Immune Checkpoints and the Immunology of Liver Fibrosis

Abstract

Liver fibrosis is a very complicated dynamic process where several immune cells are involved. Both innate and adaptive immunity are implicated, and their interplay is always present. Multi-directional interactions between liver macrophages, hepatic stellate cells (HSCs), immune cells, and several cytokines are important for the induction and perpetuation of liver fibrosis. Detailed studies of proteomics and transcriptomics have produced new evidence for the role of individual cells in the process of liver fibrosis and cirrhosis. Most of these cells are controlled by the various immune checkpoints whose main function is to maintain the homeostasis of the implicated immune cells. Recent evidence indicates that several immune checkpoints are involved in liver fibrosis. In particular, the role of the programmed cell death protein 1 (PD-1), the programmed death-ligand 1 (PD-L1), and the role of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have been investigated, particularly after the availability of checkpoint inhibitors. Their activation leads to the exhaustion of CD4+ve and CD8+ve T cells and the promotion of liver fibrosis. In this review, the current pathogenesis of liver fibrosis and the immunological abnormalities are discussed. The recent data on the involvement of immune checkpoints are identified as possible targets of future interventions.

1. Introduction

The development of liver fibrosis (LF) is characterized by the deposition of extracellular matrix (ECM) proteins produced by myofibroblasts (MFs) of various origins including epithelial cells, mesenchymal stromal cells (MSCs), and HSCs [1]. Hepatocyte damage, irrespective of etiology, leads to the recruitment of immune cells in the liver. Quiescent HSCs (qHSCs) are activated and transformed into MFs, which are the main producers of connective tissue elements. If the insult is short term, the pro-fibrotic and anti-fibrotic mechanisms of the liver are in balance, and LF is not likely to occur. The continuous activation of the heterogenous population of hepatic MFs, mostly driven by liver macrophages, is the hallmark of chronic liver disease (CLD), but several other cells of the innate and adaptive immunity are also implicated [2,3]. The advancement of liver fibrosis leads to the final stage of cirrhosis. The pathological characteristics of cirrhosis are extensive fibrosis, the development of regenerative nodules, and the distortion of the hepatic architecture leading to overt clinical manifestations [4].

Global epidemiological data [5,6] reveal that almost 1.5 billion people suffer from CLD, leading to approximately 20,000 annual deaths, half of which are direct complications of liver cirrhosis. The overall mortality from cirrhosis has increased by 47.15% in recent years [7]. The WHO’s Global Burden of Diseases reports indicate that 560.4 age-standardized disability-adjusted life-years (DALYs) per 100,000 population worldwide were due to cirrhosis. In comparison, only 151.1 DALYs were due to liver cancer [8]. The most frequent causes of CLD are viral hepatitis, alcoholic liver disease, and metabolic-associated fatty liver disease (MAFLD/MASH) with heterogeneity across geographical regions [9]. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the underlying etiology of more than 60% of cirrhotic cases worldwide [10]. Less frequent etiologies that lead to fibrosis and cirrhosis include, among others, genetic diseases of iron or copper overload, cholestatic syndromes, and autoimmune diseases [4]. An important step in the clarification of the immune modulation and the therapeutic potential of the inhibition of certain immune checkpoints practically started when James Allison described the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and Tasuku Honjo described the programmed cell death protein 1 (PD-1). They were awarded the Nobel Prize for Physiology or Medicine in 2018 [11,12]. Immune checkpoint molecules are ligand–receptor pairs that exert inhibitory or stimulatory effects on immune responses. Most of the immune checkpoint molecules that have been described so far are expressed on cells of the innate and adaptive immune system, particularly on natural killer (NK) cells and T cells, respectively. They maintain the self-tolerance and modulate the immune responses of effectors in different tissues to minimize the tissue damage. Immune checkpoint proteins (ICPs) trigger the exhaustion, senescence, or apoptosis of effector immune cells [13].

It is, therefore, imperative to delineate the mechanisms implicated in the process of fibrosis. The present review will focus on the current data on the pathogenesis of liver fibrosis with emphasis on the immunological aspects and the emerging roles of the immune checkpoints.

2. A Pathogenetic Overview of Liver Fibrosis

2.1. The Fibrotic Process

For descriptive reasons, four pathological stages of fibrosis can be identified. Initially, exogenous or endogenous elements cause damage to the local liver cells and induce inflammation, followed by the large recruitment of immune cells at the site of the initial damage, thus aggravating the inflammatory response. Damaged hepatocytes or activated sinusoidal cells, by whatever cause, produce cytokines such as tumor necrosis factor-alpha (TNF-α) and IL-1, which are responsible for the recruitment of extrahepatic immune cells. In addition, they secrete pro-fibrotic factors, generating the background for the third stage. Quiescent hepatic stellate cells (HSCs) are transformed into myofibroblast-like HSCs [14,15] that lead to the fourth stage, the deposition of large amounts of ECM, and the remodeling of the liver architecture [16,17,18].

The ECM is composed of collagens, including type I (the most abundant protein) and type III collagens, fibronectin, elastin, and smaller amounts of several other proteins. The important components of the ECM are the basement membrane proteins such as laminin. It should be noted that myofibroblasts first secrete procollagen into the tissue, while mature collagen fibers evolve at a later extra-cellular stage through modification and cross-linking [19].

An important mechanism of fibrosis is the Epithelial-to-Mesenchymal Transition (EMT). It implies the differentiation of epithelial non-mesenchymal cells that acquire a fibroblast phenotype. Their participation in liver fibrosis has been extensively studied [20,21,22,23]. Epithelial cells undergo EMT under the influence of certain stimuli. An important one is the snail family transcriptional repressor 1 (Snail1). Hepatocytes with the deletion of the snail1 gene showed a significant decrease in EMT. Snail1 affected genes known to contribute to the progression of liver fibrosis, increasing the expression of pro-fibrotic genes such as those involved in collagen and vimentin production in the liver [24]. Moreover, the expression of Snail1 increased during the TGF-β1-induced EMT in murine hepatocytes, while the expression of the miR-30 family members was significantly downregulated. miR-30 inhibited the EMT transformation in hepatocyte by targeting Snail1 [25]. Studies of cholestatic liver diseases have demonstrated the participation of EMT transformation in experimental and in human cholangiopathies such as biliary atresia. Cholangiocytes acquire mesenchymal markers and lose their epithelial characteristics [26,27,28]. The involvement of liver macrophages in EMT hepatocyte trans-differentiation has not been conclusively proved. Data from extrahepatic cancers indicate that the involvement of liver macrophages cannot be ruled out [29,30].

Recent evidence has indicated a significant role of non-coding RNAs in liver fibrosis. Myofibroblast activation is positively or negatively modulated by a number of non-coding RNAs [31]. They act on either EMT or through the ECM. ECM deposition can suppress miR-29, an important negative regulator of pro-fibrotic genes. Consequently, many such genes are recruited in the ribosomes and sustain the deposition of ECMs even in the absence of the initial stimulus. On the other hand, increased ECM stiffness activates the Hippo pathway effector Yes-associated protein 1 (YAP1), which also increases ECM deposition, thus initiating another positive feedback loop mediated by miR-21 [32]. miRNAs are implicated in liver fibrosis and stellate cell activation by targeting SMAD proteins [33]. MiR-199a promotes EMT transformation and fibrosis by increasing the expression of genes encoding procollagens and the tissue inhibitor of metalloproteinase-1 (TIMP1) [34]. MiR-32 is also pro-fibrotic in hyperglycemia in experimental conditions. Its inhibition attenuated EMT-induced liver fibrosis [35]. Several other miRNAs increase fibrosis in liver damage, affecting fibrotic pathways such as transforming growth factor-β/Smad, Wnt/β-catenin, and snail [36,37].

HSCs activation is also influenced by several anti-fibrotic miRNAs such as miR-16 and miR-19b among others that retain the quiescence of HSCs or induce either apoptosis or the de-differentiation of activated HSCs [37]. In more detail, miR-30a attenuates the EMT process by reducing TGF-β1. There is an inverse relation between mir30 and snail1, indicating that snail1 is a possible target of mir30, as mentioned before [38]. In addition, miR-30a can repress fibrosis by suppressing beclin-mediated autophagy [39].

Long non-coding RNAs (lncRNAs) are also implicated in liver fibrosis. An upregulation of lncRNA H19 in murine fibrosis activated the EMT pathway [40]. GAS5 acts as a sponge platform for miR-23a, a fact that ameliorates the progression of fibrosis [41]. The overexpression of Meg8 lncRNA was noticed during the activation of HSCs. Meg8 repressed the pro-fibrotic genes in activated HSCs and EMT, while its knockdown induced the expression of mesenchymal markers in hepatocytes [42].

In murine MASH models, the circRNA_29981 was identified as a possible regulator of HSC transformation [43]. Moreover, the mitochondrial circRNA SCAR can close the mitochondrial permeability transition pores, repressing the activation of MFs by inhibiting the mitochondrial ROS output [44].

A third important mechanism implicated in the regulation of liver fibrosis is the involvement of transcription factors such as the nuclear receptors (NRs) [45]. They mediate anti-inflammatory effects through direct interaction with other transcription factors, such as NF-κB [46,47]. They also have a fundamental role in liver regeneration and HSC activation [48,49]. The farnesoid X receptor (FXR) is better studied, and the use of FXR agonists repressed liver fibrosis in animal models by reducing HSC activation [50,51,52]. The details of nuclear receptors on liver fibrosis are found in recent extensive reviews [53,54].

A fourth and very important factor modulating liver fibrosis, and other forms of organ fibrosis, is the epigenetic modification of genes. They may lead to either the activation or repression of downstream proteins. Non-coding RNAs may act as epigenetic regulators. Other epigenetic modifications include DNA methylation, histone modification, and chromatin remodeling [55,56]. The DNA methylation pattern is critical in liver fibrosis [57] as it is in the activation of HSCs. The downregulation of the gene coding for the DNA methyl transferases DNMT3a and DNMT3b decreased DNA methylation followed by the suppression of HSC activation [58]. The activation of the hedgehog (Hh) pathway triggers liver EMT. The hypermethylation of the negative regulator of Hh, patched 1 (PTCH1), leads to its downregulation and an increase in liver fibrosis. Recent studies have established the anti-fibrotic efficacy of Salvianolic acid B (Sal B) that inhibits the Hh-mediated EMT [59]. In Sal B-treated cells, PTCH1 was increased due to the inhibition of DNA methyltransferase 1 (DNMT1), followed by a decrease in DNA methylation. The observed upregulation of miR-152 led to the hypomethylation of PTCH1, as DNMT1 was the direct target of miR-152 [60].

2.2. Cells Involved in Liver Fibrosis

2.2.1. Kupffer Cells and Liver Macrophages

Traditionally, Kupffer cells (KCs) included all macrophages in the liver, expressing surface markers such as F4/80 in mice or CD68 in humans. However, hepatic macrophages are a heterogeneous population, particularly after liver injury, and can be broadly divided into embryonic tissue resident KCs and monocyte-derived macrophages [61].

KCs are, therefore, liver resident macrophages initially generated in the embryo but also during adulthood [62,63]. Embryo-derived KCs (Em-KCs) persist in the liver throughout life by self-renewal [64]. In normal adulthood, bone marrow (BM)-derived monocytes can enrich the KC pool when Em-KCs are exhausted [65]. Monocyte-derived macrophages are recruited and accumulated in the liver after a damaging insult [66]. Em-KCs are CD49a+, a fact that distinguishes them from BM monocytes [67]. Em-KCs have a dual role in liver inflammation as they express both pro-inflammatory cytokines such as TNFa and anti-inflammatory cytokines such as IL-10. Em-KCs seem to be operational during normal homeostasis and promote tolerance, participating only in early liver injury, while BM-KCs act in chronic inflammation and fibrosis [68]. In the murine liver, only a few macrophages originate from BM monocytes under normal conditions [69]. Murine monocytes are divided into two phenotypes based on the presence of the lymphocyte antigen 6 complex, locus C (Ly6C). Ly6Chigh monocytes are recruited to the liver in liver injury and differentiated into BM-derived macrophages that are responsible for chronic inflammation and fibrosis. On the other hand, Ly-6Clow BM-derived macrophages promote damage resolution [70]. During early liver injury, damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), produced by the injured hepatocytes, interact with the Toll-like receptors (TLRs) on KCs. The activated KCs in turn recruit Ly-6Chigh macrophages through the release of chemokines such as CCL2 and CXCL1. Ly-6Chi macrophages sustain the activation and survival of HSCs [71], producing pro-fibrogenic mediators such as TGFβ, PDGF, and CCL2 [72]. Galectin-3 is also a lectin secreted by macrophages, which promotes HSC activation [73]. Liver macrophages also express receptors that bind the alarmin high-mobility group box 1 (HMGB1), released from injured hepatocytes. HMGB1 also activates HSC and stimulates the phenotypic responses of liver MFs [74].

The initial classification of KCs included classically activated pro-inflammatory M1 cells and alternatively activated anti-inflammatory M2 cells. LPS and IFNγ polarize KCs into M1 cells expressing inflammatory molecules such as IL-1, IL-12, TNFa, inducible nitric oxide synthase (iNOS), and a group of CXCL chemokines. On the other hand, Th2 helper T cells and IL-4 polarize KCs into M2-type expressing Arginase 1, IL-10, and PDL-1 and CCR2, CXCR1, and CXCR2 chemokines [75]. The M1/M2 balance is also dependent on production by the M2 cells of IL-10 that promotes M1 apoptosis [76,77]. M2 macrophages are additionally classified into distinct subtypes expressing different genes [78]. The M2 phenotype predominantly mediates tissue repair, but when the liver injury persists, M2 macrophages acquire a pro-fibrotic capacity [79].

In the diseased liver, macrophages frequently express both inflammation and regenerative markers with phenotypes that may change according to the local conditions and, therefore, the traditional model may not be relevant in liver damage [80,81]. A complex classification goes beyond the traditional distinction between M1 and M2 polarization [82,83]. It has been proposed that instead of the classical classification, a more detailed description should be made based on the activation stage, such as M (IL-10) or M (TGF-β) [84]. This classification is compatible with the changing roles of macrophages in liver disease that may be completely different or even opposite [19]. The fact that most macrophages do not comply with the M1/M2 model was recently verified. Two cytokines, the macrophage colony stimulating factor (M-CSF) and the granulocyte macrophage stimulating factor (GM-CSF) involved in the differentiation of KCs, indicate that the situation is more complex. In general, GM-CSF leads to M1 polarization and M-CSF to M2 polarization, but their combination with other cytokines may lead to a spectrum of macrophages expressing both M1 and M2 markers [85,86]. The murine models of liver fibrosis have shown an additional factor that participates in macrophage activation. The Notch signaling pathway is a significant regulator of macrophage differentiation. The suppression of the Notch1/Jagged1 signaling pathway may reverse M2 polarization [87]. In another model, the repression of the Notch signaling reduced the activation of HSCs and the polarization of macrophages into the M1 phenotype with the upregulation of anti-inflammatory genes and the reduction in liver fibrosis [88]. Several other macrophage-specific signaling pathways such as c-Jun N-terminal kinase (JNK), nuclear factor kappa-B (NF-kB), Janus kinase (JAK), and the signal transducer and activation of transcription (STAT) participate in liver fibrosis progression. On the opposite side, the activation of the Wnt/β-catenin signaling pathway in macrophages favors the resolution of liver fibrosis [19]. Murine studies, using single-cell RNA sequencing, identified a distinct type of hepatic bone marrow-derived macrophage with an inflammatory profile, particularly prominent in MASH [89].

The definition problem is far from being solved. Recently, in the murine livers, two types of KCs were characterized by single-cell RNA sequencing. KC1 represents the majority of KCs and is an endothelial cell-selective adhesion molecule (ESAM) negative with a low expression of CD 206. Functionally, it is tolerogenic, while KC2 (CD206hi) has pro-inflammatory potential [19]. An additional pro-fibrogenic subset of liver macrophages was characterized by the presence of the triggering receptor expressed on the myeloid cells 2 (TREM2+) CD9 + marker and was prominent in liver fibrosis, particularly in MAFLD/MASH patients [90,91]. In patients with liver fibrosis, findings analogous to the Ly6C murine macrophages were described.

The exact role of each of the described macrophage subtypes in individual liver diseases is not fully clarified. Murine alcohol-related liver disease (ALD) is exacerbated by infiltration of chemokine receptor positive macrophages, such as CCR2+ or CCR5+ [92]. Moreover, activated liver macrophages secrete vasoconstrictive agents that lead to the induction of portal hypertension and the development of liver fibrosis as they enhance HSCs transformation into MFs [64]. In addition, the activation of KCs and liver macrophages is metabolically re-programmed by endoplasmic reticulum (ER) stress present in MAFLD/MASH [93]. The additional implications of macrophage subtypes in MASH have been mentioned above.

2.2.2. Hepatic Stellate Cells

Under normal conditions, they contain retinoids and are the only site of vitamin A storage. Activated HSCs (aHSC) secrete more pro-fibrotic factors and a positive loop is operative, aggravating fibrosis [94,95]. aHSC may proliferate, produce ECM proteins, and generate inflammatory signals [31]. ECM accumulation is the outcome of the synthesis and degradation of ECM proteins. Matrix metalloproteinases (MMPs), including collagenase (MMP1) and Gelatinase B (MMP9) are zinc-dependent enzymes that degrade ECM components. The deposition of ECM in fibrosis depends on the balance between the MMPs and tissue inhibitors of metalloproteinases (TIMPs) [96]. An imbalance in the activity of MMPs and TIMPs can promote either the progression or the resolution of liver fibrosis [97]. Upon activation, HSCs lose vitamin A droplets and have different characteristics compared to quiescent HSCs such as increased expressions of alpha-smooth muscle actin and collagen type 1 alpha 1. The expression of the peroxisome proliferator-activated receptor γ (PPARγ) is reduced [1]. aHSCs are the main, but not the only, progenitors of MFs [98]. MFs may also originate from portal fibroblasts and bone marrow-derived cells [99] and by the already discussed EMT [100,101] or the complementary endothelial-to-mesenchymal (EndoTM) transition [102,103]. The activation of HSCs is mediated by either several extracellular growth factors, including the platelet-derived growth factor (PDGF), the transforming growth factor-β (TGFβ), the connective tissue growth factor (CTGF), the Wnt/β catenin pathway, chemokines, lipolysaccharide (LPS), DAMPs and PAMPs, or by nuclear mechanisms through the actions of miRNAs. Epigenetic mechanisms may also be implicated, as mentioned before, as well as a number of cellular factors such as oxidative stress and reactive oxygen species (ROS), ER stress, and the autophagic pathway [104,105].

Autophagy has attracted attention as it provides the necessary energy through a specialized form of autophagy called lipophagy that metabolizes the lipid droplets to maintain the activation of HSCs [106,107,108]. However, there is evidence that autophagy may also protect from liver fibrosis, acting as a double-edged sword [109,110,111,112]. Thus, PDGF inhibited autophagy, inducing the release of multivesicular body-derived exosomes and microvesicles from HSCs. Therefore, increased autophagy in HSCs represses liver fibrosis by inhibiting the release of fibrogenic extracellular vesicles [113].

HSCs are by no means a homogeneous population as believed in the past. Studies in the livers of rodents identified two transcriptomes from different populations. One was located in the portal area and the other was associated with the central vein. Interestingly, the latter was responsible for the production of collagen during centrilobular liver damage [114]. The HSC subtype of zone 1 is not transformed into MFs in liver injury but behaves as a capillary pericyte, participating in the process of sinusoidal capillarization [115]. In aging livers, HSCs with a “mixed” phenotype have been identified. They have lipid droplets indicating quiescence together with the markers of senescence and activation such as aSMA. ScRNAseq analysis indicated that even MFs are also a heterogeneous population with distinct functions [101,116].

2.2.3. The Interplay Between KCs and HSCs

As mentioned above, damaged hepatocytes release reactive oxygen species (ROS) and DAMPs. DAMPs activate Toll-like receptors (TLRs), TNFa receptors, and IL1R. The binding of DAMPs to their ligands initiates the myeloid differentiation 88 (MYD88) pathway, followed by the activation of nuclear factor kB (NF-κB) in Kupffer cells, and the transcription of the NLRP3 inflammasome. The resultant inflammatory response is due to the transcription of procaspase-1, pro-IL-18, and pro-IL-1β. ROS also initiate the transcription of NLRP3. Activated inflammasomes induce the production of IL-1β and IL-18, which in turn differentiates HSCs into myofibroblasts, promoting the development of liver fibrosis [117,118,119]. TNFa produced by Kupffer cells may promote fibrosis as it inhibits the apoptosis of HSCs and increases the production of TIMPs by activated HSCs [113,120].

Kupffer cells and HSCs in the murine fibrotic liver were able to recruit Ly6Chi monocytes by secreting CCL2, after the hepatocyte-specific deletion of NF-κB. Recruited monocytes further activated HSCs and aggravated fibrosis [121]. The deletion of CCL2 inhibited monocyte recruitment and attenuated liver fibrosis [121]. In addition, activated HSCs produced tissue inhibitors of metalloproteinases (TIMPs), which aggravated fibrosis by inhibiting the degradation of ECM by metalloproteinases [122].

MCP-1 secreted by macrophages increases fibrosis, interfering with macrophages and HSCs. CCR2, the receptor of MCP-1, is expressed on both Kupffer cells and HSCs. In Kupffer cells, the stimulation of CCR2 increases liver infiltration by macrophages, inducing early liver inflammation [123]. In HSCs, the stimulation of CCR2 causes an overexpression of fibrosis genes [124]. CXCL6 was found to be an initiator of TGF-β production by Kupffer cells [125]. In addition, the production of CCL2 and CCL5 by macrophages induced the fibrotic phenotype of HSC and initiated their movement toward the damaged area via their matching receptors in HSCs [126,127]. Stimulated HSCs also express CCL2 and CCL5, which participate in a positive feedback loop and aggravate liver fibrosis [128,129]. Increased levels of CXCL6 were demonstrated in the serum and liver of patients with advanced fibrosis. In vitro cell experiments indicated that HSCs were only indirectly stimulated by CXCL6, which induced TGF-β secretion by KCs [125]. Furthermore, the binding of PDGF produced by activated KCs transforms quiescent HSCs into activated HSCs [130].

Table 1. Macrophage cytokines and chemokines involved in the interaction with HSCs.

Molecules	Functions	References
TGF-β	Primarily produced by macrophages. Enhances ECM production in HSCs through Smad-dependent pathways and Smad-independent pathways.	[131,132]
PDGF	Produced by macrophages. It contributes to fibrosis progression through HSC activation.	[133]
TNF-α	Upregulates TIMP-1 production, prevents HSC apoptosis.	[134,135]
IL-1β,IL-18	Produced by pro-inflammatory macrophages through activation of the NLRP3 inflammasome. Activates HSCs, upregulates TIMP production.	[136,137]
IL-13, IL-4	Produced by M2 macrophages. Promotes the activation of HSCs.	[138]
MCP1	Activates CCR2 in Kupffer cells and HSCs.	[139,140]
CCL2 CCL5	Produced by macrophages and HSCs. Increases macrophage infiltration and fibrotic phenotype of HSCs.	[141,142]
CXCL6	Induces TGF β production by KCs and indirectly promotes fibrosis.	[143]

presents a synopsis of macrophage cytokines and chemokines involved in the interactions with HSCs.

HSCs secrete anti-inflammatory cytokines such as IL-10 and TGF-β that initiate the polarization of macrophages toward an anti-inflammatory phenotype, leading to fibrosis resolution. However, the same anti-inflammatory macrophages can also produce cytokines such as IL-13 and IL-4, which favor the differentiation of HSCs into myofibroblasts [144]. IL-6 from either KCs or HSCs may also differentiate HSCs toward myofibroblasts [145,146]. In a murine model of ALD, the extracellular vehicles from alcohol-damaged hepatocytes increased IL1β and IL-17 expression in macrophages followed by the activation of HSCs and the exacerbation of liver fibrosis [147].

An additional mechanism of macrophage–HSC interaction was recently proposed. Cadherin-11 (CDH11) induced intercellular junctions between activated HSCs and macrophages, forming a fibrotic niche. As a result, TGF-β that is produced by macrophages activates the connected HSC, thus inducing their prolonged activation. The repression of CDH11 could derange this niche and promote fibrosis resolution [148].

2.2.4. Liver Sinusoidal Endothelial Cells (LSECs)

Liver sinusoidal endothelial cells are liver endothelial cells with the unique characteristic of the presence of fenestrae. An additional characteristic is the minimal presence of basement membrane. LSECs maintain hepatic cell-to-cell communication and are regulators of signal transduction among cells [134]. The presence of fenestrations is a distinguishing feature useful for the distinction of LSECs from other liver endothelial populations [149]. LSECs are considered to be the actual gatekeepers of the liver microenvironment [150]. Any impairment of the intercellular communications of LSECs may lead to the development of liver fibrosis [151]. Thus, vascular cell adhesion molecule 1 (VCAM1) deletion from LSECs reduces macrophage accumulation in the liver and ameliorates fibrosis, as VCAM1 is an important mediator of LSEC capillarization and hence of liver fibrosis [152].

SECs also have loose cell junctions [136]. In analogy with Kupffer cells and HSCs, LSECs are not a homogeneous population in the normal mouse liver. Their phenotype is variable in the different zones of the liver acinus. Zone 1 LSECs are CD36hi and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) low, whereas zone 2 and zone 3 LSECs are CD36low, LYVE1hi, and CD32hi [153]. In the cirrhotic liver, seven different subpopulations of LSECs were identified by scRNA analysis [91]. The increased expression of atypical chemokine receptor 1 (ACKR1) + ve and plasmalemma vesicle-associated protein (PLVAP) + ve were described in LSECs, which are restricted to the fibrotic niche and increase the trans-migration of leucocytes [91,137]. In addition, LSECs may be transformed into endothelial–mesenchymal transition (EndMT), acquiring the phenotype of mesenchymal cells. They start producing ECM proteins that further accumulate in the sinusoids, aggravating capillarization [154,155]. The impairment of autophagy, as observed in MAFLD, increases the EndMT of LSECs and induces the inflammatory response and finally liver fibrosis [139]. The underlying molecular mechanisms involve the stimulation of Twist1 by the transcriptional regulator megakaryocytic leukemia 1 (MKL1) and the signal transducer and activator of transcription 3 (STAT3), leading to an amplification of EndMT in LSECs by TGF-β [140]. The implication of LSECs in liver fibrosis is mostly indirect through the loss of the capacity of LSECs to suppress the activation of the HSCs. This is due to the capillarization of the sinusoids that prevent secretory factors from LSECs to inhibit the activation of HSCs [31].

2.2.5. Cytokines Involved in Liver Fibrosis

Fundamental fibrogenic cytokines, such as the transforming growth factor-β (TGF-β), the platelet-derived growth factor (PDGF), the vascular endothelial growth factor (VEGF), and the connective tissue growth factor (CTGF), all act through specific receptors and participate in advanced liver fibrogenesis [16].

a. TGF-β and IL-10.

TGF-β belongs to a superfamily of 33 cytokines, including among others the isoforms of TGF-β (TGF-β1/2/3), the bone morphogenetic proteins (BMPs), the growth and differentiation factor (GDF), and activins [143].

Activated TGF-β molecules are liberated from a latent complex after liver injury. TGF-β then binds to the TGF-β type II receptor (TβRII), resulting in the recruitment of the TGF-β type I receptor (TβRI). Then, TβRII phosphorylates TβRI that in turn phosphorylates SMAD2 and SMAD3 proteins which are complexed with SMAD4, which translocate to the nucleus and regulate the transcription of target genes such as αSMA and CTGF [141,142]. TGF-β also activates non-canonical SMAD-independent pathways, such as MAPK, mTOR, PI3K/AKT, and Rho/GTPase. SMAD7 negatively regulates TGF-β, competing with SMAD3 and SMAD4 for TβRI binding [156].

There has been plenty of evidence that TGF-β is a crucial factor in liver fibrosis. The deletion or suppression of TGF-β attenuated liver fibrosis in mice, whereas the induced overexpression of TGF-β increased liver fibrosis [72,133,157]. The internalization of the type II receptor (TGFβRII) is dependent on the protein diaphanous homolog 1 (Diaph1) as the first step for the transformation of HSCs into MFs. The inactivation of Diaph1 inhibited the endocytosis and intracellular trafficking of TβRII, reducing Smad 3 phosphorylation [138]. The activation of the focal adhesion kinase (FAK) is also a vital component in TGFβ signaling. FAK protects TGFβRII from lysosomal degradation and promotes TGFβ-mediated HSC activation [158]. Almost all the secreted TGF-β1 is found in a latent form bound to the ECM, and the activation of TGF-β1 during fibrosis is site-specific [159]. The better-studied activation mechanism for TGF-β1 is the interaction of the latent complex with the αv-containing subset of integrins. Specifically, the integrins αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 bind to the latency-associated peptide (LAP) that inhibits the active molecule from binding to its receptors [160,161,162]. The deletion or blockade of the αvβ6 integrin protected mice from biliary fibrosis in the bile duct-ligated model [163,164]. Also, the blocking of αv-containing integrins by a small molecule ameliorated liver fibrosis, even after the establishment of fibrosis [165]. There is evidence that all TGF-β subtypes are involved in liver fibrosis. The increased levels of TGF-β1 have been found in the murine models of liver fibrosis [166], while increased mRNA levels of TGF-β1 have also been observed in fibrotic patients [143,166,167]. Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in isolated cell experiments. The upregulation of miR-200a downregulated smad-3 activity and mitigated the TGF-β-dependent EMT. TGF-β1 and TGF-β2 were shown to downregulate the expression of miR-200a. miR-200a also downregulated the expression of TGF-β2 via direct interaction with the 3′ untranslated region of TGF-β2 [168]. Interestingly, the serum levels of the TGF-β subtypes are different according to disease etiology. Serum TGF-β2 was significantly higher in viral cirrhosis but not in primary biliary cholangitis (PBC) patients compared to healthy controls. TGFβ-3 was increased in early and late PBC and decreased in viral cirrhosis. Hepatic vein subtype levels were similar to those in peripheral blood. All TGF-β subtypes were identified by immunocytochemistry in portal tract lymphocytes, sinusoidal cells, and cholangiocytes. TGF-β3 was only overexpressed in hepatocytes from PBC patients [169].

TGFβ and IL-10 regulate the induction of and prolongation of T cell exhaustion. IL-10 is often overproduced in chronic infections such as HIV, HBV, and HCV. The inhibition of IL-10 may prevent or even restore T cell exhaustion. IL-10 acts either directly on T cells through STAT-3 or indirectly by inducing APCs to increase T cell exhaustion and viral persistence. On the other hand, the inhibition of IL-10 in combination with PD-1 leads to the preservation of effector T cell responses, and the effective control of viral replication. Moreover, the use of neutralizing IL-10 antibodies along with therapeutic vaccination promoted CD8+ and CD4+ T cell responses, decreasing viral load [170].

TGF-β is also a suppressive cytokine involved in T cell exhaustion. TGF-β can ameliorate immune cell activation by activating downstream SMAD transcription factors. In acute viral infections, TGF-β is a negative regulator of effector function through the repression of T-bet (T-box expressed in T cells) leading to the upregulation of the pro-apoptotic factor Bim. In chronic viral infections, TGF-β expression and/or downstream SMAD2 activation lead to T cell exhaustion, thus promoting fibrosis [170,171]. HBV initiates the production of TGF-β and IL-10 by macrophages and inhibits TNF-α production [172]. Similarly, in chronic HBV (CHB) patients, monocytes produce more IL-10 and TGF-β and express high levels of PD-L1. Studies have demonstrated that HBsAg and HBV DNA directly promote PD-L1 expression and anti-inflammatory cytokines production from the monocytes of healthy people [173].

b. Activin A is expressed in murine hepatocytes, HSCs, and LSECs but not in KCs. Different activin receptor combinations are expressed in liver cells. HSCs do not respond to activin A due to the downregulation of type II activin receptors, while KCs respond by increasing the production of TNFα και TGFβ1. Conditioned medium from activin A-treated KCs led to HSC transformation into a pro-fibrogenic phenotype, expressing collagen and αSMA [174]. In addition, TGF-β itself stimulates the production of activin A by fibroblasts [131].

c. Other cytokines are implicated in the fibrotic process in the liver [132,175]. IL-1β has fibrogenic effects similar to TGF-β by inducing EMT, which can be blocked by a monoclonal antibody [176]. It should be noted that IL-6, TNFa, and IL-1-β synergistically act with TGF-β, because the deletion of these cytokines attenuates liver fibrosis [177,178,179,180]. Mechanistically, IL-1-β, and TNFa enhance TGF-β actions by downregulating the BMP activin membrane-bound inhibitor (BAMBI), which is a pseudo-receptor for the TGF-β type I receptor and a negative regulator of TGF-β signaling [120].

Whatever the mechanism of liver fibrosis might be, the end result is due to the balance of synthesis over the degradation of ECM, particularly collagens. This in turn is the balance between collagen-synthesizing enzymes and degradative factors such as collagenases and MMPs. The balance may be different according to the etiology of fibrosis. In alcoholic fibrosis and primary biliary cholangitis, it is the synthesis that predominates, while in viral fibrosis, it is the reduced degradation that is mainly responsible [181].

2.3. Resolution of Liver Fibrosis

The resolution of liver fibrosis requires a reduction in the number of activated HSCs and other MFs. This can be achieved through three mechanisms: the regression of activated HSCs to quiescence, the induction of senescence, and the elimination of activated HSCs and MFs through apoptosis and ferroptosis [2,31].

Activated HSCs can de-differentiate back to an inactivated phenotype by upregulating transcription factors such as peroxisome proliferator-activated receptor-γ (PPARγ), GATA-binding factors 4 and 6, and transcription factor 21 (TCF21) [182]. They may also enter senescence or may be eliminated by cell death. Both HSC apoptosis mediated by NK and CD8+ T cells [183] and ferroptosis have been reported during the resolution of liver fibrosis [116]. The induction of apoptosis is often mediated by natural killer cells (NK cells) through the production of interferon-γ (IFNγ) [184,185]. NK cells may also kill senescent MFs, in addition to activated HSCs [186]. The implication of natural killer T cells (NKT cells) in the induction of apoptosis is still controversial [187]. In that respect, it was recently reported that Artesunate (an ester from Artemisin) induced ferroptosis in HSCs and attenuated liver fibrosis in a murine model [188]. Another way to reduce liver fibrosis is the change in the phenotypes of liver macrophages [189]. This has been clearly demonstrated in murine models, as mentioned before. Pro-fibrogenic Ly-6Chi macrophages can change into Ly-6Clow anti-fibrotic macrophages, releasing anti-inflammatory cytokines such as IL- 10, restorative growth factors such as HGF, and ECM-degrading MMPs [61,72,83,190] including MMP12 and MMP13 [82,191]. Partial resolution is still feasible even if the fibrosis is advanced. The appearance of Ly-6Clow macrophages is associated with either the apoptosis of MFs induced by the macrophage production of TNF-related apoptosis-inducing ligands (TRAIL) [192] or by reversion to quiescent HSCs [193].

Many traditional Chinese medications (TCMs) have been reported to be effective in treating liver fibrosis. Single herbal extracts and TCM formulas may prevent or treat hepatic fibrosis. HSCs and oxidative stress, which are implicated in liver fibrosis, are common targets of TCMs [194,195]. However, caution should be exercised in the interpretation of the results as many papers on the clinical trials of TCMs do not comply with the acceptable design of trials [196].

Figure 1 summarizes the mechanisms of liver fibrosis.

BEC: Biliary epithelial cells; CB1 and 2: cannabinoid receptors 1 and 2; DAMPs: damage-associated molecular patterns; EM: epithelial-to-mesenchymal transition; EndoMT: endothelial-to-mesenchymal transition; EVs: extracellular vesicles; FGF: fibroblast growth factor; FXR: farnesoid X receptor; FGF: fibroblast growth factor; Hh: hedgehog ligand; LSECs: liver sinusoidal endothelial cells; LncRNA: long non-coding RNA; miRNA: microRNA; MMPs: matrix metalloproteinases; PAMPs: pathogen-associated molecular patterns; PDGF: platelet-derived growth factor; PPAR: peroxisome proliferator activated receptor; TIMPs: tissue inhibitors of metalloproteases; VDR: vitamin D receptor.

3. Immunology of Liver Fibrosis

3.1. The Liver as an Immune Organ

The liver, in addition to its central metabolic role, is also a significant immune organ [197,198]. It contains several components implicated in both innate and adaptive immunity. Some of them are not strictly immune cells, such as hepatocytes [199] and LSECs, but express Toll-like receptors (TLRs) and major histocompatibility complex (MHC) molecules, and participate in the maintenance of tolerance [200,201,202,203] or the biliary epithelial cells (BECs) and HSCs, which are also tolerogenic and may present antigens to T lymphocytes [135,198,204]. These cells are implicated in both innate and adaptive responses. On the contrary, liver sinusoids contain the proper cells of innate immunity such as KCs, dendritic cells, myeloid-derived suppressor cells, or lymphoid-derived cells (NKs and innate lymphoid cells). Certain cells do not comply with either the innate or adaptive immunity criteria and are defined as “innate-like”, or “unconventional” lymphocytes. They include mucosal-associated invariant T (MAIT) cells, natural killer T (NKT) cells, and γδ-T cells. In addition, the normal liver also houses the conventional T and B lymphocytes of adaptive immunity [198].

Tolerance is a main function of the liver. An important mechanism of hepatic tolerance is the expression by several liver cells of MHC molecules not accompanied by co-stimulatory molecules. Other, equally important tolerogenic mechanisms, are the secretion of suppressor cytokines such as IL-10 and TGF-β, the inhibition of professional antigen-presenting cells (APCs), and the subjection of immune cells to programmed cell death-ligand 1 (PD-L1) [205,206,207]. Liver-draining lymph nodes (LNs) are also important components of the liver immune system. Portal LNs are an area of regulatory T cells (Tregs) induction, while celiac LNs are an area of T cell responses [205,208]. Moreover, cellular metabolism is associated with immune responses. A glycolytic metabolism is involved in the effector function of T lymphocytes, while fatty acid oxidation is used by non-inflammatory immune cells such as Tregs [209]. Glycolysis induced by hypoxia-inducible factor 1-alpha (HIF-1a) and oxidative metabolism induced by IL-4/STAT6 are used by either pro-inflammatory or anti-inflammatory macrophages, respectively [210,211,212].

3.2. Immune Factors Implicated in Liver Fibrosis

Liver fibrosis is closely linked to impaired hepatic immune responses [213]. Several experimental data indicate that the immune cells can regulate both the progression and reversal of liver fibrosis [214]. Excessive alcohol consumption, viruses, western dietary habits, or MAMPs and PAMPS originating from the microbiota of a leaky gut, may impair hepatic immune homeostasis leading to liver inflammation, fibrosis, and cirrhosis. The liver must handle antigens arriving at the sinusoids from the systemic circulation and the intestinal tract. These antigens are processed by the liver through a series of pattern recognition receptors (PRRs), such as TLRs and nucleotide-binding oligomeric domain-like receptors (NOD-like receptors), which induce either a tolerogenic response or inflammation and fibrosis [215,216,217,218].

Early in the course of chronic liver disease, damaged hepatocytes release inflammatory mediators that recruit and activate inflammatory cells, such as macrophages, lymphocytes, and NK cells [183,190,219]. Inflammation leads to a disordered crosstalk between hepatic immune cells, which drives the induction and progress of fibrosis [189,220,221]. TLRs are expressed on various hepatic cells like KCs, dendritic cells, hepatic stellate cells, endothelial cells, and hepatocytes [222]. Changes to the liver immune system leading to fibrosis include a decrease in CD8+ T cells and NK cells and an increase in CD4+ T cells infiltration accompanied by the expression of certain immune-regulatory genes [223,224].

Innate and Adaptive Immunity

Innate and adaptive immune cells are involved in hepatic inflammation, fibrosis, cirrhosis, and HCC. They have distinct roles, but at the same time, they affect each other. Adaptive immunity depends on the activation signals and cytokines secreted by the innate immune system. PAMPs from damaged hepatocytes lead to the activation of intrahepatic innate cells that initiate the recruitment of circulating immunocytes to the liver. The infiltrating cells stimulate other parenchymal and non-parenchymal cells in the liver, thus creating a perpetuating circle. The induction of pro-fibrogenic mediators such as IL-10 and TGF-β encourage liver fibrosis by activating quiescent hepatic stellate cells. The continuous supplementation of these fibrogenic stimuli promotes further disease progression toward fibrosis and cirrhosis [225].

3.3. Innate Immunity in Liver Fibrosis

As mentioned above, DAMPs activate innate immunity that induces fibrosis. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a major element of the innate immunity, which functions as PRR, recognizing both PAMPs and DAMPs [226]. Kupffer cells are rich in NLRP3, and its activation leads to the secretion of several pro-inflammatory cytokines, such as IL- 1b and IL-18 [227]. Human and murine data indicate that NLRP3 activation induces caspase-1-mediated pyroptotic death and the liberation of inflammasomes that are engulfed by HSCs, leading to their activation and liver fibrosis [228].

3.3.1. Cells Involved in Innate Immunity

a. Hepatocytes. Hepatocytes may induce innate immunity as they express immune receptors that recognize PAMPs. These receptors include surface receptors such as TLR4, endosomal receptors such as TLR3, cytoplasmic receptors such as the stimulators of the IFN gene (STING) and the members of the NOD family [199,229,230]. Hepatocytes can also induce adaptive immunity. During inflammation, certain hepatocytes express MHC-II molecules and activate T lymphocytes [206,231], but as they do not express co-stimulatory molecules such as CD80 and CD86, they are not capable of generating the long-lasting activation of T cells. Importantly, hepatocytes express PD-L1 either after viral infection or under the influence of type I and type II IFN, thus mediating the apoptosis of T cells [232]. Activated hepatocytes may also induce the transformation of BM-derived monocytes into pro-inflammatory macrophages, upregulating the Yes-associated protein (YAP) and the transcriptional coactivator with a PDZ-binding motif (TAZ) [233]. YAP/TAZ is the effector in the Hippo pathway, which is a regulator of the TGF-β2-mediated fibrogenesis as indicated by data from other organs [234]. The increased expression of the transcription factor Fork head box M1 (FoxM1), and the subsequent overexpression of the CCL2 chemokine, induce hepatocyte death, leading to liver inflammation and fibrosis through macrophage recruitment [235,236].

Most of the data mentioned above come from animal experiments. However, there is evidence from liver disease patients that hepatocytes are indeed involved in inflammation and fibrosis. MHC-II molecules are expressed in the hepatocytes of alcoholic hepatitis, and activate CD4+ T cells, inducing positive lymphocytes [237]. Lipid-laden hepatocytes are more susceptible to apoptosis in patients with MASH [238]. Exosomes derived from HBV-infected hepatocyte contain miR-222 and increase fibrosis by inhibiting the transferrin receptor (TFRC) and TFRC-induced ferroptosis [239].

The autophagy pathway within the hepatocytes is also implicated in immune-mediated liver fibrosis. Autophagy protects hepatocytes from death signals [199,240] and is implicated in most chronic liver diseases [241,242,243,244]. The vitamin D receptor (VDR) promotes autophagy by regulating beclin-1, bcl-2, the mTOR elements of the autophagy pathway, and lysosomal maturation [245]. The VDR was decreased in the hepatocytes of cirrhotic patients [246]. In murine models and human cirrhosis, hepatocyte autophagy was inhibited by the miR-125a/VDR axis, leading to increased liver fibrosis [247].

Endoplasmic reticulum (ER) stress and hepatocyte senescence are two additional factors that implicate hepatocytes in the process of liver fibrosis. The accumulation of misfolded proteins in the ER activates the unfolded protein response (UPR), mediated by three ER sensors, namely PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) to counteract the protein-folding defect [248]. Massive ER stress overcomes UPR and leads to hepatocyte steatosis and death [93,249]. ER stress can trigger C/EBP Homologous Protein (CHOP) transcription factor-dependent NLRP3 inflammasome activation and the activation of IRE1A in hepatocytes, both leading to the release of pro-inflammatory cytokines and inflammatory extracellular vesicles (EVs), therefore promoting fibrosis [199]. A strong positive correlation between senescent hepatocytes and liver fibrosis severity has been described in MASH and alcoholic liver disease patients [250,251]. In chronic viral hepatitis patients, senescence is associated with telomere shortening and the absence of telomerase in hepatocytes, favoring virus replication and liver cirrhosis [252,253]. An additional confirmation that senescent hepatocytes are directly involved in liver fibrosis was recently reported. PDGF is a potent activator of HSCs, as mentioned before. PDGF levels were significantly higher in the media from cultured senescent hepatocytes compared to control hepatocytes, and similar findings were demonstrated in serum samples from patients with cirrhosis compared to healthy controls [254].

b. Kupffer cells and liver macrophages. KCs are probably the most important cells in liver innate immunity. They are professional antigen-presenting cells (APCs) to T cells and therefore participate in the initiation of adaptive immunity [83,255]. In MASH, there is a positive correlation between the severity of inflammation and fibrosis and the number of pro-inflammatory macrophages in the periportal zone [256]. As mentioned before, early liver damage activates hepatic macrophages including the KCs. In turn, they secrete several cytokines such as TGF-β1, PDGF, TNF-α, IL-1, IL-6, and IL-10, and cytokines such as CXCL1, CCL2, and CCL5. KCs produce mediators such as ROS that induce HSC transformation and attract BM monocytes and neutrophils [183,191]. Liver macrophages, irrespective of origin, are the main sources of TGFβ and one of the leading causes of increased ECM deposition in liver fibrosis. They also maintain the survival of MFs by activating NF-kB through the secretion of IL-1β and TNFa [183,189,191,219]. In addition to these functions, Kupffer cells activated by DAMPs and PAMPs, as mentioned above, induce an increased expression of vascular adhesion molecules on LSECs [82,257].

Recruited bone marrow-derived macrophages differentiate into a Kupffer cell-like phenotype [65], approximately 60 days of repopulation after liver damage [258]. It is not clear if the newly recruited macrophages live long or whether their functional role is comparable to the original Kupffer cells [65,259].

KCs have a dual role in the immune regulation of liver inflammation and fibrosis. Depending on the microenvironment, they can acquire a pro-inflammatory phenotype (referred to as M1). M1 KCs are activated by IFNγ and lipopolysaccharide (LPS) and are characterized by the ability to present antigens and produce inflammatory cytokines such as TNFα, IL-1, IL-6, IL-12, and IL-23, promoting antiviral activity. Alternatively, KCs can acquire an anti-inflammatory phenotype (referred to as M2) characterized by their ability to balance inflammatory responses and facilitate tissue repair through the release of IL-10, IL-4, IL-13, and TGF-β and the low production of IL-12, IL-6, and TNF-α [260,261]. Furthermore, IL-17 activates Kupffer cells and lead to the upregulation of pro-fibrotic cytokines like IL-6, IL-1β, and TGF- β1 [262].

KCs differentiation into the M2 phenotype upregulates PD-L1 and galectin-9 expression in the presence of HBeAg. HBcAg upregulates TLR-2 on the surface of KCs and increases IL-10 secretion, thus upregulating CD8+ T cell exhaustion [263]. The activation of the TLR4 signaling pathway promotes M1 inflammatory differentiation that leads to the upregulation of the clearance of HBV [264]. The stimulator of interferon genes (STING) is a key adaptor in DNA-initiated innate immune activation [265]. The stimulation of KCs by STING increases the hepatic expression of interferon-inducible protein 16 (IFI 16), which binds to HBV cccDNA, inhibiting cccDNA transcription and leading to its silencing [266].

In human cirrhosis, KC numbers are similar to the normal liver [91,267], in contrast to the murine liver where extensive fibrosis and cirrhosis is accompanied by a reduction in the number of KCs [268]. The presence of KCs and the differentiation of other macrophages is influenced by stromal cells through the inhibition of monocyte maturation. This is achieved by the production of IL-6 from the stromal cells. Interestingly, the local IL-6 levels are diminished in early-stage human liver injury, implicating a protective role of IL-6 [269]. Apart from the production of TGF-β and the maintenance of the viability of MFs, KCs may be transformed into fibroblast-like cells, contributing to ECM production [270]. The KCs also promote collagen cross-linking that stabilizes collagen through the action of Lysyl-oxidase (LOX) and Lysyl oxidase-like protein-2 (LOXL2) [271]. On the opposite side, KCs produce MMP9, leading to collagen degradation [272]. KC infusion attenuated liver fibrosis in a murine model [273]. Interestingly, the T-cell immunoglobulin domain and mucin domain-4 (TIM-4) expression by KCs represses liver fibrosis [274].

Although fibrosis is the final common result in all liver diseases irrespective of etiology, the underlying participation of the involved cells may be different. Hepatitis B and hepatitis C viruses (HBVs, HCVs) activate human macrophages, but the response is different in the two viral diseases. Macrophages respond to HBV by the production of inflammatory cytokines and the stimulation of NK cells [275,276]. The response to HCV proteins is the activation of inflammasomes mediated through TLR2 activation [277,278]. HBV and HCV infection lead human macrophages to secrete immunomodulatory mediators such as IL-10, TGFβ1, PD-L1, and PD-L2 that eventually mitigate antiviral T cell response [279]. In MASH, the accumulation of fat in the macrophage [280] production of EVs from fat-containing hepatocytes, [281,282] or histidine rich glycoprotein [283,284], induces an inflammatory phenotype in liver macrophages. In ALD patients, macrophages have a fundamental role in the inflammatory response during severe alcoholic hepatitis [285,286]. In murine ALD models, increased gut permeability contributes to the recruitment of pro-inflammatory macrophages [279,287,288]. In cholestatic conditions, BM-derived macrophages are influenced by the concentration and the composition of the bile acids in the liver [289]. Chenodeoxycholic acid (CDCA) activates the NLRP3 inflammasome in the macrophages of cholestatic animals with fibrosis [290]. On the contrary, KCs have the G-protein-coupled bile acid receptor 1 (TGR5), which is a sensor for bile acids, leading to the inhibition of inflammasomes [291] and the emergence of an anti-inflammatory phenotype [292,293].

c. The role of HSCs. The multiple factors implicated in the activation of HSCs are further complicated due to the interaction of HSCs with the cells of the immune microenvironment during liver fibrosis. All cells involved in both innate and adaptive immunity are communicating with HSCs either directly or indirectly [90,294]. aHSCs, apart from their fundamental contribution as ECM producers, are also pro-inflammatory cells. They produce several cytokines and chemokines such as IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). They also recruit monocytes and hematopoietic stem cells [15]. Inflammatory mediators produced by HSCs target the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), the central regulator of the inflammatory response [295,296]. Therefore, NF-κB is a critical molecule in the inflammation and resultant fibrosis but also in the apoptosis or survival of HSCs after liver injury. Indeed, NF-κB activation is associated with an increased resistance to the apoptosis of activated HSCs [104,297].

The activation of HSCs is important in the pathogenesis of MASH. There is experimental evidence that free cholesterol accumulation in HSCs leads to their activation [298,299,300,301]. Free cholesterol in hepatocytes indirectly affects HSC activation through the stabilization of the transcriptional coactivator with the PDZ-binding motif (TAZ) with subsequent over-secretion of the pro-fibrotic factor Indian Hedgehog [302]. In chronic hepatitis B, a different mechanism is operative. aHSCs recruit large numbers of Th17 cells and promote the secretion of IL-12A and IL-22 that contribute to fibrosis [303]. On the other hand, HSCs may favor the development of tolerance in mice as they favor the expansion of FoxP3+ve Tregs or myeloid-derived suppressor cells [304,305].

HSCs also influence B-cell activity. HSCs inhibit the increased expression of activation markers on B cells and immunoglobulin production. Interestingly, blocking the interaction of PD-L1 with PD-1 mitigated the inhibition of B cells by HSCs [306].

HSCs are also implicated in inflammation and fibrosis by their involvement with the adipokines leptin and adiponectin, which act through binding to their receptors on HSCs [307,308,309]. Leptin is a pro-fibrotic factor that suppresses the sterol regulatory element-binding protein-1c (SREBP-1c) and activates HSCs through the β-catenin pathway [310]. Adiponectin, on the other hand, ameliorates liver fibrosis through the induction of nitric oxide (NO) and TIMP-1 production, leading to the suppression and inactivation of aHSCs [311,312,313,314,315]. Currently, it is not yet clear under which conditions hepatic stellate cells are pro-inflammatory and under which conditions they are tolerogenic [219].

The details of the immune regulation of fibrosis by HSCs have been extensively reviewed [2].

d. The role of liver sinusoidal endothelial cells. LSECs express TLRs and MHC molecules and are implicated in the maintenance of tolerance either through the direct inhibition of T lymphocytes by PDL1 expression or through the so-called “veto” effect, consisting of inhibiting other APCs such as dendritic cells to activate T lymphocytes by physical contact without the need for the presence of MHC [200,201,202,203,316]. The early stages of MAFLD are a clear example of the immunological role of LSECs in liver fibrosis. Lipotoxicity, adipokines, and gut-derived PAMPS lead to LSECs’ de-differentiation and sinusoidal capillarization. Capillarized LSECs are transformed into a pro-inflammatory and pro-fibrotic phenotype, recruiting immune cells that cannot support the quiescence of HSCs and KCs [151,317]. Specifically, the quantity and size of LSEC fenestrae are lost, and in advanced stages they even disappear. The blood filtration in the sinusoids is impaired, and harmful components are not adequately removed, and the risk of liver injury is increased [200]. Human studies reported that the expression level of the scavenger receptor Fc gamma receptor IIb (FcγRIIb) in LSECs is negatively correlated with fibrosis and inflammation in patients with MASH. FcγRIIb is involved in the elimination of small immune complexes from sinusoids [318]. A study on patients with chronic hepatitis C showed that LSEC capillarization was observed even at the initial stages of fibrosis [319]. Moreover, there is evidence that capillarization is the result of the impaired differentiation of the bone marrow-derived LSECs [320]. An important regulator of LSECs fenestration is the bone morphogenetic protein 9 (BMP9). The role of BMP9 in liver fibrosis is controversial. The deletion of BMP9 in murine models increased liver fibrosis [321,322]. On the other hand, liver biopsies from fibrotic patients showed increased levels of BMP9 in advanced fibrosis. In murine models, BMP9 overexpression accelerated liver fibrosis, and BMP9 knockdown ameliorated fibrosis. BMP9 directly stimulated hepatic stellate cell activation via the SMAD signaling pathway to upregulate hepatic fibrosis [323]. The details of the effects of BMP9 on liver fibrosis were recently reviewed [324].

In addition to capillarization, hepatic neo-angiogenesis was also strongly related to LSECs and was associated with the development of liver fibrosis [149], possibly mediated through the vascular endothelial growth factor (VEGF) [325]. This is supported from the alterations of LSEC phenotypes made by certain drugs such as Vatalanib, a VEGFR1 and VEGFR2 inhibitor, or Lenvatinib, an inhibitor of VEGF1, VEGF2, and VEGF-A. They decreased both sinusoidal capillarization and liver fibrosis [326,327]. The beta-blocker carvedilol repressed the expression of VEGF and angiopoietin-2, leading to the attenuation of sinusoidal capillarization [328]. Moreover, HIF-1α is also implicated in LSEC capillarization and angiogenesis. MiR322/424 upregulated the expression of the HIF-1α protein in LSECs and promoted neo-angiogenesis and liver fibrosis [329]. LSECs can also directly stimulate neo-angiogenesis through the secretion of angiogenic factors. Interestingly, LSEC capillarization is a process that starts before the activation of macrophages and HSCs in liver fibrosis [134]. A detailed description of the participation of LSECs-dependent angiogenesis in liver fibrosis was recently reported [134].

LSECs are involved in the progression of liver fibrosis by two additional mechanisms. Reduced NO bioavailability has been observed in LSECs from cirrhotic rodent livers [330] due to the ROS scavenger effect of NO via the superoxide anion (O2-) and the ERK1/2-AKT axis [331]. ERK1/2 shifted the balance toward NO, favoring LSEC homeostasis, while AKT could shift the balance toward ROS, promoting liver fibrosis. A second mechanism by which NO modulates liver fibrosis is the induction of autophagy. Autophagy in LSECs increases the bioavailability of NO and eliminates the accumulation of ROS [332]. This protective action of autophagy is not sufficient at the later stages of chronic liver [333]. Autophagy acts the opposite way as well by degrading Caveolin-1 (Cav-1), leading to LECs defenestration [334]. Moreover, LSECs mediate in the transformation of macrophages into KCs. The interactions of the Notch ligand delta-like ligand 4 (DLL4) produced by LSECs binding to the Notch receptor found in macrophages is required for the induction of the KC identity. Ligands such as ICAM-1 and vascular cell adhesion protein 1 (VCAM-1) in LSECs interact with the integrins on KCs and participate in the direct interplay between KCs and LSECs [335,336].

The central role of LSECs in the regulation of liver immunology and their effects in liver fibrosis is clearly exemplified in viral diseases. In murine adenovirus infection, 90% of the virus is rapidly taken up by LSECs and only 10% is found in KCs [337]. HIV-like particles are also taken up by mouse LSECs at a rate of 100 million viral particles per minute [338]. In the duck hepatitis B virus (DHBV) model, viral particles are mostly taken up by LSECs before passing on to further infect hepatocytes [339]. In HCV, the innate sensing of the virus by LSECs leads to the release of paracrine molecules such as the pro-viral molecule bone morphogenetic protein 4 (BMP4), which promotes the viral infection of hepatocytes [340,341]. On the other hand, the direct sensing of HCV RNA in LSECs produce type I and type III interferon-containing exosomes that inhibit HCV replication [342]. The balance of the two opposite responses determines whether the HCV virus will be eliminated or will cause a sustained infection.

e. Mesenchymal stromal cells (MSCs) are fibroblast-like cells with immunomodulatory ability, as they regulate both innate and adaptive immunity and have the potential to differentiate into hepatocyte-like cells (HLCs) [343,344,345,346]. MSCs express a specific set of surface markers, such as CD73, CD90, and CD105 [347]. MSCs produce the hepatocyte growth factor (HGF) and IL-6, which inhibit monocyte differentiation into dendritic cells and the activation of KCs. They also secrete a variety of other growth factors that help the proliferation of healthy cells and protect the destruction of other cells [348,349,350]. They also produce IL-10 and mitigate the activation of T cells [351]. MSCs secrete prostaglandin E2 (PGE2) to transform M1 macrophages into M2 macrophages [352]. MSCs repress the proliferation of CD8+ T lymphocyte and promote Th1-to-Th2 conversion [353]. It should be noted that the anti-fibrotic potential of MSCs is dependent on autophagy and senescence. Intact autophagy maintains anti-fibrotic activity, while reduced autophagy that coincides with advanced age is associated with a reduction in MSC numbers and function, promoting liver fibrosis [354]. The scRNA analysis of cirrhotic human livers identified four subpopulations of mesenchymal cells. Mes (1) was identified as vascular smooth muscle cells, Mes (4) expressed mesothelial markers, Mes (2) resembled HSCs, but they were not present in the cirrhotic niche, and Mes (3) distinguished by PDGFRA were pro-fibrogenic. Mes (3) cells were increased in cirrhotic livers [91,137]. Extensive reviews on MSCs were recently published [355,356,357].

f. Natural killer (NK) cells. They bear the activating receptor NKG2D and are capable of HSC elimination, inducing apoptosis and IFNγ secretion [358,359,360]. IFNγ secreted by NK cells directly inhibits HSC activation and ECM synthesis [361] and amplifies the killing capacity of NK cells against HSCs by increasing the expression of the NKG2D receptor [362]. The decreased numbers and function of NKs have been demonstrated in the murine models of cirrhosis [363] and cirrhotic patients [364,365,366]. These findings confirmed the anti-fibrotic effects of NK cells. Four immunity-related genes in NK cells, including interferon regulatory factor 8 (IRF8) and REL, are involved in liver fibrogenesis [224].

NK cells are classified into two subpopulations. The vast majority, over 90%, express low levels of CD56 (CD56dim), while the minority express high levels of CD56 (CD56bright). The first is more cytotoxic and a better immunomodulator [367]. In acute viral hepatitis, they both exhibit an antiviral effect either by the direct killing of infected cells or by the activation of viral-specific T cells secreting IFN-γ and TNF-a [368]. NK cell function is defective in patients with chronic hepatitis B (CHB), participating in persistent HBV infection and the development of fibrosis [172]. A further mechanism of NK dysfunction in the particular group of HBeAg+ve patients with CHB is the induction by HBeAg of IL-10 secretion from Tregs, leading to an increased expression of the inhibitory receptor NKG2A on NK cells [369]. Among the many immune abnormalities found in MAFLD, a reduction in CD56bright NK cells and an elevation in CD56dim with less expression in the activating receptor NKG2D was described in patients, offering an additional explanation for the progress of liver fibrosis in MAFLD [370]. A recent observation shed more light on the role of NK cells in MAFLD. Uncoupling protein 1 (UCP1) participates in the leak of protons from the mitochondrial inner membrane. Reduced levels were found in NK cells from patients with MAFLD. Sustained high-lipid administration in mice decreased UCP1 expression and promoted NK cell necroptosis and was involved in the progression to fibrosis [371]. Patients with primary sclerosing cholangitis (PSC) showed considerably higher serum levels of IFNγ and elevated numbers of hepatic CD56bright NK cells. Murine knockout experiments confirmed that increased IFNγ turned the phenotype of hepatic NK cells into increased cytotoxicity, while its absence ameliorated liver fibrosis in PSC [372].

g. Neutrophils. There are very few resident neutrophils in the healthy liver, but there is a rapid recruitment from the circulation in the diseased liver [373]. Neutrophils participate in the liver inflammatory response through the secretion of pro-inflammatory cytokines and the production of extracellular neutrophil traps (NETs). They activate KCs and recruit other types of immune cells [374,375,376]. In mice, the elimination of neutrophils ameliorates the development of hepatic fibrosis [377]. The characteristic neutrophil infiltration into the liver during alcoholic hepatitis is associated with the upregulation of the glycoprotein lipocalin-2 (LCN2) in the neutrophils. A deficiency of bactericidal activity and myeloperoxidase secretion was also found in these patients [92]. The liver-infiltrating neutrophils are also implicated in the immune response in the fibrotic progression in MASH [378,379].

3.3.2. LSECs as Gatekeepers of Innate and Adaptive Immunity

LSECs have a central role in the regulation of both the innate and adaptive immunity being the gatekeepers of the overall liver immune response. Thus, in normal livers, LSECs prevent HSC activation through vascular endothelial growth factor (VEFG)-induced nitric oxide (NO) production. In addition, normal LSECs can reverse activated HSCs back to quiescence through unidentified mechanisms. Decapillarized LSECs isolated from normal rat livers can suppress HSC activation, but capillarized LSECs from cirrhotic rats lose this function. The interaction between LSECs and Kupffer cells is not clarified so far. However, in the fibrosis model, there is evidence that the crosstalk between LSECs and Kupffer cells results in a loss of fenestration and increased CD31 expression [380]. In addition, fenestrated LSECs inhibit liver inflammation by having antioxidant activity. In contrast, capillarized or defenestrated LSECs caused by factors such as ROS production or through lipotoxicity have low antioxidant activity and can induce liver inflammation and promote HSC activation.

It should be noted that the fenestrations of LSECs allow for effector CD8+ T cells to recognize viral antigens expressed in hepatocytes and produce antiviral cytokines by cellular protrusions that extend through the fenestration in a diapedesis-independent manner. This mechanism is obviously lost along with the fenestrae in liver fibrosis, and the effector function of antigen-specific T cells is decreased [381].

Furthermore, LSEC death produces PAMPS that can promote liver inflammation, fibrosis, and cirrhosis acting on KCs and macrophages [317]. LSECs are also implicated in adaptive immunity. They can restrict the entry of immune complexes and leucocytes into liver tissue. Most importantly, LSECs can act as antigen-presenting cells (APCs) and regulate lymphocyte action because they constitutively express MHC class I and II, CD54 (ICAM-1), CD4, CD11, and CD106 (VCAM-1) molecules as well as co-stimulatory molecules CD40, CD80, and CD86, which are necessary for the antigen presentation to T cells. LSECs express MHC- I receptors and present antigens to CD8+ cytotoxic T cells. At low antigen concentrations, this presentation leads to the tolerogenic deletion of CD8+ T cells, but at high antigen concentration, leads to a memory effector T cell phenotype. In HBV infection, CD8+ T cells attach to the sinusoids and then search for infected hepatocytes through LSEC fenestrae. CD8 T cells actively cross the LECS barrier once they sense an infected hepatocyte and release TNFa that in turn eliminates the infected hepatocyte. Moreover, LSECs also present antigens to CD4+ T cells through MHC class II receptors, inducing suppressor Treg cells [200,382].

3.4. Adaptive Immunity T Cells

Cells of adaptive immunity are the many subpopulations of the T lymphocyte family and the B cells. Virtually all lymphocytes originating from naive CD4+ cells participate in the regulation of liver fibrosis. T cells are classified into conventional T cells and innate-like T cells (unconventional T cells). Unconventional T cells consist of natural killer T (NKT) cells, γδ T cells, and mucosal-associated invariant T (MAIT) cells [383,384]. Conventional T cells can be further subdivided into CD8+ cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, T follicular regulatory (Tfr) cells, and CD4+ T helper cells, including Th1, Th2, Th9, Th17, Th22, and T follicular helper (Tfh) cells [385,386]. TNF-α-producing CD4+ T cells are dominant in HBV infection, participating in the progression of liver damage. A sequential increase in IFNγ-producing CD4 T cells characterizes patients with elevated levels of viral clearance [387].

A distinct type of T cell is the tissue resident memory (TRM) T cell that is important as a first-line defense in the liver. These consist of CD8+ and CD4+ cells and they do not circulate [385]. Their storing effector ability of hepatic TRM cells makes them critical in chronic liver diseases. The proliferation of liver TRMs is modulated by cytokines such as interleukin IL-2, IL- 15, IL-10, and TGF-β. Liver TRM cells are antiviral in chronic viral hepatitis. Importantly, the number of liver TRMs positively correlates with inflammation in patients with obesity [388]. A detailed description of the role of the T cell subclasses in liver fibrosis has been published [386].

APCs are classified into professional and non-professional. All professional APCs express the MHC-II molecules and include macrophages, dendritic cells (DCs), and B-lymphocytes. The MHC family consists of MHC-I and MHC-II receptors. MHC-I presents antigens to CD8+ cytotoxic T cells, while the MHC-II molecules induce CD4+ T cell activation [213]. Hepatocytes express MHC-II molecules and co-stimulators, and they may act as an atypical APC to promote T cell activation [389,390]. This has been demonstrated in the liver samples of patients with alcoholic hepatitis (AH) and MASH, where increased levels of MHC-II were observed in close association with MHC-II-producing hepatocytes [237]. Although DCs belong to the cells of innate immunity, they connect the innate and the adaptive immunity [391]. Hepatic DCs (HDCs) are less than 1% of total liver myeloid cells and are subdivided into plasmacytoid and myeloid subpopulations. Myeloid HDCs are further classified as type 1 and type 2 [392,393]. In the healthy liver, HDCs are tolerogenic [394], but they stimulate CD4 +T cells during liver diseases [393]. The majority of HDCs are localized at the portal vein area, with a few localized at the central vein area [395], while their numbers are significantly increased in MASH patients [396]. In the murine models of liver diseases, HDCs have no effect on the survival of HSCs in contrast to macrophages [71]. Animal and human data indicate that other cell types express the MHC-II molecules and act as atypical APCs. They include mast cells, basophils, eosinophils, neutrophils, and innate lymphoid cells (ILCs) [397].

T cell response after the presentation of antigens involves the recognition of the antigen by the T cell receptors (TCRs) on the surface of either CD4+ or CD8+ cells acting in collaboration with the CD3 co-receptor [398]. Other co-stimulatory molecules such as OX40L are required for proper antigen recognition by the T cells [399,400,401]. TCRs comprise two different heterodimers: TCRα/TCRβ or TCRγ/TCRδ [402]. A reduction in TCR subtypes was found in the liver of fibrotic animals, while the deletion of TCRβ aggravated liver fibrosis [403].

Current evidence indicates that T cell immunity influences the fibrosis process [386]. Earlier studies reported that the transfer of CD8+ve T cells contributed to liver fibrosis. CD8+ve T cells directly activated HSCs in murine models [404]. IL-21 promotes the antiviral activity of HBV-specific CD8+ T cells by promoting the production of IFNγ, granzyme B, and CD107a and decreasing PD-1 and TIM-3 production [405]. In addition, IL-2 promotes the proliferation of CD8+ T cells by activating the mTOR pathway to restore dysfunctional CD8+ T cells [406]. IL-33 initiates the proliferation of HBV-specific CD8+ T cells and upregulates PD-1 production, promoting HBV clearance. Not unexpectedly, the plasma levels of IL-33 are low in patients with CHB [407].

The severity of liver fibrosis was positively correlated with intrahepatic CD4+ve T cell apoptosis [408]. CD4+ve T cell activity is involved in the progression of liver fibrosis, by secreting cytokines such as IL-4, IL-10, and IFN-γ, and by stimulating other immune cells such as NK cells [224,366,409]. An analysis of T cell distribution in a small number of viral cirrhosis patients found that CD4+ve T cells, but not CD8+ve cells, were decreased in cirrhotic tissue. This is in contrast to a larger and more detailed study, which reported that a reduction in CD8+ve and NK cells and an infiltration of CD4+ve memory T cells contributed to immune changes in cirrhosis [224,366,409]. The impairment of CD4+ve T cells is implicated in the evolution of liver fibrosis in MASH. An accumulation of liver CD4+ve T cells was demonstrated in human disease and murine MASH models [410,411,412]. CD4+ve T cells were critical in the progression of liver fibrosis after the transfer of human T cells to a specific murine model of MASH. Moreover, the depletion of human CD4+ve T cells attenuated fibrosis in the humanized MASH mice, confirming the significance of these cells in the pathogenesis of MASH [410].

As mentioned before, the group of CD4+ve T lymphocytes includes different subgroups without a uniform behavior in liver fibrosis.

T helper 1 (Th1) cells are pro-fibrotic, producing cytokines such as IFN-γ, IL-2, and TNF-a [413]. An indirect support for the role of Th1 cells in liver fibrosis came from an INFγ knockout murine model of MASH, where the attenuation of fibrosis was observed [414]. These results are in line with the clinical observations that MASH patients have increased hepatic IFNγ-producing CD4+ve T cells [415,416].

Th2 cells are anti-inflammatory, eliciting a protective immune response [417]. Th2 cells produce cytokines such as IL-4, IL-5, and IL-13 [418,419]. Increased serum levels of IL-13, accompanied by the increased hepatic expression of its receptor IL-13Ra2, were reported in MASH patients. Moreover, the IL-13-mediated killing of IL-13Ra2+ve cells suppressed liver fibrosis in a rat model of MASH, supporting the involvement of the IL-13/IL-13Ra2 pathway in MASH [420]. Paradoxically, the administration of IL-33 increased liver fibrosis in a murine model of MASH, despite the fact that IL-33 promotes a Th2 response [421].

Th1 and Th2 cells communicate with LSECs through different adhesion molecules to exert opposite effects in liver fibrosis. The interaction of Th1 cells and LSECs facilitates the reduction in LSEC fenestrae and increases LSEC angiogenesis, finally aggravating liver fibrosis, while the interaction of Th2 cells and LSECs attenuates fibrosis [422,423].

TGF-β and IL-6 are the mediators of the differentiation of T cells into Th17 cells [179,424]. The IL-17 cytokine family consists of six members, namely IL-17A-F [425]. Murine Th17 cells have strong pro-fibrogenic and pro-inflammatory potentials [183,426,427,428]. Th17 cells can trigger hepatic inflammation possibly due to the recruitment of macrophages by the IL-17-dependent upregulation of the chemokine CXCL10 [429,430]. However, the role of Th17 cells in liver fibrosis is not clear. There are reports supporting the pro-fibrotic potential of IL-17 [417,431,432,433]. Other studies support an opposite effect after blocking IL-17 [429,434]. Th17 cells play a crucial role in inflammation, hepatic fibrosis, and HCC development. Th17 cells secrete IL-17 in the presence of IL-6, IL-1β, IL-12, and IL-23, acting through binding to its receptor [435]. Almost all liver cells including hepatocytes, HSCs, BECs, KCs, and LSECs express IL-17R [436]. Moreover, Th17 cells also secrete IL-22 and granulocyte macrophage colony-stimulating factors. These cytokines increase the production and recruitment of neutrophils. Increased numbers of Th17 cells in HBV patients are associated with fibrosis and cirrhosis [437,438]. IL-17 activates MDCs and monocytes to release inflammatory cytokines and recruit neutrophils to the liver [439]. Moreover, there is a negative correlation between disease severity and the methylation level of the IL-17 promoter [440]. The Th17/Treg cell ratio increases and positively correlates with liver injury in patients with a chronic HBV infection [441].

T helper 22 cells are characterized by the production of IL-22 in the absence of IL-17 [442]. The differentiation of the Th22 cell is mediated by IL-6 and TNFa and is inhibited by TGF-β. Evidence supports an anti-fibrotic effect for IL-22 that would be beneficial in MASH [443,444]. However, there is a concern that IL-22 treatment may be a risk for hepatocellular carcinoma development through the activation of STAT3 [445].

There is an interaction of the different cytokines produced by several sources in the modulation of liver fibrosis [446]. For example, the pro-fibrotic cytokine IL-17A was also produced by neutrophils and mast cells [427,447,448]. Th17A promotes the secretion of TGF-β but also promotes the expression of TGF-βRII on fibroblasts, therefore increasing the effect of TGF-β [426,428,449], a similar effect with the Th17-associated cytokine IL-22 [447]. TGF-β in turn induces the expression of IL-17A in collaboration with the IL-1, IL-6, or TNFa [450]. The cytokines IL-4 and IL-13 are also important inducers of fibrosis in association with an eosinophil and M2 macrophage environment [451]. IL-13 induces the production of TGF-β by macrophages [452], but it may increase fibrosis independently of TGF-β [452,453] by a direct effect on myofibroblasts [454].

One should remember that the immune mechanisms of liver fibrosis vary according to the underlying etiology, and results are often contradictory. T cells are no exception. Thus, in MAFLD, activated NK cells attenuate fibrosis progression [455,456,457]. Single-cell transcriptome analysis showed that CD4+ve T cells, CD8+ve T cells, and γδ T cells are increased in the liver with MASH [458]. Alcohol exposure impairs the balance between different T cell subpopulations, leading to a reduction in naïve CD4+ve T cells and CD8+ve T cells [459]. CD8+ve T cell activation and infiltration are considered as the effector mediators of bile duct damage in PBC. Research has demonstrated that specific cytotoxic CD8+ T cells are indeed increased in PBC patients [460,461]. A comprehensive review on the role of Th cells in liver fibrosis has been recently published [430].

One of the most important players in the process of inflammation and fibrosis is a subset of CD4+ve cells originating from the thymus and peripheral organs that are called regulatory T cells (Tregs). They suppress the proliferation and activity of CD4+ve and CD8+ve T cells through co-inhibitory molecules such as the cytotoxic T lymphocyte antigen 4 (CTLA-4) or by secreting suppressor cytokines such as IL-10 and TGF-β [462,463]. Tregs express the transcription factor Fork head box protein 3 (FOXP3). Within the liver, both myeloid and plasmacytoid HDCs are responsible to transform naive CD4+ve T cells into Tregs by expressing the membrane checkpoint programmed cell death 1 ligand 1 (PD- L1) and by releasing IL-10 and kynurenine [462].

In hepatic steatosis, increased oxidative stress leads to the apoptosis and reduction in hepatic Treg cells, and leads to a lowered suppression of inflammatory responses [464]. Moreover, Tregs were reported to be more sensitive to apoptosis in steatohepatitis [412]. Decreased numbers of hepatic Tregs were described in the animal models of MAFLD [411,464,465,466]. An additional explanation is that in fatty livers, adipokines affect Treg cells. Increased leptin production from adipose tissue reduces Treg differentiation, stimulating dendritic cells to polarize CD4+ve cells into Th1 and Th17 cells instead of Tregs [467]. In disagreement with these findings, a recent study found increased numbers of Tregs in the livers of high fat- and high carbohydrate-fed mice. Moreover, the elimination of Tregs inhibited the progression of MASH [468]. In another model of MASH, increased intrahepatic Tregs were found, but when Tregs were transferred, they aggravated MASH, indicating that Tregs increase the metabolic inflammation [469]. In the bile duct ligation model, the elimination of Tregs aggravated liver fibrosis in association with the decreased production of IL-10 [470]. However, Tregs also secrete TGF-β, a well-known promoter of liver fibrosis [142]. To make things more complicated, Tregs were increased in chronic HCV and repressed liver fibrosis [471] but promoted fibrosis in another study of chronic HCV [472]. Despite these contradictory findings, most available evidence indicates that Treg cells are anti-fibrotic, secreting the immunosuppressive IL-10 [419]. An earlier study may offer some explanation. In chronic HBV, it is the significance of the balance between Tregs and Th17 cells that is important and not the absolute number of the individual cells. Both Tregs and Th17 cells in the peripheral blood were increased, but it was the ratio of Treg/Th17 that was correlated with liver fibrosis. Moreover, experiments with isolated human HSCs indicated that Tregs from HBV patients inhibited the activation of HSCs, while recombinant IL-17 increased HSC activation [473].

Tregs are increased during persistent HBV infection [474], downregulating the effector T cells and recruiting innate immune cells to the infected liver, leading to incomplete viral clearance. IL-1β upregulates Treg activation and produces inhibitory cytokines such as IL-10, IL-35, and TGF-β, which are key mediators of Treg function. IL-10 inhibits host anti-HBV activity, leading to the increased replication of HBV. Increased IL-10 levels are correlated with HBV DNA and liver inflammation [475]. HLA-DQ promotes the suppressive function of Tregs [476], while decreased PD-1 expression mitigates the immunosuppressive ability of Tregs and promotes the antiviral activity of effector T cells [477].

HBsAg-specific Tregs intervene with Tfh-dependent HBsAb dysregulation by limiting the differentiation of HBsAg-specific Tfh cells, resulting in insufficient HBsAb production [478]. Tfh cells are implicated in B cell response. They participate in the development of germinal centers from which high-affinity memory B and long-lived plasma cells originate. B cells and plasma cells are required for a protective antibody response [479].

IL-35 is mainly secreted by regulatory T cells and regulatory B cells, which contribute to immune tolerance and viral persistence during chronic HBV infection [480]. IL-35 modulates CD4+ and CD8+ T cells, and induces immunosuppression in chronic HBV infection and non-viral hepatitis-related HCC [481,482]. IL-35 increases PD-1 expression through the JAK1/TYK2/STAT1/STAT4 pathway [483].

In summary, it is clear that Tregs are implicated in the development of liver fibrosis. Tregs activity may either be protective or promotive at different stages of fibrosis development or at different combinations with other interleukins, acting as a two-edged sword. Signaling through the mammalian target of the rapamycin (mTOR) pathway is involved in the protective function of Tregs [484].

Liver B cells are also involved in the immunological response during liver fibrosis through the production of antibodies and the presentation of antigens [485]. It seems that B cells favor the induction and progression of liver fibrosis [484], but most data are derived from pulmonary fibrosis. However, the elimination of B cells attenuated CCl4-induced fibrosis progression in mice [486], while B cell accumulation in the livers of MASH patients correlates to hepatic inflammation and fibrosis [487].

A particular subset of B cells are the B regulatory cells. Bregs in patients with CHB are high, reaching a peak at the immune-active stage. There is a negative correlation with the levels of IL-17 and IFN-γ-secreting Th1 and Th17 cells and CD8+ cells, and a positive correlation with IL-4-producing Th2 cells [488,489]. Bregs can dysregulate T cell function through IL-10, TGF-β, and IL-35. IL-35 levels correlate with the deterioration of liver cirrhosis. The progression of inflammation favors the elevation of Bregs to prevent excessive immune responses, but this may prove detrimental contributing to the persistence of HBV [480,490,491].

Table 2. A synopsis of cytokines involved in immune responses in liver diseases.

Cytokines	Functions	References
TNF-α	Inhibit HBV replication, provide antiviral immunity, induce inflammation.	[432,461]
TGF-β	Impair NK cell function, promote fibrosis and HCC.	[131,132,177]
IL-10	Inhibit cytokine production, regulate T cell immunity, develop persistence of HBV infection.	[481,482,484]
IL-13	Induce inflammation, liver fibrosis. and cirrhosis.	[471,472]
IL-6	Produced by macrophages. Induce inflammation and fibrosis. Inhibit HBV replication; inhibits HBV entry.	[200,202,203]
IL-18, IL-1β	Pro-inflammatory. Activate HSCs. IL-1β induces the phosphorylation of Smad2/3 to promote the transformation of hepatocytes to EMT. IL-18 rs187238 GG genotype increases the risk of HCC in a healthy population and the risk of cirrhosis in CHB carriers.	[137,199,202]
IL-17	Exacerbate inflammation, induce liver fibrosis and cirrhosis.	[436,450,451]
IL-21	Produced by activated CD4+ T cells. Activate T and B cells. Maintenance of specific CD8+ T-cell functions and control of viremia. Increased levels may promote cirrhosis and exacerbate liver injury.	[424]
IL-22	Inhibit liver inflammation and fibrosis.	[462,463]
IL-27	Higher levels in patients with liver cirrhosis or hepatocellular carcinoma. Compensate the function of IL-21 by supporting Tfh-B cell function, required for protective antibody response.	[424]
IL-33	Induce liver damage and fibrosis, activate Tfh cells, and enhance humoral immunity; suppress HBV replication.	[426,433,440]
IL-35	Development of fibrosis, cirrhosis, and HCC. Inhibit HBV-specific CD8 T cells cytotoxicity. Inhibit cytokines and induce antiviral immunity.	[492,493,494]
IFN-γ	Antiviral immunity. Inhibit HBV replication; induce inflammation.	[432,434]

presents a synopsis of cytokines involved in immune responses in liver diseases.

3.5. Unconventional T Cells

They are a heterogeneous group of lymphocytes belonging to the immune system of the liver. The better-studied subpopulations of unconventional T cells include mucosal-associated invariant T (MAIT) cells, γδ T cells, and NKT cells. In the peripheral circulation, they represent almost 10% of T cells. In the liver, however, they are the majority of T cells [379,495]. There are plenty of MAIT cells in the human liver (15–45% of the total T cells), but they are scarce in the liver of mice. On the other hand, invariant NKT (iNKT) cells are <1% of the total T cells in the human liver as opposed to 30–50% in the murine liver [68]. From a functional point of view, NKT cells can be considered as the murine equivalent of human MAIT cells [496].

NKT cells are subdivided into type I NKT (iNKT) cells and type II NKT cells [497], with the former being important in the pathogenesis of several liver diseases [367,498]. In HBV-related cirrhosis, peripheral iNKTs are over-activated and may be partly responsible for the progression of fibrosis [499]. High cholesterol uptake destroys the function of NKT cells through lipid oxidation during the evolution of MAFLD toward cirrhosis. At the early stages of MASH, a reduction in NKT cells has been reported, while in advanced MASH, NKT cells are anti-fibrotic [500,501]. Patients with PBC have increased numbers of Il-17A-producing iNKT cells. The levels of 17A correlate with fibrosis severity [492]. However, this suggestion has been recently disputed in a murine model of PBC, where it was IL-21 and not IL-17A that was associated with disease progression [502]. But again, one should remember the differences in liver NKT cells between humans and mice in every effort to explain these differences.

γδ-T cells have a TCR with two γ and δ chains instead of α and β and comprise 15–25% of all intrahepatic T cells. They are mostly located in portal tracts and areas of bile duct fibrogenesis [493,495]. The activation of γδ T cells does not require an antigen presentation by MHC molecules in contrast to αβ T cells. Therefore, they are referred to as MHC-unrestricted, which may not be absolutely true as some targets of γδ-TCR include the class I MHC molecules [494]. These cells are IL17A producers [503]. γδ-T cells were increased in the liver of the murine models of MAFLD, and their deletion or depletion ameliorated steatohepatitis and accelerated damage repair [504,505]. Interestingly, the gut microbiota may act synergistically with γδ-T IL17+ve cells in disease progression [506]. In contrast to these findings, a transfer of normal γδ T cells ameliorated liver inflammation by increasing the apoptosis of activated HSCs in the methionine–choline-deficient diet of chronic liver disease [507].

Innate lymphoid cells (ILCs). They are subdivided into three groups based on cell surface markers, the transcription factors that regulate their function, and the production of characteristic cytokines [508]. ILC1s consist of IFNγ-producing cells, and they are T-bet dependent, while ILC2s express type 2 cytokines such as IL-5 and IL-13 and are dependent on GATA-binding protein 3 (GATA3) for their function. ILC3s produce IL-17 and IL-22 and depend on the transcription factor retinoic acid receptor-related orphan receptor γt (RORγt) for their function [508,509,510,511]. Recently, a revision has been proposed to include conventional NK (cNK) cells and lymphoid tissue-inducer cells [512,513]. An intrahepatic accumulation of ILC3 cells with pro-fibrotic activity was reported in the CCl4-induced liver fibrosis model. The transfer of ILC3s after the elimination of resident ILC3s increased ECM deposition and liver fibrosis, indicating a pro-fibrogenic role of ILC3 [119,510,512,514]. In addition, a positive relation between the severity of liver fibrosis and the proportion of intrahepatic ILC2 was described. The pro-fibrotic effect of ILC2 was mediated by the overproduction of IL-13, which in turn was induced by IL-33 production from hepatocytes and Kupffer cells [515].

Mucosal-associated invariant T (MAIT) cells in circulation vary between 1 and 10% of total T cells but in the liver may increase up to 45% of intrahepatic T lymphocytes [516]. In patients with either alcohol-related or MAFLD cirrhosis, circulating MAIT cells were reduced, but they were increased in the fibrous septa. Most MAIT cells (80%) from both healthy controls and cirrhotics were CD8+ve, while 20% were double negative (CD8−CD4−). In animal models, the enrichment of mice with MAIT cells promoted liver fibrosis. MAIT cells also enhanced the fibrogenic functions of MFs and MB-derived macrophages [517]. Decreased peripheral MAIT cells with an impaired production of IFN-γ and TNF-α were also described in MAFLD patients. MAIT cells were also increased in the liver and were positively correlated with MAFLD severity. But in contrast to the previous findings, a protective role of MAITs was suggested, as activated MAIT cells in vitro induced M2 macrophage phenotype, and in MAIT-deficient animals, steatosis and inflammation was aggravated [518].

3.6. Extrahepatic Factors

The first and most important extrahepatic factor that is implicated in inflammation and fibrosis is the lymphocyte and monocyte recruitment in the liver. This is dependent on an adhesion cascade influenced by intercommunications between parenchymal and non- parenchymal cells. An example is the liberation of DAMPs by damaged hepatocytes leading to the overproduction of pro-inflammatory mediators by Kupffer cells, which increase adhesion molecule expression by LSECs. Lymphocyte recruitment across activated LSECs involves a firm adhesion on the LSEC surface. Lymphocytes then move along the luminal endothelium until a signal makes them migrate through LSECs through either a paracellular or a transcellular route. Chemotactic factors secreted from activated HSCs direct lymphocytes into the final position within the liver tissue [200].

The dysfunctional gut–liver axis is the second extrahepatic factor that is seriously involved in liver fibrosis. It leads to a “leaky gut” through which bacterial products obtain access to the portal blood and activate liver macrophages, leading to fibrosis. The intestinal barrier is the first line of defense against human intestinal microbiota. The translocation of bacteria is further inhibited by the junctional complex of the intestinal epithelium and by immune cells infiltrating the lamina propria. In gut dysbiosis, as found in chronic liver diseases, all these elements are compromised, allowing abnormal translocations to the liver [519,520]. Different receptors expressed in liver cells can discriminate between gut commensal and pathological antigens. When dangerous signals are detected, APCs, including hepatocytes, recruit immune cells to eliminate pathogen,s maintaining immune homeostasis. On the other hand, when the massive translocation of PAMPs and DAMPs from the impaired intestinal barrier reach the liver, tolerance is replaced by an inflammatory and fibrogenic microenvironment. Innate immunity has the leading role, while adaptive immunity may sometimes be protective. Most relevant research has been based on investigations contacted in relation to MAFLD/MASH [519,521,522]. Other extrahepatic factors include the regulation of liver immunity by other organs. Thus, the spleen affects the composition of liver immune cells. Spleen lipocalin-2 represses the macrophage-induced activation of HSCs. The lung may also affect liver immune regulation, possibly via TNFa modulation of the inadequately studied lung–liver axis. The role of adipose tissue has already been presented. Activated adipose macrophages can migrate to the liver in MASH. Finally, the brain regulates liver immune responses through the liberation of catecholamine and acetylcholine from efferent sympathetic and vagus nerve fibers. They respond to hepatic inflammatory signals transmitted to the central nervous system [523].

3.7. The Interaction of Innate and Adaptive Immunity

The extensive interaction between innate and adaptive immunity was already mentioned in the subchapters of individual cells. However, there are certain discrete bridges that mediate the interplay between innate and adaptive immunity in liver fibrosis.

A first bridge is the activation of γδT cells acting as a connecting point between the innate and adaptive immunity, as they express TCRγδ that recognizes antigens and also produce inflammatory cytokines such as IL- 17A after stimulation [524]. The second bridge of the interplay between innate and adaptive immunity became evident in MASH investigations. Lipid toxicity and oxidative stress damage the hepatocytes, as mentioned before. Both innate immune response and adaptive immunity contribute to MASH-associated inflammation. Innate immunity may lead to fibrosis via PRRs, including TLRs and NLPR3 inflammasomes, that recognize PAMPs and DAMPs. T cell-mediated adaptive immunity also promotes fibrosis in MASH via cytotoxicity and cytokines. KCs are the bridge between the innate and the adaptive responses here. The third bridge is provided by hepatocytes, which, in addition to their functions as innate cells, also express MHC-II molecules and co-stimulators, acting as atypical APCs to induce CD4+ve T cell activation and their Th1 or Th17 cell polarization [213]. IFNγ and other Th1 cell cytokines provide the fourth bridge, as they increase the stimulation of liver macrophages to release M1 pro- inflammatory cytokines and chemokines that further increase the recruitment of monocytes and lymphocytes. Macrophages and dendritic cells release B cell-stimulating cytokines, such as the B cell-activating factor (BAFF), which are fundamental for B cell maturation into plasma cells. The fifth bridge is the secretion by both the hepatocytes and macrophages of IL-15 that improves the survival of CD8+ve T cells and in association with CXCL16, promotes liver NKT cell survival [412,484,525].

Figure 2 summarizes the immune mechanisms implicated in liver fibrosis.

4. Immune Checks in Liver Fibrosis

4.1. Immune Checkpoint

Immune checkpoint proteins (ICPs) have attracted extensive interest, as they are critical suppressors of the immune responses in a variety of tumors. However, they have a wider potential because they maintain immune tolerance in health by repressing T cell activation and proliferation [526,527,528,529]. ICPs which are expressed in tumor cells trigger the exhaustion, senescence, or apoptosis of effector immune cells [530,531]. The best-studied inhibitory immune checkpoints are CTLA-4, PD-1, and PD-L1. CTLA-4 is a molecule that is upregulated on the surface of activated T cells to break their over-stimulation by the TCRs. CTLA-4, also known as CD152, is a strong competitor of the TCR co-stimulatory molecule CD28 and binds to CD80 (B7-1)/CD86 (B7-2) with a stronger binding affinity compared to CD28, thus inhibiting T-cell activation [532]. CTLA-4 is mostly located in intracellular vesicles and is translocated to the cell membrane after T cell activation [533].

The upregulation of PD-1 has also been reported on activated T cells. PD-1 is mostly located at the membrane of cells [533] and binds to its ligand, PD-L1, transmitting inhibitory co-stimulatory signals that prevent T cell activation. The pro-oncogenic and immunosuppressive phenotype of the tumor microenvironment is characterized by the overexpression of PD-L1 by cancer cells and the overexpression of PD-1 and CTLA-4 by T cells [534,535]. Anti-PD-1 and anti-CTLA-4 monoclonal antibodies are two types of extensively used inhibitors of immune checkpoints (ICBs) [536].

PD-1 is expressed in several immune cells, including almost all subtypes of T cells, B cells, NK cells, macrophages, DCs, and monocytes [537,538,539,540]. High levels of PD-1 were an important characteristic of T cell exhaustion [537]. Resting effector T cells do not express PD-1, but after stimulation by antigens, there is a strong expression of PD-1 [541]. PD-1 downregulation follows the elimination of stimulation signals, otherwise the high levels are maintained [542]. Cytokines such as IL-10 and TGF-β also initiate the expression of PD-1 [543,544]. In addition, PD-L1 is induced by several inflammatory mediators such as IFN-γ, a fact that links PD-L1 expression with persistent inflammation [545,546]. The binding of PD-L1 to PD-1 inhibits the proliferation and differentiation of T cells into inflammatory populations, including Th1, Th2, and Th17 cells. This interaction also inhibits the function of CD8+ve cells. Moreover, this interaction upregulates the differentiation of T cells into Tregs [547].

CTLA-4 molecules were upregulated in CD4+ve and CD8+ve T cells in CHB according to recent studies, while the constitutive expression of CLTA-4 was reported in Tregs. CTLA-4 inhibition produced inconclusive results, as T cell proliferation and cytokine production were upregulated in some studies, while others confirmed this only after the inhibition of CTLA-4 in combination with other inhibitory receptors. CTLA-4 is also involved in T cell exhaustion in CHB, but the existing evidence is inadequate at this moment [548].

Macrophages are also modulated by ICPs. PD-1 expression in macrophages is negatively correlated with the presence of M1-polarized tumor-associated macrophages (TAMs) and their phagocytic capacity against tumor cells [549,550].

An important aspect of ICP function is the effect of post-translational modification by glycosylation. During glycosylation, glycan molecules are covalently attached to proteins or lipids by an enzymatic site-specific mechanism that affects the functions of ICPs such as biosynthesis and interactions [551]. No studies have addressed the significance of the glycosylation of ICPs in liver fibrosis and cirrhosis, but there is evidence that this may be important. Epithelial–mesenchymal transition (EMT) is a process that is involved in liver fibrosis, as mentioned before. The glycosylation of PD-1 has been studied in cancer stem cells, where the roles of EMT and N-glycosyl-transferase STT3 were explored. EMT upregulated PD-L1 expression in cancer stem cells by the EMT/β-catenin/STT3/PD-L1 signaling axis. The elimination of both STT3 isoforms suppressed EMT-mediated PD-L1 induction [552].

4.2. Association of Immune Checkpoints with Liver Fibrosis

4.2.1. The PD-1/PD-L1 Axis and CTLA-4

In recent years, the role of the PD-1/PD-L1 axis in liver fibrosis has attracted attention, but the data are still scarce and not conclusive. Most data are coming from the fibrosis progression in other organs, mainly the lung. However, they confirm that a close relationship exists between PD-1/PD-L1 signaling and liver fibrosis. The PD-1/PD-L1 interaction increases fibrosis by promoting important fibrogenic mechanisms such as macrophage polarization, T cell activation, and the trans-differentiation of epithelial cells. The upregulation of PD-L1 induces EMT, and signals that initiate EMT can also promote the expression of PD-L1, creating a positive loop [553]. Recent data indicated that an immune dysregulation of the PD1/PD-L1 immune checkpoint may be implicated in liver fibrosis [554]. There is evidence that PD-L1 inhibitors, such as pembrolizumab and nivolumab, used to treat several cancers, have a potential effect on fibrosis treatment as they reduce fibroblast activation and ECM deposition [555]. Murine studies reported that the Golgi membrane protein 1 (GOLM1) is highly upregulated in carbon tetrachloride-induced liver fibrosis. GOML1 triggered PD-L1 expression and increased fibrosis by activating the EGFR/AKT/STAT3 signaling pathway [556]. The above data indicate that the PD-1/PD-L1 signaling favors the progression of liver fibrosis. On the other hand, reports have suggested that the indirect activation of PD-L1 signaling attenuates liver fibrosis [557].

Blocking PD-L1 inhibits the production of IL-10 and differentiation into Tregs and restores in part the function of CD4+ T cells [558] and HBV-specific CD8 T cells [559]. Continuous HBsAg and HBeAg exposure led to the exhaustion of many CD8+ve T cells and a gradual upregulation of PD-L1 and CTLA-4 expression. PD-1 inhibition alone could not completely restore the exhaustion, which was achieved only after a combined PD-1/CTLA-4 inhibition [559,560]. Interestingly, lower TLR2+ve monocytes and increased PD1 + CD8 + T cell proportions may contribute to viral breakthrough (VBT) in HBV patients switched to IFNa after the failure of nucleoside/tide (NUC) analogs. The combination of TLR2 activation and the PD1/PDL1 pathway blockade may repress HBV replication and prevent VBT through increased cytokine production and the recovering of CD8 T-cell function [561]. PD-L1 antagonists may block HBV replication by initiating cytokine production and by promoting the cytotoxic effects of CD8+ T cells, mostly in patients with low HBV DNA and negative HBeAg [562].

The investigation of specific disease entities offered more evidence that ICPs are implicated in liver fibrosis. In acute viral hepatitis, PD-1 and CTLA-4 are increased during the symptomatic phase, and decreased during recovery. PD-1 and CTLA-4 have protective effects as inhibitory molecules to stop the destruction by cytotoxic T cells in self-limited viral hepatitis. In HBeAg-negative chronic asymptomatic HBV carriers, ICPs are highly expressed on Th1, Th2, Th17, and Tregs [439,563].

Moreover, in chronic HCV, hepatocytes express high levels of PD-L1, leading to the generation of Tregs and follicular regulatory T cells and the liberation of extracellular vesicles rich in TGF-β [564]. The T cell response to chronic infection is suppressed, and liver fibrosis is promoted [565]. In addition, PD-L1 expression mediates the transformation of M2 macrophages in liver fibrosis [566]. A recent clinical study demonstrated that serum PD-1 levels were higher in patients with HCV infection compared to normal controls and gradually increased along with the severity of liver fibrosis [567]. In another clinical study, peripheral blood and splenic CD4+ve and CD8+ve T-cells expressed higher levels of PD-1, mucin domain-containing protein 3 (Tim-3), and CTLA-4 in HCV patients with cirrhosis and portal hypertension compared to normal [568].

Investigations in other organs confirm the association of checkpoints with the process of fibrosis. Several findings link the PD-1/PD-L1 axis with idiopathic pulmonary fibrosis (IPF), as abnormalities of this axis were reported in many cells implicated in IPF pathogenesis [569]. Interestingly, a recent report indicated that anti-PD-L1 antibodies mitigated the ECM deposition of TGF-β1-induced lung fibroblasts by downregulating the PI3K/Akt/mTOR signaling pathway, which is critical in autophagy regulation [570]. These findings are important as the mechanism of PD-L1 in hepatic fibrosis has many similarities with pulmonary fibrosis, mainly in connection with EMT induction in the lung and the liver. PDL1 can induce the production of TGF-β in liver fibrosis. In agreement with pulmonary fibrosis, EMT pathways involving TGF-β, such as Smad and PI3K/AKT, are also active in liver fibrosis [156,571,572,573]. PDL1 also activates HSCs, leading to the production of several factors involved in hepatic fibrosis, as presented above [574]. Finally, PD-L1 favors the transformation into M2 macrophages, which in turn suppresses E cadherin and increases vimentin in hepatocytes, thus promoting EMT [575] in direct analogy with drug-induced pulmonary fibrosis, where the upregulation of PD-L1 promoted fibrosis through the inhibition of vimentin degradation [576]. Mechanistically, PD-L1 directly upregulated the serum and glucocorticoid kinase 2 (SGK2) and activated the SGK2/β-catenin signaling pathway to induce EMT and the transformation of liver cancer cells into a stem cell phenotype [577].

However, there are differences between liver and pulmonary fibrosis. PD-L1 on liver fibrosis is mostly immunomodulatory. Thus, in an earlier paper on chronic persistent HCV disease, it was demonstrated that HCV-specific CD8 T cells from the liver expressed high levels of PD-1 and a significant impairment of their function. CTLA-4 was also upregulated in PD-1+ve T cells from the liver but not from the blood of persistently infected HCV patients. Interestingly, the impaired function of CD8+ve cells was synergistically reversed by a combined PD-1/CTLA-4 blockade, but not by blocking PD-1 or CTLA-4 alone, indicating that both PD-1 and CTLA-4 pathways participate in the virus-specific T cell exhaustion in chronic HCV [578]. Although reported data suggests a similar role of CTLA-4 in T cell exhaustion, the documentation is weak to support the role of CTLA-4 in T cell exhaustion in chronic HBV infection, as mentioned above [548].

Another approach to clarify the role of ICPs in liver fibrosis is to delineate the association of ICPs with the functions proved to participate in the fibrotic process. ICPs are implicated in the function of critical cells in the regulation of liver fibrosis such as MCS, macrophages, and HSCs.

Thus, PD-L1 expression is involved in the immunomodulation mediated by the mesenchymal stromal/stem cells (MSCs) as well. PD-L1 on the surface of MSCs interacts with PD-1 on the surface of T cells through direct cell-to-cell communication, inhibiting the functions of T cells. PD-L1 may also be secreted, inhibiting T cell function without the close contact of cells. MSCs may transfer PD-L1 in extracellular vesicles, again affecting T cells from a distance. Signal transmissions from MSC PD-1 create a positive loop, enhancing their immunomodulatory potential. On the other hand, anti-PD-L1 antibodies can reduce immunomodulation mediated by MSCs [579].

Further immunomodulation by IPCs in liver fibrosis may be mediated via PD-1 induction in monocytes and macrophages through TLR signaling and cytokines such as TNF-α, IL-1β, and IL-6 [580,581]. Furthermore, PD-1 expression in macrophages may repress innate inflammatory responses [582,583]. PD-L1 activation sends negative signals to macrophages, inducing an immunosuppressor cell phenotype [584]. The overexpression of PD-L1 in macrophages and peripheral monocytes has been demonstrated in chronic viral infections in the liver [232,585]. A study of patients with cirrhosis showed that liver macrophages overexpressed the immune-suppressive proteins PD-L1, MARCO, and CD163. Monocytes from patients also overexpressed PD-L1, which was related to disease severity and the presence of infections. A blockade of PD-L1 with anti-PD- L1 antibodies restored liver macrophage functions [267]. These findings have been confirmed in an acetaminophen-induced acute liver injury murine model. Reduced bacterial clearance by KCs expressing PD-1 was observed during liver injury. During resolution, KCs expressed higher levels of PD-1 and lymphocytes expressed higher levels of PD-L1. The suppression of PD-1 expression by anti-PD-1 improved KC bacterial clearance. Increased PD-1 expression in monocytes and increased PD-L1 expression in lymphocytes of peripheral blood were found in patients with acute liver failure. Moreover, PD-L1 plasma levels were positively correlated with sepsis and mortality. Interestingly, PD-1 in vitro blockade restored monocyte functionality [586,587].

Activated hepatic stellate cells from human livers induce the apoptosis of activated T cells through the expression of PD-L1. Human HSCs have strong immunoregulatory activity via the B7-H1-mediated induction of apoptosis in activated T cells [588]. Murine HSCs suppressed the upregulation of activation markers on B cells together with the repression of their proliferation and their cytokine production. Interestingly, the elimination of the interaction of PD-L1 with PD-1 decreased the ability of HSCs to suppress B cell activation [306]. Several recent reports have clearly demonstrated that senescent HSCs display an increased expression of PD-1/PD-L1 proteins. An increase in the level of the PD-L1 protein in senescent cells is able to suppress their immune surveillance and inhibit their elimination by cytotoxic CD8+ve T cells and NK cells [186,589,590,591,592]. ICPs also affect TGF-β function. PD-L1, produced by HSCs, is necessary for HSC activation by protecting the two TGF-β receptors from degradation. The extracellular domain of PD-L1 protects the TβRII protein, while the 260-RLRKGR-265 motif on PD-L1 protects the TβRI mRNA [593].

4.2.2. Other ICPs Involved in Liver Fibrosis

Apart from the better-studied PD-1/PD-L1 and CTLA-4, additional ICPs have the potential to be involved in fibrosis, although the data are inadequate.

The B7 homolog 3 protein (B7-H3), also designated as CD276, is a critical ICP of the B7 immunoglobin superfamily [594]. B7-H3 is expressed on APCs and is involved in T-cell-mediated immunity. The aberrant expression of B7-H3 in several cancers is associated with a poor prognosis and increased angiogenesis [595]. B7-H4 is also a member of the same B7 superfamily. It was also found on professional APC, preventing T cell activation and was also associated with poor prognosis [596,597].

ICPs such as LAG-3, TIM-3, and CD39 on CD8+ve T cells were increased in patients with chronic HBV. The ability of CD8+ve cells to secrete TNFa, IFNγ, and perforin was downregulated [598]. TIM-3 levels positively correlated with HBV DNA levels [599]. Lymphocyte activation gene 3 (LAG-3), also designated as CD223, has been a promising target in the treatment of hepatocellular carcinoma (HCC). In patients with HCC, LAG-3 expression in Tregs and NK cells is implicated in tumor immune evasion by interacting with MHC-II molecules. Its overexpression is associated with T cell exhaustion in synergy with PD-1 and increased angiogenesis [600].

The increased expression of TIM-3 and galectin-9 also led to the inhibition and apoptotic deletion of T cells. Th cells expressing TIM-3 have a limited production of IFN-γ and TNF-α after the recognition of HBV peptides and are sensitive to galectin-9-initiated cell death. The expression of TIM-3 on peripheral T cells parallels disease progression and markers of liver damage including increases in ALT, AST, bilirubin, and international normalized ratio (INR) [600]. The inhibition of TIM-3 initiated the proliferation of HBV-specific CD8 T cells and upregulated antiviral cytokine secretion [601].

Kynurenine (Kyn) is another important IPC modulator of immune responses via its aryl hydrocarbon receptor (Ahr). For Kyn synthesis, two enzymes are implicated, the indoleamine 2,3-dioxygenase (Ido) and the tryptophan 2,3-dioxygenase (Tdo). Ido is responsible for 90% of tryptophan catabolism. Although Kyn is increased in various liver disorders, the exact involvement of Kyn in liver damage has not been clarified as Ido1, Ido2, and Tdo are activated in several cell types. However, Ido1 deficiency aggravated liver fibrosis in the CCL4-induced liver injury murine model [602]. Moreover, liver fibrosis in the same model was mitigated in Ido2−/−, indicating that the inhibition of kyn or ido 2 may ameliorate hepatic fibrosis [603].

4.3. Therapeutic Implications of Checkpoint Inhibitors in Liver Fibrosis

Despite the increasing evidence that checkpoint inhibitors are involved in the regulation of inflammation and liver fibrosis, there are very few clinical data and some experimental observations on their use in these two conditions.

A blockade of inhibitory checkpoints including PD-1, CTLA-4, 2B4, TIM-3, and galectin-9 alone or in combination has emerged as a potential therapeutic approach to restore T and B cell functions in CHB [478,558,604,605,606,607]. In studies of HBV-infected mice and blood from patients with a chronic HBV infection, a Tfh cell response to HBsAg was required for HBV clearance, and this response was blocked by Treg cells. Inhibiting Treg cell activity using neutralizing antibody against CTLA4 restored the ability of Tfh cells to clear HBV infection. This approach might be used in future clinical trials for the treatment of patients with chronic HBV infection [478].

However, there are sufficient data from the extensive use of ICPs in the treatment of HCC. It is known that the great majority of HCC cases have a background of fibrosis and cirrhosis. Therefore, data on HCC treatment may offer an overview of the use of these drugs in advanced liver disease. The results of clinical trials for HCC may be extrapolated in the future treatment of liver fibrosis, at least as far as safety is concerned.

The published results of anti-PD1/PD-L1 monotherapy for HCC with nivolumab, pembrolizumab, durvalumab, and camrelizumab indicate a non-impressive overall survival of 13.2–16.9 months. More favorable were the results of the combinations of ICPs with tyrosine kinase inhibitors. Atezolizumab, a PD-L1 inhibitor, combined with bevacizumab showed a 56% reduced risk of death compared to sorafenib. Camriezumab combined with alpatinib had an overall survival of 22.1 months compared to 15.2 months for sorafenib [608]. The Himalaya trial evaluated the STRIDE regimen consisting of a single dose of tremelimumab with a dose of durvalumab every 4 wks. A significant but again not impressive increase in overall survival of 16.4 months vs. 13.8 months for sorafenib was reported [609]. Other combination treatments of anti-PD-1 with ipilimumab and tremelimumab (CTLA-4 IPCs inhibitors) are in progress [610].

Interestingly, a systematic review of systemic therapies in HCC from 2002 to 2020 revealed that immunotherapies were more effective in viral etiologies as compared to non-viral etiologies, possibly because the immune responses are more vigorous in viral infections compared to non-viral etiologies [611].

An additional interesting approach was recently reported. Coagulation factor Xa (FXa) and its receptor proteinase-activated receptor-2 (PAR-2) promote tumor metastasis in several forms of cancer. The combination of the anti-coagulation drug rivaroxaban and an anti-PD-1 antibody induced synergistic antitumor effects in experimental models. Most importantly, rivaroxaban improved the objective response rate of HCC patients and prolonged the overall survival time [612].

Whatever the survival benefits might be, adverse events (AEs) do happen, including immune-related adverse events (irAEs) such as rash and pruritus, diarrhea and colitis, hypothyroidism and hypophysitis, pneumonitis, and psychiatric disorders [613,614]. The reactivation of HBV has also been observed [615]. ICP inhibitors combined with angiogenesis inhibitors may reduce incidence and mortality for most irAEs [616].

5. Conclusions

Liver fibrosis is the end result of almost all chronic liver diseases. However, the underlying mechanisms are different in many respects according to etiology. There has been great progress in the cellular and molecular biology of liver fibrosis, and it is now accepted that the sinusoids are a fundamental field in fibrosis induction and progress. Kupffer cells and the hepatic stellate cells are the most important cells of innate immune response implicated in fibrosis. They are the masterminds of the immune regulation of fibrosis as they interact with each other, assisted by other immune cells such as lymphocytes and liver endothelial cells. Kupffer cells are implicated in the activation of HSCs that in turn are the producers of ECM through a complex network of cytokines and chemokines. At the same time, both Kupffer cells and HSCs are responsible for the resolution of fibrosis through a network of inhibitory cytokines and the production of degrading metalloproteases. It is also well documented that adaptive immunity and its very many cells are critical components in the regulation of fibrosis as are cells of an intermediate nature such as MAIT cells and γδ T cells that interact with elements of both innate and adaptive immunity. Recently, a hotspot of research is the role of the immune checkpoints (ICPs), as they are the main controllers of the excessive immune responses, and their inhibition is currently in clinical use in an effort to overcome the immune evasion masterminded by several cancers. There is increasing evidence that ICPs are involved in the regulation of liver fibrosis; therefore, a new chapter in anti-fibrotic therapy may start as many new ICPs are described and the role of the better-studied PD-1/PD-L1 and CTLA-4 is intensively researched. They are effective in non-fibrotic viral liver disease as well, but their application will be limited. The current antivirals are very effective and more cost-efficient, and it is not reasonable to replace them. There are sufficient data on the safety of ICP inhibitors, alone or in combination with other drugs, from extensive trials on the treatment of HCC, indicating that trials on the treatment of liver fibrosis are justified, and possibly a new era on immunotherapy of this difficult-to-treat liver disease is ahead.