Liver immunology: Biological role and clinical significance

Abstract

Liver diseases are of growing interest to clinicians and researchers due to their high prevalence, difficulty in early diagnosis, and limited treatment options. The liver is an important organ at the intersection of many metabolic and immune pathways. To this end, it contains a large number of immune cells of both the innate and adaptive immune system that perform multiple functions, detecting and destroying pathogens that enter the body through the intestine, as well as recognizing endogenous antigens. Immune cells in the liver have a complex regulation that can be impaired in various diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD), liver cancer, and biliary diseases. A growing body of evidence reinforces the realization that not only impaired metabolism but also many immune mechanisms underlie MASLD. The liver has complex bilateral immune and metabolic links with the gut microbiota, and disruptions of these links underlie the development and progression of both gastrointestinal and other organ diseases. In this regard, acting on immune mechanisms is a promising therapeutic target for liver diseases.

Key Words: Liver; Immunology; Immunometabolism; Innate immune system; Adaptive immune system; Immune cells; Metabolic dysfunction-associated steatotic liver disease; Biliary diseases; Gut microbiota

Core Tip: The liver is an important organ at the intersection of many metabolic and immune pathways. Immune cells in the liver have a complex regulation that can be impaired in various diseases such as metabolic dysfunction-associated steatotic liver disease, liver cancer, and biliary disorders. Interactions with the gut microbiota play an important role in liver immunology. Effects on immune mechanisms are considered a promising therapeutic target for liver diseases.

INTRODUCTION

Liver diseases are an urgent medical problem due to their high prevalence, as well as significant underdiagnosis and underestimation in real clinical practice. The problem of liver diseases is also reinforced by the difficulty and expense of diagnosing and monitoring the disease course, which contribute to many patients going for long periods of time without a confirmed diagnosis[1,2]. In addition, patients may not seek medical attention due to mild or no symptoms for long periods of time. Metabolic dysfunction-associated steatotic liver disease (MASLD), for example, is often not diagnosed in a timely manner, although it is one of the most common metabolic diseases, occurring in 17%-46% of the adult population, and is particularly common in obese patients[3,4]. In this regard, the study of the pathogenesis of liver diseases and the identification of new biomarkers for early diagnosis and monitoring of the course of these diseases is an urgent task for clinicians and researchers.

The liver is the largest gland in the body, representing about 3% of total body weight, and performs many different functions, the full consideration of which is beyond the scope of this review[5,6]. According to current thinking, the liver is a central regulator of many metabolic and immune processes. Indeed, the liver acts as an important barrier to various toxic substances and pathogens that may enter the bloodstream from the intestine. At the same time, on the one hand the liver must detoxify and neutralize the harmful substances that enter the body through the bloodstream; on the other hand the liver needs to maintain immune tolerance in order to ensure adequate immune responses to commensal microorganisms and food antigens[7-10]. Thus, the liver is an important defense organ, and impaired immune function is associated with various diseases. It is important to note that this complex combination of immune and metabolic functions characteristic of the liver is not random, but rather is a product of evolutionary pressures that have sculpted the liver into a vital immune organ[11].

Therefore, the aim of the current review is to discuss the immune function of the liver and how these functions are impaired in various noninfectious liver diseases.

STRUCTURE AND IMMUNE FUNCTION OF THE LIVER

The liver is located at the crossroads of blood flow pathways, and consequently, has a very intensive blood supply. It receives about one-quarter of the cardiac output, and the blood volume in the liver is about 14% of the total blood volume in the body. The liver has a dual blood supply: Approximately 20%-30% comes from the hepatic artery, primarily supplying oxygen, while the remaining 70%-80% arrives via the portal vein, which is mainly responsible delivering nutrients[12,13].

The structural and functional units of the liver are hepatic lobules (Figure 1), which are hexagonal structures with a central vein[14-16]. Each lobule consists of hepatocytes arranged as plates diverging from the central vein; sinusoids, i.e. capillary-like vessels between the plates of hepatocytes through which blood flows from the portal triads into the central vein; and portal triads, which are located in the corners of the lobules and consist of a branch of the hepatic artery, a branch of the portal vein, and the bile duct[14-16]. The intra-lobular blood capillaries are lined with liver sinusoidal endothelial cells (LSECs). Numerous Kupffer cells are located between the endothelial cells. The space between hepatocytes and LSECs is known as the space of Disse, where hepatocytes exchange substances with the blood[13].

Figure 1 Structure of liver hemodynamics.

The liver is composed of parenchymatous cells (hepatocytes) and non-parenchymatous cells, which include LSECs, Kupffer cells, hepatic stellate cells (HSCs) (Ito cells), lymphocytes, and a number of other cells. Parenchymatous cells account for about 70% of its mass, and non-parenchymatous cells make up the remaining 30%. The liver contains many cells of innate and adaptive immunity, including Kupffer cells, dendritic cells, natural killer (NK) cells and T cells, which are essential for detecting and responding to pathogens.

Hepatocytes

Hepatocytes constitute about 70% of all liver cells. They have an irregular polygonal shape and are about 20-25 μm in diameter[14,15]. Many of these cells (up to 20%) contain two or more nuclei. The number of these cells depends on their functional state. Hepatocytes form a cellular barrier separating the sinusoidal blood from the bile ducts. Hepatocytes are characterized by tissue polarity in which their basal membrane faces the endothelial cells of liver sinusoids, and the apex of hepatocytes forms numerous bile tubules directly adjacent to neighboring hepatocytes[13].

The main function of hepatocytes is to participate in the metabolism of substances, protein synthesis, and the neutralization of toxins. Hepatocytes also produce fibrinogen, a key factor in the blood coagulation system[17-19]. It should be noted that the blood coagulation system is evolutionarily linked to immunity, as it is designed to ensure that the bloodstream is sealed in the event of damage. In addition, hepatocytes are directly involved in immunity by synthesizing acute phase proteins and complement, as well as producing opsonins that promote phagocytosis of foreign pathogens including C-reactive protein, serum amyloid A, and serum amyloid P[19]. Hepatocytes also express a number of immune receptors such as Toll-like receptors (TLRs), retinoic acid-inducible gene 1 and nucleotide-binding and oligomerization domain (NOD)-like receptors. In addition, they are a major source of lipopolysaccharide (LPS) binding protein, soluble cluster of differentiation 14 (sCD14), and soluble myeloid differentiation factor 2 required for LPS signaling[19-22]. In response to cytokine action during liver injury or in response to bacterial infection, hepatocytes can produce acute phase proteins and also produce several chemokines, such as monocyte chemoattractant protein 1 (MCP-1) and C-X-C motif chemokine ligand 1, involved in the recruitment of immune cells (e.g., macrophages and neutrophils)[19].

Hepatocytes are also involved in efferocytosis, the engulfment of other cells, including dead immune cells such as apoptotic T lymphocytes[23,24]. In addition to engulfment by hepatocytes, autoreactive CD8+ T lymphocytes can themselves actively infiltrate hepatocytes where they undergo lysosomal degradation in a process known as suicidal emperipolesis[23,25,26]. In addition to being involved in inflammasome activation, hepatocytes also exert important anti-inflammatory functions, for example through the production of peptidoglycan recognition protein 2. This immunomodulatory protein promotes the hydrolysis of bacterial peptidoglycan to smaller fragments, which impairs its recognition by TLR of immune cells[19].

In addition to mechanisms of the innate immune system, hepatocytes may participate in adaptive immunity by expressing major histocompatibility complex (MHC) I molecules and molecules associated with antigen presentation. During inflammation, some hepatocytes may express MHC II molecules and interact with lymphocytes and induce their activation. MHC II expression on hepatocytes, for example, has been found in alcoholic and non-alcoholic hepatitis[13,27]. Hepatocytes do not express costimulatory factors, cluster of differentiation 80 (CD80) and CD86, so they cannot induce sustained activation and survival of T cells.

Interestingly, the function of hepatocytes depends on their anatomical location. Analysis of hepatocyte populations by single-cell RNA sequencing, identified several clusters of hepatocytes that differ in function, including hepatocyte clusters found to express genes involved in immunity[28].

Thus, hepatocytes, as the main parenchymatous cells of the liver, perform a number of immune functions, which are of importance in various diseases.

LSECs

LSECs are highly specialized cells that play a critical role in maintaining liver homeostasis and are involved in a variety of physiological and pathological processes. LSECs have a unique fenestrated structure that is essential for the exchange of nutrients and waste products between the blood and liver parenchyma[29,30]. To perform this function, LSECs do not have a dense basal membrane (unlike normal endothelium), which facilitates the exchange of molecules. Fenestrae are small pores 50-300 nm in diameter, and their total area is approximately 10% of the entire surface area of the LSEC[31-34]. Fenestrae are absent in other types of endothelium, and in the liver provide direct filtration of substances between blood and hepatocytes. Fenestrae allow large lipoproteins (e.g., chylomicrons) to penetrate into the space of Disse for subsequent metabolism. In addition, LSECs, like vascular endothelial cells, are involved in the control of vascular tone through the production of vasoactive substances (e.g., nitric oxide [NO]), regulating sinusoid diameter and pressure in the portal system. In addition, NO produced by LSECs is involved in the regulation of LSECs differentiation and hepatic lipid homeostasis[35,36].

In addition to transport and vasoregulatory functions, LSECs play an important role in liver immunology. They act as scavenger cells, removing potentially harmful macromolecules from the blood via receptor-mediated endocytosis[30,37-40]. LSECs also remove soluble colloidal or macromolecular wastes via clathrin-mediated endocytosis[32,39,40]. Thus, LSECs act together with Kupffer cells to remove bacteria as well as dead and dying cells and insoluble waste by phagocytosis. LSECs have, for example, been shown to promote the uptake and destruction of enterovirus bacteria[41]. A peculiarity of LSECs is that they contain about 15% of the total volume of lysosomes and about 45% of the total volume of pinocytosed vesicles in the liver, although they represent only 2.8% of the total liver volume[42,43].

To exert immune function, LSECs express a wide range of pattern recognition receptors (PRRs) including TLR-3, TLR-4, TLR-7, TLR-9, and MHC and costimulatory molecules[44]. LSECs are also involved in immune mechanisms through cytokine secretion. By secreting interleukin 10 (IL-10), transforming growth factor beta (TGF-β), and other mediators, LSECs modulate the activity of macrophages (Kupffer cells) and liver stellate cells. LSECs interact with other cells, including Kupffer cells, hepatocytes, and other immune cells, playing a central role in the liver's response to injury and disease[45-47]. In liver injury, such as in MASLD and alcoholic liver disease (ALD), LSECs alter the expression of chemokines, attracting immune cells and inducing inflammation[48]. On the other hand, LSECs can promote immune tolerance by presenting antigens and interacting with immune cells, thereby preventing excessive immune responses under normal conditions[38,46,49]. LSECs also play a role in the tumor microenvironment, contributing to immune tolerance and the development of hepatocellular carcinoma (HCC)[50].

In addition, LSECs participate in liver regeneration by secreting growth factors (hepatocyte growth factor [HGF] and vascular endothelial growth factor [VEGF]) that support hepatocyte proliferation after injury. LSECs secrete angiogenic signals that are crucial for liver regeneration after injury[51]. On the other hand, LSECs are involved in the development and progression of liver fibrosis. They interact with HSCs and also contribute to the fibrotic process through capillarization, i.e. loss of their fenestrated structure[52]. In chronic damage (alcohol, viruses), LSECs activate HSCs via TGF-β, promoting collagen deposition and fibrosis.

Disruption of the fenestrated structure and function of LSECs plays an important role in the pathogenesis of liver disease. The appearance of the basal membrane and loss of fenestra transform sinusoids into capillaries (a process known as capillarization). Capillarization of LSECs impairs the ability of LSECs to absorb and transport various substances, which impairs many liver processes and function. In cirrhosis, alcoholic disease or MASLD, defenestration impairs metabolism and contributes to portal hypertension.

Thus LSECs, although they are endothelial cells, have a number of structural and functional features, which are associated with their diverse functions in the liver.

Kupffer cells

Kupffer cells are the population of resident macrophages in the liver. Kupffer cells account for 80%-90% of the total number of fixed macrophages in mammals. They have an amoeboid shape and are usually found in the lumen of the liver sinusoids, attaching to the endothelial lining, and are also capable of migrating along the sinusoid walls[43]. Kupffer cells and macrophages differentiated from monocytes (monocyte-derived macrophages) are distinct subgroups of macrophages in the liver that can be distinguished from each other by differential expression of cell surface markers[53,54]. Kupffer cells have their origin in the yolk sac, from where germinal macrophages migrate to the fetal liver and complete differentiation into Kupffer cells.

Kupffer cells serve as the first line of defense against pathogens, effectively recognizing and phagocytosing pathogens[55,56]. Liver macrophages play differential roles in inflammation depending on their immunometabolic polarization: Pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype. The phenotypes are switched due to changes in cell metabolism. The M1 phenotype is characterized by increased glycolysis, reduced oxidative phosphorylation, and a disrupted tricarboxylic acid cycle; by contrast, the M2 phenotype is characterized by normal oxidative phosphorylation and the tricarboxylic acid cycle and moderate glycolysis. M1 polarization is induced by interferon gamma (IFN-γ) and LPS and characterized by the production of pro-inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α). In addition, macrophages with M1 polarization also produce reactive oxygen and nitrogen species and are involved in phagocytosis and antigen presentation. M2 polarizing macrophages are induced by IL-4 and IL-13 and are characterized by the production of anti-inflammatory cytokines such as IL-10. M2 macrophages are involved in tissue repair, angiogenesis, and immune regulation. This is due to the switch of arginine metabolic pathways from NO production (in M1 macrophages) to ornithine and proline production (in M2 macrophages) required for tissue repair after inflammation[57-60].

Disruption of macrophage polarization has been reported in a variety of diseases including cancer, metabolic and inflammatory diseases. In cancer, tumor-associated macrophages promote tumor growth by stimulating angiogenesis and suppressing the immune response. In metabolic disorders such as obesity and type 2 diabetes, macrophages in adipose tissue undergo M1 polarization and contribute to chronic inflammation, insulin resistance, and glucose intolerance.

Kupffer cells are major effector cells that damage hepatocytes by producing pro-inflammatory cytokines, lysosomal enzymes, phospholipase, reactive oxygen species (ROS) and NO. In addition to participating in inflammation activation, Kupffer cells may play an important role in immunological tolerance by suppressing the activity of effector T cells and activating regulatory T cells (Tregs)[54,61-63]. Kupffer cells are also the major antigen-presenting cells (APCs) in the liver.

Kupffer cells play an important role in cholesterol metabolism. They take up and transport low-density lipoprotein (LDL)-derived cholesterol into hepatocytes and are involved in the regulation of high-density lipoprotein (HDL) and LDL cholesterol levels by the cholesterol ester transfer protein (CETP) surface receptor[53,64]. Kupffer cells produce CETP in plasma[64]. CETP is a plasma protein that transfers cholesterol from HDL to the atherogenic LDL fraction[65]. CETP plays an important role in reverse cholesterol transport, a homeostatic pathway to maintain cholesterol balance. During reverse cholesterol transport, excess cholesterol is transported from macrophages to HDL and then to the liver for utilization. It is important to note that reverse cholesterol transport can be used not only for cholesterol transport but also as a pathway for utilization of LPS in the liver. In this regard, the pathways of cholesterol metabolism are closely related to the immune function of macrophages. Thus, Kupffer cells are important participants in the innate immune system of the liver.

HSC

HSC are non-parenchymatous cells located in the Disse space between hepatocytes and endothelial cells of liver sinusoids. The most characteristic feature of these cells is the presence of cytoplasmic perinuclear droplets containing retinol (vitamin A) esters, as HSCs carry out storage of this vitamin[66].

In the quiescent state, HSCs can produce cytokines and growth factors such as HGF[67]. Liver stellate cells participate in liver regeneration by producing growth factors necessary for liver development and repair, and they also maintain the balance of the extracellular matrix (ECM), which is essential for maintaining liver structure and function. In addition, HSCs can interact with LSECs to regulate hepatic blood flow[66].

Upon liver injury, HSCs switch from a quiescent state to an activated myofibroblast-like phenotype. This activation includes proliferation, increased ECM production, and secretion of pro-inflammatory and profibrogenic cytokines, all of which contribute to the development of hepatic fibrosis. HSC activation involves loss of lipid droplets and expression of contractile fibers, increased proliferation and chemotactic activity of HSCs, and signaling for leukocyte recruitment[67]. Upon activation, human HSCs express smooth muscle α-actin and begin to produce an ECM composed of collagen (e.g., collagen type I alpha 1 chain, collagen type I alpha 2 chain). Alpha smooth muscle actin production during HSC activation contributes to vascular deformation, increased vascular resistance, and the development of portal hypertension[66]. Activated HSCs are a major source of collagen in liver fibrosis, leading to excessive ECM deposition and tissue scarring. HSCs contribute to the inflammatory response and participate in the development of HCC by interacting with cancer cells and promoting tumor growth and angiogenesis. NK cells can induce remission of liver fibrosis by destroying HSCs and producing IFN-γ in a mouse model. At the same time, activated hepatic star cells reduce the ability of NK cells to fight fibrosis through TGF-β-dependent emperipolesis, which has been shown in patients with hepatitis B virus (HBV)-induced cirrhosis[68].

Lymphocytes

Lymphocytes are a major cell population in the liver and are represented by many different subtypes. Both resident and migratory T-cell subtypes are present in the liver and play a critical role in regulating inflammation and antimicrobial defense of the liver. Given the peculiarities of the liver structure, circulating T cells in the blood may interact with antigen-presenting liver cells in the sinusoids. As already mentioned, APCs in the liver are dendritic cells, LSECs, and Kupffer cells, which are readily accessible to circulating naive T cells in the blood. In addition, hepatocytes can also perform antigen-presenting functions in inflammation[69,70].

CD4+ T cells in the liver coordinate the immune response by secreting cytokines and are also involved in chronic liver inflammation and fibrosis[70,71]. Cytotoxic CD8+ T cells directly destroy infected or cancerous cells and play an important role in viral hepatitis (e.g., HBV, hepatitis C virus [HCV])[72,73]. Recognition and destruction of APCs is performed by CD8+ T cells through binding of MHC class I molecules and the T-cell receptor (TCR) on the cell surface[74]. Upon activation, CD8+ T cells produce IFN-γ and other cytokines and exhibit cytotoxic activity through the release of perforin and granzyme B[72,75,76].

CD8+ T cells play an important role in liver inflammation, affecting the liver in a variety of ways and contributing to both direct and indirect liver damage. CD8+ T cells can exert a direct cytotoxic effect on hepatocytes through the production of perforin, leading to the release of signaling molecules such as IL-33, which can activate other immune cells and promote further liver inflammation[75,77,78]. CD8+ T cells can cause secondary liver injury by stimulating Kupffer cells to produce TNF-α, which enhances the inflammatory response and promotes liver damage[79]. CD8+ T cells can travel to the liver sinusoids where they interact with platelets and detect antigens in hepatocytes, leading to their activation and subsequent inflammatory responses[72,80,81].

Another important subgroup of liver lymphocytes is represented by NK cells. NK cells account for 25%-50% of the total number of liver lymphocytes and play a central role in innate immunity, including early antiviral and antitumor properties[82-84]. The number of NK cells in the liver is altered in various diseases due to an increase in the influx of circulating NK cells into the liver[85,86]. These cells include conventional NK cells (circulating subset), which mediate antiviral and antitumor responses and resident liver NK cells (tissue-specific), which exhibit adaptive properties and regulate fibrosis[87,88]. NK cells can increase liver inflammation by secreting pro-inflammatory cytokines and cytotoxic action on hepatocytes and cholangiocytes. At the same time, NK cells can also attenuate liver inflammation by killing activated T cells and pathogenic cells located in the liver. Thus, the effect of NK cells on liver disease is mediated by their direct cytotoxic function against hepatocytes, cholangiocytes or immune cells in the liver, including T cells and APCs, as well as by the secretion of cytokines, including IFN-γ[84].

NK T cells (NKT cells) are another unique subset of lymphocytes that link innate and adaptive immunity and play a critical role in liver homeostasis, inflammation, and disease. In the liver, NKT cells are particularly abundant compared to other organs, making them key players in liver immunology. NKT cells are divided into two major subsets according to their TCR diversity and antigen specificity: (1) Type I invariant NKT cells recognize lipid antigens via CD1d and express a semi-invariant TCR[89,90] and play diverse roles in liver diseases including viral, autoimmune, and toxic liver diseases[82,91,92]; and (2) The second group consists of type II NKT cells, which have a variety of TCRs. Thus, two different subtypes of NKT cells recognize antigens differently and may play opposite roles in inflammatory liver diseases[91,93,94]. Type I NKT cells, when activated, secrete T helper 1 (Th1) cytokines (IFN-γ, TNF-α) and Th2 cytokines (IL-4, IL-13) and destroy infected or stressed cells with cytotoxic granules (perforin, granzyme). These cells modulate immune responses in viral hepatitis (HBV, HCV), fibrosis, and HCC, which contribute to liver protection against viral infections and tumors. On the other hand, overactivation of these cells leads to liver damage[95-97]. Type II NKT cells often counteract the effects of type I NKT cells by promoting tolerance or suppressing inflammation[90]. Type II NKT cells are associated with autoimmune liver diseases[98]. When activated, type II NKT cells can produce cytokines such as IL-13 and IFN-γ, although their cytokine profile may vary depending on the stimulus[99].

Mucosal-associated invariant T (MAIT) cells comprise about 10%-40% of all T lymphocytes in the human liver[100]. These cells express TCR-α and TCR-β, but differ from conventional T cells in that this receptor has a limited TCR diversity, which consists of a semi-invariant TCR-α chain linked to a limited repertoire of TCR-β chains. In addition, MAIT cells are restricted not by MHC but by the nonpolymorphic antigen-presenting molecule MHC class I-related protein 1[101,102]. MAIT cells exert protective effects on chronic viral hepatitis, alcoholic hepatitis, MASLD, primary sclerosing cholangitis (PSC), and decompensated cirrhosis[103]. A decrease in the number of MAIT cells in the blood and liver of patients with primary biliary cholangitis (PBC) compared with the control group has been shown. Moreover, MAIT cells in patients with PBC are activated, depleted, and persistently deficient, including after ursodeoxycholic acid treatment[104].

γδ T cells are another important cell population that accounts for about 3%-5% of the total number of lymphocytes in the liver[105]. γδ T cells continuously populate the liver, especially in the sinusoids and periportal regions. γδ T cells can function as an innate immune system because their TCRs function as PRRs and can also produce cytokines such as TNF-α and IFN-γ in response to infection or tumor antigens and are capable of phagocytosis[106-108]. At the same time, γδ T cells can also participate in the adaptive immune response due to the ability to rearrange TCR genes, providing greater diversity potential in putative ligand binding sites, and also exhibit the development of a memory phenotype[109-112]. Because of this dual function, γδ T cells are considered an evolutionarily early lymphocyte population that is functionally intermediate between innate and adaptive immunity[113,114]. The immune functions of γδ T cells are also ensured by the production of various biologically active substances, including representatives of the C-type lectin family (antimicrobial peptide RegIIIγ, which kills Gram-positive bacteria, and its related lectin RegIIIβ), granzymes and perforins, which have cytotoxic effects against infected and tumor cells, as well as immunosuppressive cytokines such as TGF-β or IL-10 that regulate other cells involved in innate immunity mechanisms[115-119].

γδ T cells show different activities against tumors. On the one hand, they are characterized by antitumor activity, as they recognize stress ligands (e.g., MHC class I polypeptide-related sequence A and B) on tumor cells and mediate cytotoxicity, as well as produce IFN-γ to enhance antitumor immunity and inhibit angiogenesis[120-122]. On the other hand, tumor-infiltrating γδ T cells can acquire immunosuppressive phenotypes (e.g., programmed cell death 1 [PD-1], IL-17), contributing to impaired immunity. IL-17 produced by γδ T cells[120,123] can stimulate angiogenesis and tumor growth[124-128]. It was also shown that HCC-infiltrating γδ T cells were cytotoxic but depleted. HCC-infiltrating γδ T cells show reduced proliferation and cell cycle arrest at the G2/M stage[129].

In general, γδ T cells may play important roles in a variety of diseases and conditions, including liver infections, MASLD, autoimmune hepatitis, fibrosis and cirrhosis, cholangitis, and in the development of liver cancer and regeneration[130-133]. γδ T cells are involved in the progression of metabolic dysfunction-associated steatohepatitis (MASH). The γδ T cells are activated through the NK receptor group 2 member D receptor and secrete IL-17A, which promotes inflammation and fibrosis in the liver. IL-17A allows hepatocytes to produce chemokines that attract pro-inflammatory cells to the liver, leading to MASH and fibrosis[134,135].

Thus, liver lymphocytes are represented by a vast group of functionally heterogeneous cells that maintain immune balance by linking innate and adaptive immunity.

CLINICAL IMMUNOLOGY OF THE LIVER

Chronic liver diseases involve a number of common links regardless of etiology. Repetitive liver injury leads to fibrosis, cirrhosis, and eventually terminal disease leading to liver failure. All these processes involve closely overlapping immune links, a better understanding of which will improve approaches to the diagnosis and treatment of liver disease.

MASLD

MASLD is a widespread disease that is closely associated with other metabolic disorders including metabolic syndrome, obesity and diabetes mellitus. MASLD ranges from simple steatosis, to MASH with progressive hepatic fibrosis and possible outcome to cirrhosis[136]. The term MASLD was recently proposed and it replaced the previously used term non-alcoholic fatty liver disease, as MASLD more accurately reflects the contribution of risk factors. The key pathophysiologic and histologic characteristic of MASH is excessive fat accumulation in the liver, which has complex developmental mechanisms involving closely intertwined immune and metabolic mechanisms. The classical “two-hit hypothesis” proposed in 1998 by Day and James[137] described the pathogenesis of nonalcoholic steatohepatitis as a two-stage process. The first stage is related to the negative impact on the liver of metabolic risk factors such as dyslipidemia, obesity, and type 2 diabetes mellitus[137-139]. The first stage involved an increased supply of free fatty acids (FFAs) to the liver, which promoted excess triglyceride formation in hepatocytes. The subsequent second “hit” is due to the effects of oxidative stress and the action of pro-inflammatory cytokines that lead to hepatocellular damage, inflammation and liver fibrosis[137].

The current understanding of the pathogenesis of MASLD corresponds to the concept of “multiple parallel-hit”, i.e. it is believed that the disease develops as a result of a complex chain of events, many links of which are cross-linked, including insulin resistance, de novo lipogenesis, local and systemic inflammation, disturbances in the structure of gut microbiota, and oxidative stress. The gut microbiota has a close bilateral relationship with the liver, and disruption of the microbiota may contribute to the development and maintenance of inflammation in the liver. The liver-microbiota relationship on the one hand involves the production of primary bile acids (BAs) by the liver, which can regulate the composition of the microbiota; on the other hand, the microbiota can metabolize primary BAs into secondary BAs, which after reabsorption in the intestine, can re-enter the liver and partly the general bloodstream, where they have a regulatory effect on some immune and metabolic processes[140-142]. Secondary BAs are ligands for many receptors, such as the nuclear farnesoid X receptor (FXR), which regulates bile acid synthesis and metabolism[143]. In addition, disruption of the intestinal epithelial immune barrier may lead to an increase in its permeability to bacterial LPS, which contribute to the activation of hepatic immune cells, which is relevant for the development of MASLD. The gut microbiota under normal conditions is also a source of various substances that are involved in various immune and metabolic processes in the human body. For example, short-chain FAs (SCFAs) produced by the gut microbiota are transported to the portal vein, liver and partly to the general bloodstream, where they have various immunomodulatory effects. Disturbances in the composition of the gut microbiota may lead to decreased production of SCFAs, which has important clinical implications, including for liver function[144-147]. In addition, changes in the intestinal microbiota may lead to other metabolic processes, such as excessive ethanol production. Thus, in recent years, there has been increasing interest in the gut-liver axis, in which the gut microbiota plays an important role. Disruption of the function of this axis is considered to be an important link in the development of liver diseases such as MASLD[148-150].

A complex of mechanisms results in the accumulation of excessive amounts of lipid droplets containing triglycerides (triacylglycerols [TAGs]) in hepatocytes in MASLD (Figure 2)[151-153]. Lipid accumulation results from an imbalance between lipid influx, de novo lipogenesis, and lipid export. The most widely accepted model of lipid droplet formation to date suggests that excess TAG accumulates on endoplasmic reticulum membranes, forming vesicles of a lipid bilayer of TAG and cholesterol esters in the center and a monolayer of phospholipids and sphingomyelin on the outside[154]. TAG biosynthesis utilizes FAs, which may enter hepatocytes from the blood or be formed by de novo lipogenesis and endocytotic recycling of lipoprotein residues. Moreover, the main source of FAs used for TAG synthesis is FAs from blood[152,155,156]. Insulin resistance contributes to increased delivery of FFAs to the liver, leading to triglyceride accumulation in hepatocytes. It should be noted that data suggest that TAG accumulation is not toxic to liver cells and is even considered s a certain protective mechanism against lipotoxicity induced by FFAs[157,158].

Figure 2 Mechanism of the development of metabolic dysfunction-associated steatotic liver disease.

Excess lipids in liver cells leads to organelle dysfunction, which contributes to mitochondrial dysfunction and endoplasmic reticulum stress[159]. The endoplasmic reticulum performs an important cellular function, namely, it is involved in protein and lipid synthesis. Therefore, endoplasmic reticulum stress against the background of excess lipid accumulation is characterized by the accumulation of misfolded or unfolded proteins and leads to the activation of an adaptive cellular response called the unfolded protein response (UPR). This mechanism restores endoplasmic reticulum homeostasis and promotes cell survival. However, if recovery fails, prolonged endoplasmic reticulum stress and UPR activation promote cell death[160]. In addition, endoplasmic reticulum stress is also associated with activation of inflammatory mechanisms through excessive production of ROS and activation of nuclear factor kappa B and c-Jun N-terminal kinase pathways[157,161].

Mitochondrial dysfunction is another important part of the pathogenesis of MASLD, as these organelles have an increased ability to oxidize FAs, producing ROS. The resulting imbalance between ROS and defense mechanisms, contributes to oxidative stress[159]. Oxidative stress can lead to hepatocyte death and is another link in the mechanisms of the development of MASLD[162].

A growing body of evidence is increasing the understanding of the importance of the innate immune system in the development of MASLD. Innate immune cells such as Kupffer cells, neutrophils, dendritic cells, and NK cells play an important role in the pathogenesis of MASLD. In MASLD, the liver is affected by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) such as bacterial LPS and some metabolites such as FFAs and microparticles including mitochondrial DNA derived from steatotic hepatocytes. PAMPs and DAMPs are recognized by PRRs such as TLRs and NOD-like receptors, leading to activation of proinflammatory signaling pathways[163-166].

The transition from steatosis to MASH is associated with the development of liver inflammation (Figure 2). In steatohepatitis, there is an increase and aggregation of Kupffer cells in perivenular areas. Scattered large fat vacuoles are found within the Kupffer cells[167]. Kupffer cells take up large amounts of FFAs, which contributes to their proinflammatory activation, in which Kupffer cells are involved in the production of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, which contribute to the progression of inflammation and development of MASH[168]. TLRs are important contributors to inflammation in MASH. Saturated FAs can activate proinflammatory TLR-4 pathways, mediating the link between lipid metabolism and the innate immune system[169-171].

LSECs are another important player in the pathogenesis of MASLD. Disruption of the normal LSEC phenotype promotes capillarization of sinusoidal spaces, a critical step in the fibrotic process in the liver. LSECs are also involved in the regulation of HSC activation and play an important role in liver immunology and the development of MASLD[172].

Liver-derived CD4+ T cells may be involved in the pathogenesis of MASH. Naive CD4+ T cells can be activated in the liver by various APCs and then transform into effector T cells with several polarization variants, such as Th1, Th2, and Th17. The different profile of cytokines produced by these effector cells is associated with the development of MASH. Th1 cells are involved in the production of IFN-γ and predominate in the liver of patients with MASH, while Th17 cells are present in the liver and peripheral blood of mice with MASH and are characterized by the production of IL-17A, IL-17F and IL-22. The IL-17A and IL-17F axis has been shown to play an important role in the development and progression of MASH, whereas the use of monoclonal antibodies against IL-17 in the experiment improved liver function, reduced hepatic lipid accumulation, decreased Kupffer cell activation, and decreased proinflammatory cytokine levels in a model of MASH induced by a high-fat diet[70,173-176]. In addition, effector T cells can form effector memory T cells (TEMs) and central TEMs, whose cytokines are also associated with the development of MASH[70].

CD8+ T cells in the liver may also be involved in the development of MASH. The number of CD8+ T cells is increased in MASH and they directly activate HSCs, promoting inflammation and fibrosis[77]. CD8+ T cells produce pro-inflammatory cytokines such as IFN-γ and TNF-α, which play a key role in the development of inflammation and liver damage[77,79,177,178].

Inflammation and fibrosis are important steps in the progression of MASH (Figure 2). Inflammatory mediators such as cytokines and chemokines enhance the inflammatory response. This cascade of events triggers the activation of HSCs and deposition of ECM, leading to hepatic fibrosis. Importantly, increasing the stage of fibrosis significantly increases the risk of liver-related mortality, emphasizing the importance of fibrosis[179]. Liver stellate cells, which differentiate into a myofibroblast phenotype, play an important role in the development of liver fibrosis and are involved in the synthesis and deposition of ECM proteins[180,181]. Accumulation of free cholesterol in HSC also exacerbates hepatic fibrosis[182,183].

Thus, the development and progression of MASLD is a complex process in which immune and metabolic mechanisms are intertwined and occur not only in the liver but also at the systemic level, which affects the course of comorbidities.

The role of immunotherapy in the treatment of MASLD and its complications is an emerging area of research, although it is still in its early stages. Immune checkpoint inhibitors (ICIs), including anti-PD-1/programmed death-ligand 1 (PD-L1) therapy, are promising avenues for immunotherapy. Immunotherapy using antibodies to PD-1 and PD-L1 has shown efficacy in slowing the progression of MASLD and treating HCC associated with MASLD. However, the efficacy of these therapies is still under investigation, and results regarding patient prognosis are inconsistent[184].

Therapies targeting immune cells, such as macrophage polarization, are another promising strategy. Understanding the role of immune cells in MASLD is crucial for the development of targeted immunotherapy[185].

Mesenchymal stem cells (MSCs) have shown promising results in preclinical and early clinical studies in liver diseases. It has been shown that MSCs can improve liver histology, reduce fibrosis, and restore liver functional capacity. Extracellular vesicles (EVs) and exosomes derived from MSCs may find applications in treating liver failure and mitigating post-transplant complications. Autologous MSC-derived hepatocytes are of interest for the treatment of cirrhosis, and allogeneic MSCs may have applications in liver failure and liver transplantation. However, a number of issues, including long-term safety, need to be addressed for the use of MSCs[186,187].

Immunotherapy is a promising treatment for liver fibrosis by modulating the immune response and affecting the immune microenvironment of the liver. Regulation of the immune microenvironment is considered a promising therapeutic strategy because the immune microenvironment of the liver plays a critical role in the development of fibrosis. Regulation of this microenvironment has the potential to reverse fibrosis. For example, targeting immune cells and their mediators can modulate the activation of HSCs, which play a key role in fibrosis. Cenicriviroc, a C-C chemokine receptor type 5 (CCR5) and CCR2 antagonist, is being tested for the treatment of liver fibrosis in adults with MASH[188,189].

FXR agonists are a promising therapeutic approach for the treatment of MASLD, including its more severe form, MASH. FXR is a nuclear receptor that plays a key role in the regulation of bile acid synthesis, lipid metabolism, glucose homeostasis and inflammation, making it an important target for the treatment of MASLD[190].

Activation of FXR lowers hepatic lipid levels by reducing FA synthesis and absorption. This is achieved by suppressing genes involved in lipogenesis, such as sCD1, diacylglycerol O-acyltransferase 2, and lipin-1, and by reducing bile acid absorption[191,192]. FXR regulates bile acid synthesis and transport, which is critical for maintaining lipid and glucose homeostasis. This regulation helps prevent bile acid-induced cytotoxicity. FXR agonists also inhibit inflammation and fibrosis, which is particularly important in preventing the progression of MASLD[193,194]. In this regard, drugs targeting FXR are of clinical interest[191].

The clinical use of fibroblast growth factor 21 (FGF21) analogs is also of interest. FGF21 is a hormone-like growth factor that is produced by several organs, including the liver, and plays an important role in regulating glucose and lipid metabolism, insulin sensitivity, and energy expenditure[195,196]. FGF21 increases hepatic insulin sensitivity, reduces lipogenesis, triggers β-oxidation of FAs, reduces endoplasmic reticulum stress of hepatic cells, and reduces LDL entry into the liver[195,197]. FGF21 analogs have shown promising results in reducing fasting insulin levels, body weight and total cholesterol levels in patients with metabolic syndrome[198]. They also improve insulin sensitivity and promote weight loss in animal models[199]. Clinical studies have shown that FGF21 analogs can improve conditions in dyslipidemia, especially hypertriglyceridemia, and in hepatic steatosis. They also reduce inflammation, levels of biomarkers of fibrosis and liver damage, and eliminate steatohepatitis associated metabolic dysfunction[200-202].

Thus, MASLD is an immunometabolic disease in which various cells are involved in the development and progression, and immunotherapy is considered as a promising strategy for the treatment of MASLD (Table 1)[78,157,184,190-228].

Table 1 Prospective directions of immunotherapy of metabolic dysfunction-associated steatotic liver disease.

Therapeutic approach	Mechanism	Current state	Ref.
Anti-programmed cell death 1/programmed death-ligand 1 therapy	Inhibition of immune checkpoint	Early phase, mixed results	Lombardi et al[74], Jeong et al[184], Zhang et al[203], Pinto et al[204]
MSCs	Immunomodulatory, anti-inflammatory agent	Promising preclinical results. MSC heterogeneity and long-term safety issues remain serious obstacles	Moayedfard et al[157], Niu et al[205], Yang et al[206], Hu et al[207]
Extracellular vesicles	Modulation of the immune response	Promising preclinical results	Ipsen et al[208], Abreo Medina et al[209], Niu et al[205], Yang et al[206]
Probiotics (Lactobacillus plantarum, Bifidobacterium bifidum)	Modulation of intestinal microbiota, anti-inflammatory agent		Lu et al[210], Zhang et al[211], Wen et al[212], Tang et al[213]
Targeting cytokines (e.g., IL-17), IL-1β inhibitors tumor necrosis factor-alpha inhibitors nucleotide-binding and oligomerization domain-like receptor protein 3 inflammasome inhibition	Reducing inflammation	Preclinical studies have shown mixed results	Rafaqat et al[214], Paquissi et al[215], Kucsera et al[216], Duan et al[217], Bakhshi et al[218], Mridha et al[219]
Natural anti-inflammatory agents, astaxanthin. Apigenin, ginsenoside Rg1	Reducing inflammation	Preclinical studies have shown promising results	Lv et al[220], Yang et al[221], Xu et al[222]
Inhibition of apoptosis signal-regulating kinase 1 (selonsertib [GS-4997])	Reduction of inflammation and fibrosis	Patients with MASH may develop liver scarring (fibrosis), including cirrhosis, which increases the risk of liver failure and liver cancer	Harrison et al[223], Loomba et al[224]
Caspase inhibition (Emricasan [IDN-6556])	Reduced hepatocyte apoptosis in a mouse model of MASH liver injury and fibrosis are suppressed by inhibiting hepatocyte apoptosis.	Emricasan treatment did not improve liver histologic pattern in patients with MASH fibrosis. Emricasan administration was not associated with improved hepatic venous pressure gradient or clinical outcomes in patients with MASH-related cirrhosis and severe portal hypertension	Harrison et al[225], Barreyro et al[226], Frenette et al[227], Garcia-Tsao et al[228]
FXR agonists	Regulation of bile acid synthesis, lipid metabolism, glucose homeostasis and inflammation	FXR agonists have shown promising results	Gandhe et al[190], Tang et al[191], Clifford et al[192], Adorini et al[193], Xu et al[194]
FGF21 analogs	Regulation of glucose and lipid metabolism, insulin sensitivity	FGF21 analogs have shown promising results	Su et al[195], Harrison et al[196], Falamarzi et al[197], Carbonetti et al[198], Corbee et al[199], Raptis et al[200], Chui et al[201], Theofilis et al[202]

FGF21: Fibroblast growth factor 21; FXR: Farnesoid X receptor; IL: Interleukin; MASH: Metabolic dysfunction-associated steatohepatitis; MSCs: Mesenchymal stem cells.

Alcoholic liver damage

Alcoholic liver damage is an important medical and social problem. Ethanol is metabolized in the liver mainly by alcohol dehydrogenase and the microsomal ethanol–oxidizing system, which includes cytochrome P450 2E1. The resulting acetaldehyde is a toxic metabolite that contributes to liver damage. In addition, ethanol metabolism leads to the formation of ROS, which causes oxidative stress. ROS can damage hepatocytes, Kupffer cells, and HSCs, causing inflammation and fibrosis[229,230].

In alcoholic fatty liver disease, hepatocytes are damaged by chronic alcohol consumption and may activate Kupffer cells through the production of EVs. Alcohol can also damage the intestinal barrier by increasing its permeability to Gram-negative bacteria. LPS from these bacteria is involved in the activation of Kupffer cells and promotes their production of proinflammatory cytokines, including IL-9, IL-1β, TNF-α, and MCP-1. In addition, a population of macrophages differentiated from recruited blood monocytes increases in the liver. These macrophages may play various roles in inflammation, which may influence the course of ALD[231]. Chronic ethanol exposure enhances the ability of LPS to increase ROS production in Kupffer cells[232].

A significant accumulation of CD4+ T cells, exceeding the number of CD8+ T cells, was found in the liver of patients with ALD, in contrast to patients with MASLD and healthy subjects, whereas the number of NK cells and γδ T cells in the intrahepatic infiltration was reduced[233]. Patients with alcoholic hepatitis showed marked immunosuppression, increased production of proinflammatory cytokines/chemokines, lower levels of anti-inflammatory cytokines, and more activated but dysfunctional immune cells[234]. In addition, alcoholic fatty liver disease, alcoholic cirrhosis, and mixed cirrhosis (alcoholic + viral) have significantly reduced the levels of MAIT cells. This is due to the tendency to apoptosis of these cells in patients with alcoholic cirrhosis. Moreover, the decreased number of circulating MAIT cells correlated with liver function in patients with cirrhosis may play a role as an indicator of disease severity[235,236]. The reduced number of MAIT cells in the blood in severe alcoholic hepatitis, corresponded to their hyperactivation and impaired antibacterial cytokine/cytotoxic responses[237]. Reduced MAIT cells in patients with ALD contribute to fibrogenesis and bacterial translocation due to impaired antibacterial activity[237,238]. Alcohol withdrawal does not completely eliminate MAIT cell impairment in the blood of patients with alcoholic hepatitis[239]. In addition, MAIT cells in alcoholic liver cirrhosis patients produce more IL-17 and express more early and late activation markers CD69, PD-1, and lymphocyte-activation gene 3 compared to healthy individuals. In addition to MAIT cell deficiency, patients with alcoholic liver cirrhosis exhibit a deficiency of circulating NKT cells and NK cells, as well as altered cytokine production and activation status. NKT cells in alcoholic liver cirrhosis patients showed a decreased ability to produce IFN-γ and IL-4[235]. Alcohol consumption suppresses the antifibrotic function of NK cells, thereby inducing liver fibrosis[240].

Chronic alcohol consumption also affects the composition of the intestinal microbiota. Alcohol-induced dysbiosis affects the production of microbiome metabolites, including SCFAs[241].

Thus, alcoholic liver damage is characterized by a complex combination of impaired metabolic and immune mechanisms.

HCC

HCC arises from hepatocytes and is one of the leading causes of cancer mortality worldwide[242-244]. The main risk factors for HCC are chronic infection with HBV or HCV, ALD, and MASH. Moreover, 70%-90% of patients with HCC have chronic liver disease and cirrhosis[245]. Thus, HCC is usually associated with chronic inflammation that disrupts the immune homeostasis of the liver, including leading to immunosuppression[246]. The immune system attempts to control tumor progression through tumor-infiltrating lymphocytes, which does not always prevent tumor growth, due to failure in immune control mechanisms[247].

Several immune cells are involved in the mechanism of hepatocarcinogenesis: (1) Macrophages; (2) Myeloid suppressor cells; (3) Kupffer cells; (4) Neutrophils; (5) Dendritic cells; and (6) NK cells[248]. The immune microenvironment of the liver is a multicomponent system that plays an important role in tumor development and progression. The tumor microenvironment (TME) can contribute to both protumor and antitumor activity[249,250]. Macrophages play an important role in oncogenesis. Tumor-associated macrophages (TAMs) are one of the most common tumor-infiltrating immune cell types and contain two polarized phenotypes: (1) Tumor-suppressive M1; and (2) Oncogenic M2. The function of M1 macrophages promotes tumor cell apoptosis, reduces drug resistance, and suppresses invasion, migration, and tumor development. By contrast, M2 macrophages promote tumor development, progression, and metastasis through various mechanisms[251-254]. Various cytokines such as ILs, TNFs, and chemokines are involved in the immune response to HCC. They can have both stimulatory and suppressive effects on the tumor depending on their specific role[255].

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early myeloid precursors and immature granulocytes at different stages of differentiation and are known for their immunosuppressive properties. These cells play an important role in the TME in HCC, promoting tumor progression, angiogenesis, metastasis, and resistance to therapy[256,257]. They accomplish this by creating an immunosuppressive TME that promotes cancer cell survival and proliferation. These cells can suppress the innate immunity of NK cells and the adaptive immune response of CD4+ and CD8+ T cells, which are critical for antitumor immunity[252,258]. MDSCs also induce an increase in Treg and TAM, further suppressing the immune response against HCC[259,260].

Tumor-associated neutrophils (TAN) infiltrate the TME and may exert antitumor N1 and protumor N2 functions. The accumulation of infiltrating TANs in the peritumoral region is negatively correlated with the number of T cells, and the ratio of these cells correlates with the patients' life expectancy after surgical treatment, which may have prognostic significance[252,261].

HCC utilizes multiple mechanisms to circumvent the immune system, including the suppression of effector T cells by Tregs and alteration of immune cell functions through metabolic reprogramming and cytokine production[262]. Tregs play an important role in immune evasion by suppressing the functions of other immune cells and thereby supporting tumor growth[247,262]. Interestingly, HCC cells are able to engulf neighboring cells through entosis, a pathological process involving E-cadherin and β-catenin[23,263]. Internalized cells primarily undergo non-apoptotic death and undergo lysosomal degradation; however, they can survive and be released. The significance of entosis is a subject of research, but it may support tumor cells with energy substrates[23].

Immunotherapy has emerged as a promising treatment for HCC. ICIs involve the use of cytotoxic T-lymphocyte-associated protein 4 and PD1/PD-L1 inhibitors. These drugs block inhibitory pathways in T cells, enhancing the immune response against tumor cells. Clinical trials have shown that immune response checkpoint inhibitors can improve survival in patients with HCC[264,265]. The PD-1 receptor is a negative regulator of T-cell activity. Binding of PD-1 to PD-L1 and PD-L2 ligands, which are capable of being expressed by tumor cells or other cells in the TME, results in inhibition of T cell proliferation and cytokine secretion. In studies in mouse models, blocking PD-1 activity resulted in reduced tumor growth. Therefore, studies of drugs that block the PD-1 system are of clinical interest[184,266-269].

Combining ICIs with other therapies such as anti-VEGF drugs such as bevacizumab has demonstrated significant improvement in patient outcomes compared to monotherapy[270,271]. The combination of the PD-L1 inhibitor atezolizumab and the monoclonal anti-VEGF antibody bevacizumab intravenously is considered as first-line systemic therapy for HCC to improve tumor growth control and patient survival[272]. In patients with initial signs of decompensated cirrhosis or contraindications to the use of protein kinase inhibitors, nivolumab immunotherapy is recommended as an alternative to improve tumor growth control and patient survival in one of the recommended treatment regimens[273]. Nivolumab potentiates the immune response through blockade of PD-1 binding to PD-L1 and PD-L2 ligands.

Adaptive cell therapy involves the use of chimeric antigen receptor (CAR) T cells. These T cells with a CAR are designed to target specific antigens in HCC cells and are showing promising results in clinical trials[271,274,275]. Vaccines designed to elicit an immune response against HCC-specific antigens are under investigation and have shown efficacy in enhancing antitumor immunity[276,277]. Immunotherapy is being studied as a preoperative and postoperative treatment to reduce the risk of recurrence and improve long-term outcomes. This includes shrinking tumors to make them resectable and controlling tumor growth after surgery[278,279].

Thus, HCC is characterized by complex immune abnormalities involving various cells in the TME, which is a promising target for therapy.

Biliary disorders

The biliary immune system is a complex and specialized system that plays a critical role in maintaining homeostasis and defending against infection. Biliary epithelial cells (BECs) have TLRs that recognize PAMPs. Disruption of TLR regulation, especially TLR-3 and TLR-4, leads to hypersensitivity to PAMPs, contributing to chronic inflammation and cholangitis in PBC[280-282]. BECs produce MCP-1/C-C motif ligand 2, fractalkine (chemokine [C-X3-C motif] ligand 1), and macrophage inflammatory protein-3α (MIP-3α), which attract inflammatory cells and promote liver fibrosis[281]. The bile ducts are also home to various immune cells, including macrophages, T cells and neutrophils. Kupffer cells and NK cells interact with BECs to modulate the immune response. NK cells stimulated by TLR ligands can destroy BECs in the presence of IFN-α produced by monocytes[280,283]. The innate immune responses in the bile ducts trigger the production of two chemokines, fractalkine and MIP-3α, which induce migration of inflammatory cells and APC populations and lead to chronic cholangitis specific to the bile duct epithelium in PBC[281]. Secretory immunoglobulin A and BAs exert local antimicrobial effects and protect the biliary tract from damage by signaling through receptors, mainly the Takeda G protein-coupled receptor 5 receptor on cholangiocytes[284].

The biliary system, traditionally considered sterile, contains a complex microbiota even in non-pathologic conditions. Dysbiosis, or microbial imbalance, in the biliary or intestinal microbiota may influence the development of biliary diseases[285]. Bacterial translocation and molecular mimicry are mechanisms by which the gut microbiota can lead to liver inflammation and immune system activation, contributing to AILD such as PBC and PSC[286,287]. BAs synthesized in the liver are modified by the intestinal microbiota, which may affect their reabsorption and enterohepatic circulation. This interaction is critical in the development of biliary tract diseases, including cholelithiasis and cholestasis[286].

Biliary diseases such as PBC and PSC involve complex immune mechanisms that contribute to their pathogenesis. PBC, also known as PBC, is a chronic autoimmune disease characterized by progressive non-purulent destructive inflammation of the small intrahepatic bile ducts, their further destruction followed by fibrosis formation up to cirrhosis[288-290]. PSC is a chronic progressive disease of unknown etiology, characterized by inflammation, fibrosis and development of strictures of medium and large intrahepatic and extrahepatic bile ducts[291,292]. Thus, both diseases result from chronic immune-mediated damage to the biliary system of the liver, lead to impaired bile outflow from the liver, which ultimately leads to chronic liver damage, fibrosis, and cirrhosis.

The etiology and pathogenesis of PBC, as well as other AILD, are not fully understood. An important part of the pathogenesis of PBC is the formation of anti-mitochondrial antibodies (AMA), which target the pyruvate dehydrogenase complex-E2 (PDC-E2) on the inner mitochondrial membrane of BECs[293,294]. These antibodies play a key role in the autoimmune response against BECs[295].

CD4+ T cells also play an important role in the pathogenesis of PBC, producing cytokines that create an inflammatory environment that activates other immune cells, including autoreactive CD8+ T cells that directly cause BEC damage [282,296,297]. B cells are responsible for the production of AMA and other autoantibodies, contributing to the autoimmune response[294,296,298]. Macrophages, NK cells and dendritic cells are also involved in immune-mediated BEC damage. In particular, macrophages contribute to the initiation and progression of hepatic fibrosis and cirrhosis.

In PBC, changes in the gut microbiome may influence the development and progression of the disease. Patients with PBC show significant changes in the composition of the gut microbiota compared to healthy controls. These changes include decreased bacterial diversity and specific shifts in microbial populations[299,300]. Multivariate analysis has shown that overgrowth of Sphingomonadaceae and Pseudomonas is an independent risk factor for PBC[301]. In addition, the common alpha-proteobacterium Novosphingobium aromaticivorans, a common alpha-proteobacterium, was previously found to be a potential factor causing PBC. N. aromaticivorans shares epitopes with the human PDC-E2, which is a major target of AMA in PBC. This molecular mimicry may lead to impaired immune tolerance and autoantibody production[302,303].

As already mentioned, PSC is characterized by inflammation and fibrosis of the bile ducts, leading to cholestasis, cirrhosis and an increased risk of cholangiocarcinoma. The key immune mechanisms involved in PSC is genetic susceptibility. PSC is associated with certain haplotypes of human leukocyte antigen, indicating a genetic predisposition[304]. The pathogenesis of PSC involves both innate and adaptive immune responses. Cells of innate immunity are activated by molecular patterns associated with bacterial pathogens, leading to the production of pro-inflammatory cytokines[305]. Adaptive immune responses include recruitment of gut-activated TEMs to the liver, which contribute to peribiliary inflammation and fibrosis[306]. Cholangiocytes lining the bile ducts play a critical role in PSC. They can be activated by infection, inflammation and cholestasis, leading to increased expression of adhesion molecules and antigen-presenting molecules. Activated cholangiocytes release inflammatory and fibrogenic mediators, promoting immune cell recruitment and fibrosis progression[305]. Adaptive immune responses include recruitment of gut-activated TEMs to the liver, which contribute to peribiliary inflammation and fibrosis. Cholangiocytes lining the bile ducts play a critical role in PSC. They can be activated by infection, inflammation and cholestasis, leading to increased expression of adhesion molecules and antigen-presenting molecules. Activated cholangiocytes release inflammatory and fibrogenic mediators, promoting immune cell recruitment and fibrosis progression[305]. An important feature of PSC is the association with inflammatory bowel disease. Translocation of bacterial products from the inflamed gut to the liver and migration of TEM activated in the gut to the bile ducts are key processes in the pathogenesis of PSC[307].

Thus, the biliary immune system is a complex system of interactions between cholangiocytes, Kupffer cells, T cells and the biliary microbiota. Understanding these interactions is critical for the development of effective therapies for biliary tract diseases and cancer.

CLINICAL PERSPECTIVES

Liver disease is a clinical problem whose awareness of its importance has been increasing in the last decade. This has been matched by an increasing number of studies focusing on liver immunology and the cross-talk between metabolism and immunity. The results of these studies indicate that most liver cells have diverse immune functions, increasing the understanding of the complexity of maintaining normal liver immune homeostasis. Importantly, many of the liver diseases share common pathogenetic immune mechanisms or may overlap in the same patient. Therefore, the immune mechanisms of liver diseases are of clinical interest to improve the diagnosis and treatment of these diseases.

Of interest are the data on the role of the gut-liver axis, in which microbial metabolites and components from the gut can influence liver function and immune responses[308]. On one hand gut microbiota components such as LPS and lipoteichoic acid activate TLR on liver immune cells, which triggers the production of pro-inflammatory cytokines, contributing to liver inflammation and immune regulation[150]. On the other hand, the gut microbiota and its metabolites, such as SCFAs and secondary BAs, play an important role in regulating liver immune responses and influence the development of liver diseases such as MASLD, MASH, and liver cancer[148,308]. It has been suggested, for example, that secondary BAs such as deoxycholic acid, produced by gut bacteria as a result of 7α-dehydroxylation of primary BAs, cause DNA damage and contribute to alterations in the TME[150]. The gut microbiota may influence the efficacy of ICIs used in the treatment of liver cancer, emphasizing their role in the therapeutic effects[309]. In this regard, dietary adjustments aimed at maintaining a normal microbiota are a promising therapeutic strategy. Dietary fibers, especially prebiotic fibers such as inulin, promote the growth of beneficial intestinal bacteria such as bifidobacteria and fecal bacteria that produce SCFAs[310,311].

Fecal microbiota transplantation (FMT) is another promising therapeutic approach for the treatment of various liver diseases, utilizing the gut-liver axis to restore microbial balance and improve liver function. FMT has been shown to be effective in the treatment of chronic liver diseases, including cirrhosis and MASLD, by improving microbial diversity and reducing disease severity[312,313].

The role of lipid mediators of inflammation is also a promising area of future research for the clinical application of liver immunology knowledge. Pro-resolution mediators (SPMs) are involved in the resolution of inflammation. These mediators are synthesized from ω-3 and ω-6 polyunsaturated FAs by various cells. SPMs are currently classified into four classes: (1) Lipoxins; (2) Resolvins (D-series and E-series); (3) Maresins; and (4) Protectins. They inhibit neutrophil infiltration, reduce the production of pro-inflammatory mediators (both lipid mediators and cytokines), stimulate the capture of apoptotic neutrophils by macrophages as well as phagocytosis and removal of bacteria, and promote phenotypic switching of macrophages[314-318]. SPMs can modulate insulin signaling pathways, increase insulin sensitivity and glucose uptake, as a consequence of which they are a promising agent for the correction of insulin resistance and in the treatment of obesity as well[319,320]. In this regard, SPMs and their synthetic analogs are attractive therapeutic targets in many inflammatory diseases, including liver diseases[321-323].

Participation of ATP-binding cassette (ABC) transporters in immune mechanisms, which is realized through regulation of lipid transport, transport of LPS, xenobiotics, and interaction with gut microbiota, is also of clinical interest[324]. ATP subfamily A member 1 (ABCA1) gene polymorphisms are considered as a predictor of the development of MASLD[325]. ABCA1 may also be a therapeutic target in fatty liver disease. It has been shown, for example, that the glucagon-like peptide-1 agonist exendin-4 stimulates ABCA1 expression in the liver and reduces lipid accumulation through the Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase/CaM-dependent protein kinase IV pathway[326].

Targeting immune cells such as macrophages and Kupffer cells is another promising therapeutic strategy. Various natural components and drugs such as CCR2/CCR5 antagonists and galectin-3 inhibitors are being investigated in animal experiments[327,328]. The use of glucagon-like peptide-1 receptor agonists has shown promise as a potential therapeutic strategy for MASLD, which is achieved through several known mechanisms[329]. Liraglutide has been shown to protect against inflammation in MASLD by modulating Kupffer M2 cell polarization through the cyclic adenosine monophosphate-protein kinase A-signal transducer and activator of transcription 3 signaling pathway[330]. Elafibranor, an agonist of peroxisome proliferator-activated receptors α and δ, increases insulin sensitivity, normalizes glucose levels and lipid metabolism, and reduces inflammation by acting also in an anti-inflammatory manner on macrophages and Kupffer cells[331,332].

CONCLUSION

In this regard, studies that identify novel diagnostic biomarkers and targets for immunotherapy are of interest. A better understanding of the links between immune and non-immune functions of liver cells may improve diagnostic and treatment approaches.