages display a remarkable plasticity and can adapt their phenotype according to signals derived from the hepatic microenvironment, and differentiate into functionally diverse subsets. 8In the past few decades, more information on the significant heterogeneity of the function and genetic characteristics of hepatic macrophage has been revealed, [9][10][11] further highlighting these specialized cells as attractive targets for treating liver diseases.Recently, the growing number of publications have revealed that the immune status and function of monocytes/macrophages vary in ACLF precipitated by different chronic liver diseases.Revealing this phenomenon should be able to explain the differences in clinical outcomes between HBV-ACLF and non-HBV-ACLF.Considering the heterogeneous definitions of ACLF have been proposed in different regions of the world and the impact of the tissue microenvironment of different chronic liver diseases on hepatic macrophages, in this review, we summarize current knowledge about the different definitions of ACLF, and focuses on the dynamic polarization phenotype and functional status of hepatic macrophages from the stage of HBV infection to ACLF development.The role of macrophages in pathogenesis of chronic hepatitis B (CHB), liver fibrosis (LF), cirrhosis and ACLF will be the focus of this review.

| HETEROGENEITY IN DEFINING ACLF
Globally, there is no uniform definition of ACLF, mainly because the phenotype of this condition shows regional specificity, mainly as a result of differences in disease aetiology and inducing factors.At present, different regional research collaboration groups follow varying ACLF definitions and relevant standards, including the Asian Pacific Association for the Study of the Liver (APASL), 12 North American Consortium for the Study of End-stage Liver Disease (NACSELD), 13 European Association for the Study of the Liver-chronic liver failure (EASL-CLIF) consortium 14 and the World Gastroenterology Organization (WGO) 15 (Table 1).In brief, consortia in western countries developed definitions that are applicable to patients with cirrhosis, while consortia in Asia developed definitions that apply to patients with chronic liver diseases with or without cirrhosis.Even in the same continents, country-specific ACLF definitions are sometimes used.HBV-ACLF is the most common type of ACLF in China. 16The Chinese hepatitis B surface antigen (HBsAg)-positive population is estimated to be 86 million, and this accounts for 30% of all HBsAg carriers worldwide and 60% in HBV high-endemic areas.Based on these statistics, China is an optimum source of data that is representative of HBV-ACLF. 17To clarify the clinicopathological characteristics of patients with HBV-ACLF, the COSSH team established diagnostic criteria and prognosis scores of HBV-ACLF in China (Table 1). 5

OF HEPATIC MACROPHAGES
Liver macrophages consist of ontogenically distinct populations.According to the differences in sources, hepatic macrophages are divided into two categories: tissue resident macrophages and infiltrating macrophages.Liver-resident macrophages, namely Kupffer cells (KCs), originate from yolk sac and embryonic liver tissue, accounting for about 35% of non-parenchymal cells in liver and 90% of all tissue-resident macrophages, 18 and are self-sustaining by having independence from haematopoietic stem cells in adult tissues. 19Liver infiltrating macrophages are derived from circulating blood monocytes, peritoneal macrophages 20 or splenic monocytes 21 (Figure 1).KCs are self-renewing, resident and principally non-migratory phagocytes and serving as sentinels for liver homeostasis, as well as central to the initiation of hepatic inflammatory responses.Because the liver is constantly exposed to antigens from the intestine as well as low levels of bacterial endotoxins, many mechanisms suppress an 'accidental' activation of the immune system.KCs play a major role in maintaining immunological tolerance in the liver and in providing an anti-inflammatory micromilieu during homeostasis. 22uring liver injury, the KCs on the inner surface of hepatic sinuses can scan any foreign body skimming across the cell surface, constantly engulf dead cells and clear the blood borne pathogens reaching the hepatic sinuses. 8,23,24Pattern recognition receptors (PRRs) on KCs first respond to invading pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), provoking cytokine and chemokine cascade reactions while recruiting a large number of immune cells to infiltrate the liver. 7,25This fosters the infiltration of monocytes into the liver, which give rise to large numbers of inflammatory monocyte-derived macrophages.
Circulating monocytes are important contributors to hepatic macrophage replenishment and regeneration, as circulating monocytes have been shown to contribute to the KCs pool when a significant number of KCs die or depleted. 26Interestingly, when liver injury occurs, peritoneal macrophages have the ability to complete self-renewal and relocate from the peritoneum into the liver parenchyma by migrating across the mesothelial layer that covers the liver and invades deep into the tissue, 20 and drive hepatic immunopathogenesis. 27In addition, as the spleen is the storage and distribution site of monocytes, splenic monocytes can also be recruited to the liver for immune regulation during liver injury 28 (Figure 1).

| THE HETEROGENEITY OF HEPATIC MACROPHAGES
Hepatic macrophages have emerged as central players in liver injury, considering their manifold and even opposing functions during disease.These central functions include the perpetuation of inflammation and hepatocyte injury, activation of hepatic stellate cells with subsequent fibrogenesis and support of cirrhosis development by angiogenesis.If liver injury ceases, specific molecular signals trigger hepatic macrophages to switch their phenotype towards reparative phagocytes that promote tissue repair and regression of fibrosis. 25This seems like a pair of contradictory phenomena, but it involves the 'face-changing' attribute of hepatic macrophages at various stages, ranging from injury to repair.Macrophages are activated under the stimulation of pathogenic microorganisms, inflammatory responses or some cytokines, and differentiate into different phenotypes depending on the state and changes of the microenvironment. 29Simply put, the phenotypes and functions of the macrophages change throughout the whole process of liver injury.
Intensive research on animal models of liver injury and on samples from liver disease patients elucidated the complex heterogeneity of macrophages in the liver.Traditionally, macrophages are known to exhibit inflammatory or immunoregulatory properties as determined by their surface marker phenotype or cellular functions. 30Irrespective of the cellular origins of hepatic macrophages, they differentiate into various phenotypes, depending on the stimulatory signals in the tissue environment. 31Generally, activated macrophages have been categorized into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages), with M1 macrophages being predominantly involved in pro-inflammatory responses while M2 macrophages are mainly taking part in anti-inflammatory responses 32 (Figure 1).Depending on the activating stimulus and complex cytokines produced, M2 macrophages can be further classified into M2a, M2b, M2c or M2d subtypes. 33,34At present, the M1/M2 paradigm has provided a useful framework, especially for selected immune responses in vitro.Importantly, because macrophages can develop mixed M1/M2 phenotypes under pathological conditions in vivo, a more comprehensive classification is clearly required.We need to focus not only on macrophage populations but also at the single-cell level.The advent of single-cell analysis techniques has rapidly advanced the comprehension of the activation states and intricate phenotypes of macrophages.Recent data acquired through singlecell RNA sequencing unravelled a previously unrecognized complexity of human hepatic macrophage polarization abilities. 35Obviously, there is limited understanding on the phenotype of macrophages at present, and aspects surrounding the polarization and phenotype switch by macrophages in the liver still warrants extensive research.

| Limiting HBV infection and promoting hepatic inflammation
CHB is a dynamic infectious disease with a progression mode that strongly depends on the interaction between the host immune response and the virus. 36The presence of the HBsAg for at least 6 months can be regarded as chronic infection. 37HBV behaves like a 'stealth' virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. 38When exposed to high-titre HBV, macrophages can sense nucleic acids that are derived from viral pathogens or self-constituents and then initiate an innate immune response to HBV. 38 Some PRRs that are expressed by macrophages can recognize HBV capsid/core proteins such as Toll-like receptors (TLRs) or RIG-I/MDA-5, as well as some cytoplasmic DNA sensors. 39Within 3 h, this recognition transitions to the activation of nuclear factor kappa B (NF-κB) and subsequently, the release of pro-inflammatory cytokines, including interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)-α, IL-1β and others.Pro-inflammatory cytokines that are released by macrophages control HBV gene expression and replication in hepatocytes at the transcription level, shortly after infection. 40HBV is unable to replicate in macrophages, though some researchers detected antigen of this virus in macrophages and monocytes.This possibly suggests that macrophages or monocytes may directly engulf HBV. 41,42acrophages can be induced by the debris of dead hepatocytes and HBV to undergo M1-like pro-inflammatory polarization.Anti-HBV cytokines that are secreted by M1 macrophages mediate virus clearance.Type I interferon that is synthesized by M1 macrophages is one of the critical anti-HBV factors.][45] Moreover, the TNF-α, 46,47 IL-1β 48,49 and IL-6 40 produced by M1 macrophages have also been identified as effective anti-HBV factors as they are capable of suppressing HBV gene expression and replication at the transcript level.The pro-inflammatory factors that are secreted by macrophages not only directly inhibit HBV transcription but also trigger specific immune response.IL-12 initiates terminally differentiated effector memory responses that have strong, long-term anti-HBV effects, and enhances HBV-specific cluster of differentiation (CD) 8 + T and CD4 + T cell responses. 50IL-18 is a determinant factor in controlling the balance of helper T cell (Th) 1/2 during the antiviral response, 51 in addition to enhancing the response of Th1 to viral infection. 52BsAg-induced pro-inflammatory cytokines production by macrophages may be early events in viral containment.This event not only support the induction of HBV-specific immunity upon infection by the virus, but also aggravate liver immunopathology in the process. 41ncreased CD16 + monocyte/macrophage subsets in the peripheral blood and liver exacerbate inflammatory microenvironment and liver injury in patients with CHB. 53ther cytokines like IL-23 are worth noting because they promote inflammatory injury in patients with CHB.IL-23 cytokines regulate innate and adaptive immunity processes and are critical mediators of the pro-inflammatory effects that are exerted by Th17 cells.In biopsied liver tissues from HBV-infected patients, the expression levels of IL-23 and IL-23R were remarkably elevated.HBsAg induces the production of IL-23 in macrophages and causes liver damage via the IL-23/IL-17 axis. 54In conclusion, M1 macrophages can recognize and engulf HBV, thereby establishing an effective immune control of the viral infection.This helps to prevent, control and delay chronic infections.However, the anti-infectious immune response of M1 macrophages causes some degree of pathological lesions to the host.

| HBV evade immune clearance by modulating macrophage polarization
The persistence of HBV infection and the pathobiology of CHB were caused by viral replication and interactions that are associated with the host immune.Chronic HBV infection has a suppressive effect on the innate immune response, which is more obvious in cases of high level of HBsAg. 55Emerging evidence suggests that HBV tend to evade immune clearance by modulating macrophage polarization.M2 type macrophages are more common in CHB patients, and they have anti-inflammatory and immunosuppressive effects.When HepG2.2.15 was cocultured with macrophages, HBV-inhibited M1, while enhancing M2 markers.This observation was accompanied by reduced pro-inflammatory TNF-α and augmented expression levels of anti-inflammatory IL-10. 56IL-10 and transforming growth factor (TGF)-β that are predominantly derived from M2 macrophages are considered as prototypical pleiotropic anti-inflammatory and immunosuppressive cytokines.IL-10 and TGF-βproducing monocytes induced by HBV initiated immunosuppressive cascade reactions.Such suppressive monocytes trigger the differentiation of regulatory natural killer (NK) cells to produce IL-10, ultimately resulting in T-cell inhibition. 57In a mouse model for persistent HBV infection, levels of serum TGF-β remained elevated at 6 months post-infection. 58,59fter exposure to HBV, M2 macrophages secreted more IL-10, thereby promoting hepatocyte infection. 60M2 macrophages that secrete IL-10 inhibited the production of anti-HBV cytokines (IL-6 and TNF-α) through an autocrine negative feedback loop. 61lthough the effects of HBV on macrophage polarization remain unclear, several in vitro studies suggested that HBsAg/HBeAg may be involved. 57,62,63Mechanistically, HBsAg and HBeAg could up-regulate the SIRT1/ Notch1 pathway, a scenario that leads to increased Akt phosphorylation and decreased NF-κB nuclear translocation in macrophages, ultimately contributing to M2 macrophage polarization. 56Interestingly, HBV-triggered phosphatase and tensin homologue signalling can also enhances programmed death ligand 1 (PD-L1) expression and promotes HBV immune evasion. 64PD-L1, as a pivotal immune checkpoint, is a negative regulator of the immune response and binds to its receptor programmed death 1 (PD-1).PD-L1 is expressed in macrophages, has been shown to be a major obstacle to antiviral immunity in CHB. 65,66PD-L1 silencing in liver enhances efficacy of therapeutic vaccination for CHB. 67The available data indicate a close relationship between high expression of PD-L1 and infiltrating M2 macrophages.9][70] Moreover, there have been more reports on the contribution of several miRNAs to macrophage polarization as induced by HBV-associated antigens. 71,72hese findings suggest that the mechanisms that cause macrophage polarization that is induced by HBV are highly complex, and co-regulated by multiple signalling pathways.This may be an important explanation for the notion that macrophage polarization plays multiple roles in HBV infection.In short, interactions between HBV and macrophages may be key to explaining the long 'window period' during acute infection and HBV's propensity to chronic infection.HBV-activated macrophages initiate an inflammatory response and immune clearance mechanisms.On the other hand, HBV is also capable of developing strategies for evading immune responses by regulating the phenotype and functions of macrophages.

| Promoting LF progression
LF is the final common pathway for chronic or repeated liver injury, and it is an inevitable stage for the development of CHB.During chronic HBV infection, the ongoing inflammatory response results in persistent hepatocellular damage.The process is accompanied by extensive vascular remodelling and disintegration, which eventually leads to LF and cirrhosis. 73Evidence from clinical and animal studies has shown that hepatic macrophages play important roles in the process of LF.In the initiation of LF, KCs are activated by small molecules generated by hepatocytes injury.KCs initiate immune responses through rapidly secreting cytokines and chemokines, such as IL-1β, TNFα, chemokine (C-C motif) ligand (CCL) 2 and CCL5, and then recruit other types of immune cells such as monocytes to infiltrate the site of injury, which aggravates the pro-inflammatory immune responses in the liver. 25,74owever, the number of KCs decline rapidly in the initiation stage of fibrosis, and gradually recover with the regression of inflammation and resolution of fibrosis. 75By contrast, monocyte-derived macrophages are significantly accumulated after liver injury.
The pathophysiology of LF is multifactorial and involves the activation of hepatic stellate cells (HSCs), which is considered a driver of hepatic fibrosis.Macrophages have diverse, complex and important pathways that influence HSCs.Signals, such as inflammatory stimulation and cytokines, can transform HSCs into myofibroblasts and promote LF, while producing extracellular matrix (ECM). 76,77ecent research found that HBeAg induced a transient inflammatory reaction and promoted early LF in a macrophage-dependent manner.HBeAg activated macrophages via the TLR2/NF-κB signalling pathway and further exacerbated hepatic fibrosis by facilitating the motility, proliferation and contraction of HSCs, with the help of soluble factors that are secreted by macrophages. 78eljaars et al. 79 localized and quantified different macrophage phenotypes in hepatic tissue with cirrhosis and they observed increased numbers of M1 and M2 macrophages during the formation stage of LF.Nevertheless, the phenotype of the hepatic macrophages during injury depends on the nature of the damage, be it infectious, metabolic or toxic.M1 and M2 macrophages co-existed in carbon tetrachloride-induced LF, which suggested that both of them might be involved in fibrosis. 79And in fibrosis induced by non-alcoholic fatty liver disease, hepatic macrophages show a M2 phenotype at the early stage and a M1 phenotype at the late stage. 80,81However, M2 macrophages predominate in LF that is induced by virus, 82,83 which suggested that HBV appears to continue to play a role in immune escape by regulating macrophage phenotype.Soluble CD163 (sCD163) and CD206 is a marker for the cells of the M2 macrophages.In cases where the liver is highly affected by fibrosis, the organ has been shown to have a higher density of CD206 + macrophages.Moreover, blocking the accumulation of CD206 + macrophages mitigated LF in HBV-infected humanized mice. 84sCD163 originates from the proteolytic cleavage of CD163 + macrophage receptors by matrix metalloproteinases (MMPs). 85s the numbers of CD163 + macrophages increased, so did the severity of HBV-related LF. 83 Highly specific diagnosis of substantial fibrosis could be made using sCD163.The levels of sCD163 were significantly reduced by antiviral therapy, and the degree of LF was remarkably lower.Therefore, the activation of M2 macrophages was crucial in the pathogenesis of HBV-related LF. 83 M2 macrophages induced Th2 immune response and secreted antiinflammatory cytokines such as TGF-β, thereby inhibiting immune response, dampening inflammation, as well as promoting healing of wounds and fibrosis. 86Interestingly, M2 macrophages could be activated by autocrine production of TGF-β, which aggravated hepatocirrhosis. 87 couple of studies have demonstrated that reducing the overexpression of TGF-β, blocking the signalling pathway or deleting genes could alleviate fibrosis.Interventions that align with these findings have been considered as promising therapeutic strategies against LF. 88-91

| Orchestrating fibrosis regression in liver
The liver has an extensive regenerative capacity, and LF is not completely irreversible.Evidence in humans and rodents implies that LF is a reversible process if the causative factors are eliminated. 59,92In the dynamic and reversible process of LF, macrophages not only promote fibrosis but also reverse its progression. 93,94Macrophages regulate the balance of fibrosis in the liver by secreting matrix-degrading enzymes, such as MMPs, which are critical in the regression of LF.6][97] Increased activity of MMPs could downregulate the activation of HSC and expression of MMPs inhibitors. 98ne study found that ingestion of cellular debris could influence the phenotype of macrophages.Lymphocyte antigen 6C low (Ly6C lo ) macrophage was observed to be the dominant phenotype after engulfing the cellular debris. 95his was critical for enhancing the regression of LF and restoration of normal structure.This Ly6C lo macrophage subset was most abundant in livers during fibrosis resolution and represented the principle MMPs-expressing subset.Increased active MMPs expression, loss of proinflammatory gene expression and engulfment gene enrichment were induced by macrophage polarization from the lymphocyte antigen 6C high (Ly6C hi ) phenotype to the Ly6C lo one. 95A recent study supported the notion that exosomal miR-30a-5p derived from macrophages causes activated HSCs to become quiescent HSCs by targeting apoptosis signal-regulating kinase 1, while also changing macrophage phenotype from Ly6C hi to Ly6C lo . 99Although Ly6C has shown use in defining distinct macrophage subsets in tissue.Indeed, Ly6C hi macrophages and Ly6C lo macrophages could express markers of both M1 and M2 macrophages. 21ven more interesting, contrasting results have been obtained from human and animal studies.M2 macrophages almost completely disappeared in livers that were undergoing resolution, while the numbers of M1 macrophages remained almost unchanged. 79Research clearly indicates that experimental results with regard to the role of macrophagepredominant phenotype in the regression of LF have been controversial.The exact macrophage/monocyte phenotype in LF regression is still not fully characterized.The macrophage phenotype identified in humans or rodents during the LF process cannot be easily assigned to the M1 or M2 subphenotypes.This reflects the limitations of the M1/M2 classification of macrophages in interspecies and in vivo studies in complex environments.Chronic fibrosis eventually progresses into cirrhosis, which not only represents scar deposition, but also exhibits anomalies of hepatic parenchymal cells and vascular structures.During LF, elevated shear stress and remodelled liver sinusoidal endothelial cells (LSECs) caused LSECs to lose their fenestrations.Moreover, the basement membrane appeared under the endothelium, assuming a structure that is similar to that of continuous capillaries, 100,101 and this was termed hepatic sinusoidal capillarization.Sinusoidal capillary structure compromises the bidirectional exchange of materials between sinusoids and hepatocytes, leading to hepatocellular dysfunction.DAMPs that are released by damaged hepatocytes could initiate sterile macrophage activation.During the initial stage of hepatic sinusoidal capillarization, LSECs released angiocrine signals and up-regulated levels of the chemokine CCL2 and human bone morphogenetic protein-9, which facilitated macrophage recruitment to the liver and promoted pro-inflammatory responses. 102,103ver-activation of macrophages might trigger stenosis or even block the sinusoidal lumen.Accumulating evidence indicated that crosstalk between macrophages and LSECs was critical for the capillarization of hepatic sinusoids and the phenotype transition of LSECs.At the cellular level, human LSECs completely lost their fenestrations, while platelet endothelial cell adhesion molecules increased and LSECs were dedifferentiated by being co-cultured with rat KCs. 104Additionally, activated KCs produce various cytokines like resulting in neovascularization. 1057][108] KCs were involved in inducing oxidative stress and generated numerous vasoconstrictor substances, such as leukotrienes and thromboxane A2, which raised portal pressure in a cirrhotic liver. 109,110A recent study reported that sCD163 levels were elevated in patients with cirrhosis but were normal in patients with non-cirrhotic portal hypertension. 111This suggested that the primary driver for hepatic macrophage activation was the underlying liver disease with cirrhosis, and portal hypertension might be caused by activation of macrophages.Therefore, targeting hepatic macrophages might be an effective therapy for portal hypertension in cirrhosis.

| Immunodeficiency of macrophages in cirrhosis
The normal liver architecture is disrupted and the ability to synthesize proteins is impaired in cirrhosis.These events damaged liver-immune surveillance by impairing the reticulo-endothelial system, as well as innate immune protein and PRRs synthesis.Thus, immune dysfunction describes a range of anomalous outcomes that are caused by cirrhosis.These abnormalities infect the cellular and soluble components, typically from liver or systemic immune responses.3][114] The term cirrhosis-associated immune dysfunction (CAID) was used to describe the distinctive spectrum of immune alterations that are associated with cirrhosis.Systemic inflammation and immune deficiency are the key features of CAID. 114In line with this notion, immune dysfunction in cirrhosis can be treated as a continuous process that proceeds from chronic hepatitis and deteriorates with the progress of cirrhosis and portal hypertension.Additionally, the situation may be further exacerbated due to bacterial translocation, resulting in complete immune depletion in ACLF.
CAID involved components of the innate immune system, especially monocytes/macrophages, which were pivotal effectors and target cells in CAID. 115During cirrhosis, the tolerance of macrophages is reduced, and subpopulations of KCs are substituted by pro-fibrotic macrophages. 116he activated KCs expressed TLR4/CRIg and lost tolerogenic phenotypes, but the effector response was not markedly compromised.KCs secreted pro-inflammatory cytokines and chemokines like CCL2 to amplify the immune response.They also recruited bone marrow-derived monocytes and neutrophils into the liver. 117The study found that increased gut-dysbiosis, gut barrier dysfunction and bacterial translocation exacerbated cirrhosis. 118nnate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). 115acrophages exposed to an array of bacterial products, especially of LPS, for a long period could inhibit subsequent inflammatory responses via TLR4 signalling. 119,120It was unacceptably monocytes/macrophages to be further stimulated by LPS or microbial stimulus in this state, indicative of immune tolerance.In addition, the anti-inflammatory cytokine IL-10 may contribute to the endotoxin tolerance of monocytes/macrophages in liver cirrhosis. 121unctional reprogramming of macrophages is also accompanied by cell phenotypic alteration.The results of this study were consistent with reports that suggested an increased expression of scavenger receptor CD163 and reduced expression of human leucocyte antigen-DR (HLA-DR) in monocytes/macrophages.The scavenger receptor CD163 was regulated by a variety of inflammatory mediators and represented an anti-inflammatory function.High sCD163 level was useful in identifying patients who are at a high-risk of death during an acute decompensation episode that is complicated by a bacterial infection. 122HLA-DR on monocytes/macrophages is a prerequisite for effective antigen presentation and processing, presents antigen to CD4 + T cells, which is an important component of the immune response to infection. 123Decreased HLA-DR expression is a biomarker of immunosuppression, a condition that might contribute to impaired antigen presentation and the development of an adaptive immune response.
As confirmed by some studies, bacterial translocation, endotoxemia, inflammation and T-cell activation in cirrhosis were accompanied by an increase in circulating anti-inflammatory cytokines IL-10 and reduced HLA-DR expression. 121,124Other studies demonstrated that decreased monocytic HLA-DR expression was attributed to the amplification of monocytic myeloid-derived suppressor cells which have a highly immunosuppressive capacity.During end-stage liver disease, HBV reactivation serves as the main precipitating factor in the ACLF population.It is also possible that it may be a spontaneous event driven by immunosuppression.HBV reactivation is defined as the loss of HBV immune control in HBsAg-positive, anti-HBc-positive or HBsAg-negative, anti-HBc-positive patients receiving immunosuppressive therapy for a concomitant medical condition. 37The notion that based on the immunological characteristics of ACLF, intense systemic inflammation and immunosuppression exist in parallel. 126Genomic analysis revealed that patients with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils and macrophages M0-like monocytes, as well as depletion of several lymphocyte subsets like memory B cells, resting memory CD4 T cells, CD8 T cells and NK cells. 127onocyte/macrophage dysfunction and reprogramming plays a central role in immune dysfunction.Immune dysfunction in ACLF is a complex and multifactorial form, with the pathophysiology involving defects and abnormalities of cellular and soluble immune components. 128,129Transcriptional profiling of isolated monocytes in ACLF revealed the up-regulation of an array of immunosuppression parameters, which compromised antibacterial and antigen presentation machinery. 130Numerous studies have shown that ACLF patients have lower expression of HLA-DR and higher expression of CD163/Mer tyrosine kinase (MerTK) monocytes.1][132][133] During the progression of HBV-related liver diseases, including hepatitis, compensated cirrhosis, decompensated cirrhosis and ACLF, the percentage of CD163 + CD206 + macrophages increased, while that of CD80 + HLA-DR + macrophages significantly decreased. 134Monocytes/macrophages mainly exhibited high expression of CD163 + CD206 + in patients with HBV-ACLF, which polarized towards an immune tolerance state. 134In addition, PD-L1 on monocytes/ macrophages have been demonstrated to be involved in regulating the immunosuppressive mechanism of ACLF.The CD68 + macrophages and CD16 + monocytes of these patients have highly suppressive markers PDL1 expression increasing susceptibility to infections. 135Monocytes of patients with ACLF with systemic inflammation and sepsis showed reduced HLA-DR but increased PD-L1 expression. 136Up-regulation of monocytic PD-L1 expression predicts 28-day mortality of HBV-ACLF. 137unctionally reprogramming monocytes/macrophages reveals numerous acquired defects in ACLF that impair anti-microbial abilities of these cells, thereby contributing to enhanced susceptibility to sepsis.Patients with ACLF have an increased number of peripheral blood monocytes, a scenario that displayed significant defects in phagocyte and oxidative burst capacity. 125,130,138The serum levels of anti-inflammatory cytokines like IL-10 and IL-1RA gradually increased and the responsiveness of pro-inflammatory cytokines to microbial attacks reduced during ACLF development. 139oreover, monocyte/macrophage that were repeatedly exposed to PAMPs exhibited reduced responses to second stimulation with LPS.This attenuated innate response is considered as a refractory state that favours the development of secondary infections and even increased mortality. 128,132

STRONG INFLAMMATION
Systemic inflammation is currently recognized as one of the pathophysiological characteristics for ACLF.][142] Bacterial infection plays a major role in inducing strong systemic inflammation and is associated with the adverse clinical course and high mortality rate of ACLF. 143epatophilic viruses such as HBV are involved in the entire process from hepatitis to LF, cirrhosis and the ultimate liver failure.As the disease progresses, bacterial infection is a key driving factor or life-threatening complex in ACLF.The short-term (28/90 days) mortality of patients with HBV-ACLF was significantly higher than that of patients with non-HBV-ACLF, 5 and this result was possibly related to the strong innate immunity that is enhanced by activated monocytes/macrophages.Systemic inflammation in seven was characterized by an excessive innate immune response, which was associated with disease progression and mortality. 144imilarly, in patients with HB-ACLF, monocytes significantly increased the expression of HLA-DR, receptor C-C chemokine receptor type 2 and CD80, in addition to enhancing LPS-stimulated secretion of TNF-α, IL-6 and IL-1β. 145However, phagocytic capacity remained unaffected.It was reported that acute liver failure caused by HBV is associated with high levels of NK cells and macrophages infiltration, while fierce immune inflammatory response is identified based on gene expression profiles. 146These interesting research findings suggest that HB-ACLF enhances innate-immune activation, while HBV-ACLF exhibits immune suppression.
Currently, relatively few studies have focused on HB-ACLF.This could be because the number of patients with cirrhosis in ACLF is high.Another reason could be that most clinical files on ACLF contained no detailed descriptions regarding characteristics of chronic liver diseases.In fact, the heterogeneity of diagnostic criteria for ACLF may mask the immune differences between HB-ACLF, HBV-ACLF and non-HBV-ACLF.Therefore, subgroup analysis of different chronic liver diseases in ACLF is aimed at generating hypotheses for future research.

| CONCLUSIONS
The effects of HBV infection on the phenotype-switching tendency by macrophages may favour initial virus replication.However, in the long run, macrophages also contribute to the pathogenesis of liver diseases that include fibrosis and cirrhosis, eventually leading to ACLF.Macrophages show distinct phenotypic and functional characteristics in HBV-ACLF precipitated by different chronic liver diseases.The difference in the immune status of hepatic macrophages is related to different chronic liver disease tissues microenvironments, and affects the clinical outcome of ACLF.Reports from existing studies do not completely shed light on the mechanisms associated with this discrepancy.Inspired by recent research, these problems are yet to be explored to determine whether specific regulation of the hepatic macrophage phenotype is beneficial in inhibiting the progression of HBV-related liver disease or not.More research needs to be focused on the subgroup analysis of different chronic liver diseases in ACLF.A significant effort should be made to better understand the correlation between the clinical prognosis of ACLF precipitated by different chronic liver diseases and the immune status of hepatic macrophages.In summary, understanding the immunophenotype and functional status of monocytes/macrophages in different chronic liver diseases and ACLF will provide information that helps to restore liver immune homeostasis.

F I G U R E 1
Origins and functions of hepatic macrophages in healthy and diseased livers.The figure depicts the diverse origins and functions of hepatic macrophages in healthy and diseased livers.Hepatic macrophages are composed of hepatic resident macrophages and infiltrating macrophages.Hepatic-resident macrophages (Kupffer cells, KCs) originate from yolk sac and nascent foetal liver.Erythro-myeloid progenitors develop in the yolk sac, migrate into the nascent foetal liver and differentiate into haematopoietic stem cells.Haematopoietic stem cells expand/differentiate into resident KCs in the foetal liver.KCs fill the sinusoidal lumens, scanning for any pathogens flowing through hepatic sinusoid.KCs initiate inflammatory signals of hepatic injury while recruiting immune cells to infiltrate the liver.Infiltrating macrophages can arise from circulating blood monocytes, peritoneal macrophages or splenic monocytes.Hepatic macrophages mediate different functions in organ homeostasis, impairment, restoration, immunomodulation and in fibrosis.Traditionally, macrophages are classified as M1 or M2, as determined by their surface marker phenotype or cellular functions.Arg-1, arginase-1; CD, cluster of differentiation; iNOS, inducible nitric oxide synthase; LSECs, liver sinusoidal endothelial cells; Ly6C, lymphocyte antigen 6 complex locus C1; MHC-II, major histocompatibility complex-II; MSC, myeloid stem cell.

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Macrophages results in abnormal vascular structure
T A B L E 1