Pathogenesis of Alcohol-Associated Liver Disease

Abstract

Excessive alcohol consumption is a global healthcare problem with enormous social, economic, and clinical consequences. While chronic, heavy alcohol consumption causes structural damage and/or disrupts normal organ function in virtually every tissue of the body, the liver sustains the greatest damage. This is primarily because the liver is the first to see alcohol absorbed from the gastrointestinal tract via the portal circulation and second, because the liver is the principal site of ethanol metabolism. Alcohol-induced damage remains one of the most prevalent disorders of the liver and a leading cause of death or transplantation from liver disease. Despite extensive research on the pathophysiology of this disease, there are still no targeted therapies available. Given the multifactorial mechanisms for alcohol-associated liver disease pathogenesis, it is conceivable that a multitherapeutic regimen is needed to treat different stages in the spectrum of this disease.

As an accessory but vital digestive organ, the liver serves crucial functions in the body’s metabolic regulation. It is the first organ exposed to blood-borne substances absorbed by the gastrointestinal (GI) tract. The liver is most prominent among other (esophagus and stomach) “first responder” organs that enzymatically oxidize imbibed alcohol/EtOH (the active ingredient in alcoholic beverages) to acetaldehyde. The latter compound is more toxic than EtOH but is relatively short-lived, as it is rapidly oxidized to acetate. Chronic, heavy alcohol (EtOH) consumption disrupts normal liver function and eventually causes hepatic structural damage, resulting in alcohol-associated liver disease (ALD). Although the liver efficiently eliminates EtOH, studies with rodents, nonhuman primates, and humans demonstrate that long-term (weeks to months in rodents; months to years in humans and primates) excessive alcohol consumption seriously damages the liver to eventually cause liver failure. This review presents updated findings on ALD development in experimental animals and humans. It will describe how EtOH metabolism initiates injury at the cellular level and how such damage initiates the spectrum of liver injury from simple fatty liver to hepatitis to decompensated cirrhosis. It will briefly describe potential new therapies and targets for ALD treatment. Throughout this review, we will use the terms “alcohol” and “EtOH” interchangeably.

The scope and severity of problem drinking

Excessive drinking of EtOH-containing beverages, called alcohol use disorder (AUD), is a significant global health concern. AUDs can be mild, moderate, or severe, depending on the frequency and the amount of alcohol consumed.¹ In 2016, heavy drinking contributed to an estimated 3 million preventable fatalities (5.3% of all deaths) worldwide.² For that year, the economic burden of caring for people with AUDs totaled $249 billion.³ Currently, in the United States. over 20 million Americans (about six percent of the US population) have AUDs.² It is predicted that by 2025, per capita alcohol consumption will rise from 8 to 8.4 L of alcoholic beverages per person.² Heavy drinking essentially injures all organs of the body. Among these, the liver sustains the greatest degree of injury because it is the largest internal organ that receives blood-borne nutrients, toxins, and xenobiotics (foreign chemicals, both natural and artificial) from the GI tract. The liver is also the principal site of alcohol metabolism and has the highest levels of enzymes that catalyze these oxidative reactions.⁴ Inside liver cells (hepatocytes), EtOH oxidation directly (and indirectly) generates toxic intermediates and byproducts, which, if their levels are maintained by heavy drinking, cause hepatocyte damage and death. Liver cell death gives rise to hepatic inflammation (hepatitis), a lethal and acute form of ALD, which, without medical intervention, can cause significant morbidity (illness) and/or mortality (death) from liver failure, which requires a liver transplant. Recent estimates indicate that ALD causes 50% of all deaths due to liver disease.⁵

The impact of heavy drinking is clearly illustrated by historical events, including the prohibition of alcoholic beverages in the United States beginning in 1920, after which liver disease incidence significantly declined. After prohibition ended in 1933, cirrhosis incidence, began to rise again.^{6, 7, 8} One hundred years later, in 2020, during the first peak of the COVID-19 pandemic in March, higher-than-normal numbers of people stayed home to avoid viral transmission. During that and subsequent months into the year 2021, retail sales of beer, wine and/or distilled spirits exceeded the January 2020 sales by 17% to 25%. According to the United Network for Organ Sharing Standard Transplant Analysis and Research, beginning in May 2020 and continuing through January 2021, the number of people with alcoholic hepatitis (AH) who registered for liver transplants rose two-to-three fold over the previous months. Comparable increases were also recorded for AH patients who received liver transplants. The latter patient numbers exceeded those who received transplants for alcohol-induced cirrhosis or for liver diseases unrelated to alcohol misuse. The latter patient numbers did not change over the eight-month time period from May to December 2020.⁹

Alcohol metabolism

Gastrointestinal tract (GI)

The GI tract is the principal site of absorption of ingested alcohol and hence, plays a significant role in mediating the toxic effects of alcohol on the liver and other organs. It is also a site of metabolism of the ingested alcohol, as the major alcohol metabolizing enzymes are present in GI mucosal cells, including isozymes of alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and catalase. While GI metabolism of EtOH is quantitatively lower than the liver, it does contribute to local toxicity by generating acetaldehyde. In addition to affecting the systemic availability, the stomach lining (mucosa) is the principal site of the “first pass” metabolism of the ingested alcohol, catalyzed by various isoforms of gastric ADH. Gender, age, genetics, and gastric morphology modulate gastric ADH activity. Gastric ADH levels are significantly lower in younger women compared with age-matched men.^10,11 This difference likely accounts for greater alcohol-induced liver toxicity in women. When ingested with a meal, gastric absorption rates of ethanol reportedly fluctuate from 30% to 100% among healthy individuals, with only a small amount of ethanol detected in the distal small intestine. Ethanol in the small and large intestines is principally metabolized by the microbiome that inhabits this GI region. Along its GI course, EtOH becomes bioavailable through its rapid transit into the portal system through the gastric and small intestinal mucosa. GI microbial flora also anaerobically generate EtOH by fermentation in the distal intestine. Multiple factors, such as GI motility, absorption, dilution by GI secretions, and rediffusion of alcohol, all influence ethanol metabolism in the GI tract.

Hepatic EtOH metabolism and its consequences

Over 80% of ingested EtOH is oxidatively metabolized in liver parenchymal cells (hepatocytes), which comprise ∼80% of the liver’s mass and which express the major alcohol metabolizing enzymes. The EtOH oxidation pathways are presented. EtOH hepatotoxicity is mostly attributed to its conversion to acetaldehyde, which is more toxic than EtOH because acetaldehyde is highly reactive and covalently binds to proteins,^12,13 phospholipids,¹⁴ and nucleic acids.^15,16 Such binding, called adduct formation, significantly alters the biological functions of specific proteins.^{17, 18, 19}

The cytosolic alcohol dehydrogenase is the primary enzyme that most efficiently catalyzes EtOH oxidation to acetaldehyde (Figure 1), while, in the same reaction, ADH catalysis reduces the cofactor NAD⁺, forming NADH. It is important to note that during chronic, heavy drinking, the continuous reduction of NAD⁺ to NADH in the reactions catalyzed by ADH and aldehyde dehydrogenase (ALDH), causes a significant decline in the ratio of intrahepatocyte NAD⁺/NADH, also called the cellular redox potential.⁴ The decreased redox potential causes a significant metabolic shift in hepatocytes, as EtOH metabolism generates reducing equivalents (NADH) that participate as cofactors in reductive synthesis, which shifts hepatocyte metabolism toward fatty acid synthesis (as well as increased production of lactate to cause metabolic acidosis). If the reductive fatty acid synthesis is upheld by continued drinking, hepatocytes continue to increase their fatty acid contents, which condense with glycerol to form triglycerides, stored in intracellular lipid droplets. Steatosis (fatty liver) is also maintained/augmented by significant intrahepatocyte switches in gene transcription that favor induction of transcription factors (e.g., SREBP-1c and Egr-1) to further enhance fatty acid biosynthesis.^20,21 Conversely, there is a decline in the levels of the transcription factor PPAR-alpha that regulates genes of fatty acid oxidation (breakdown).²² Enhanced production of humoral factors, including tumor necrosis factor α (TNFα) and adiponectin, further exacerbate this condition to propagate steatosis.²³

Figure 1.

Ethanol oxidation pathways in the liver. Ethanol (i.e., ethyl alcohol) is oxidized principally in hepatocytes of the liver. Alcohol dehydrogenase (ADH), a major enzyme in the cytosol, and aldehyde dehydrogenase 2 (ALDH2), located in the mitochondria, catalyze sequential oxidations that convert ethanol to acetate, producing two mole equivalents of reduced nicotinamide adenine dinucleotide (NADH⁺). Cytochrome P450 2E1 (CYP2E1) is a major inducible oxidoreductase in the endoplasmic reticulum that oxidizes ethanol, in the presence of molecular oxygen (O₂), to acetaldehyde and converts reduced NAD phosphate (NADPH) to its oxidized form, generating water. Peroxisomal catalase is a minor hepatic pathway of ethanol oxidation that uses hydrogen peroxide (H₂O₂) to oxidize ethanol to acetaldehyde and water.

The other major EtOH-metabolizing enzyme is cytochrome P450 2E1 (CYP2E1), a mixed-function oxidase that resides in the membranes of the smooth endoplasmic reticulum (ER). Its Km for EtOH is ∼10 mM or 50 mg per dL. Like ADH, CYP2E1 catalyzes EtOH oxidation to acetaldehyde and uses molecular oxygen (O₂) and NADPH as additional substrates/cofactors. Despite having a higher substrate capacity than ADH for EtOH, CYP2E1 has a significantly lower catalytic efficiency (K_cat). However, CYP2E1 is inducible, which means its intracellular content rises in hepatocytes because EtOH, its substrate, protects CYP2E1 from being degraded by the ubiquitin-proteasome system.^24,25 The resulting increase in hepatic CYP2E1 content escalates its V_max, thereby boosting the rate of hepatic EtOH oxidation and clearance by both ADH and CYP2E1. While accelerated alcohol metabolism by higher levels of CYP2E1 may appear to protect an individual drinker from liver injury, the broad substrate specificity of CYP2E1 and its catalytic cycle negate any such protection. This is because faster ethanol oxidation catalyzed by higher CYP2E1 content generates not only more acetaldehyde, but the enzyme also produces higher levels of other reactive oxygen species (ROS), including free radical forms of ethanol (i.e., hydroxyethyl radicals), superoxide anions, and hydroxyl radicals. Thus, greater production of ROS in heavy drinkers causes oxidant stress. This occurs when the rate of free radical production exceeds the hepatocyte’s ability to neutralize them with natural antioxidants, including reduced glutathione and/or vitamins E, A, or C. Liver cells are also equipped with antioxidant enzymes, including the copper/zinc (Cu/Zn) and the manganese (Mn) superoxide dismutases (SODs), catalase, glutathione peroxidase (GSH Px), glutathione reductase (GSSG Rdx)), and glutathione-S-transferase (GST). Studies using rodents report that four of these six enzymes (Cu/Zn SOD, Mn SOD, GSSG Rdx, and GST) lose activity and/or content after chronic EtOH feeding, while GSH Px activity is unaffected and catalase activity rises after alcohol administration.^{26, 27, 28} Thus, the oxidant burden in hepatocytes worsens as enzymatic antioxidant defenses are weakened by EtOH administration. Recently, the importance of these antioxidant enzymes was further underscored by in vivo studies, showing that treatment of EtOH-fed rodents with nanoparticle-bound SOD-1 alleviates liver as well as adipose tissue injury.^29,30

It is important to note that ROS spontaneously react with unsaturated lipids, forming lipid peroxides, including malondialdehyde (MDA), 4-hydroxynonenal (HNE), and acrolein that exacerbate oxidant stress. MDA can further react with acetaldehyde and then with proteins, forming larger-sized malondialdehyde-acetaldehyde (MAA)-protein adducts.³¹ Such adducts are capable of eliciting immune responses.^{32, 33, 34, 35} Acrolein is a strong electrophile and quickly reacts with amino acids lysine, histidine, and cysteine and form adducts, which can alter the protein functions and have severe pathological consequences.³⁶ A recent study documented higher levels of acrolein metabolite, 3-hydroxypropylmercapturic acid (HPMA) in patients with severe acute AH, compared with controls or nonsevere acute AH suggesting that HPMA may be a novel selective, noninvasive biomarker for severe acute AH.³⁷ Consistent with the multifactorial etiology of alcohol-associated liver disease, these authors also identified strong combined effects of HPMA and proinflammatory cytokines (IL-1β, IL-8, and TNFα) on the extent/pattern of liver cell death, further supporting the pathogenic role of acrolein. Finally, because CYP2E1 possesses broad substrate specificity, the enzyme metabolizes other substrates, including the analgesic acetaminophen. When CYP2E1 is induced by heavy drinking, it accelerates the conversion of acetaminophen to a hepatotoxic intermediate, which forms adducts with hepatocyte proteins to cause hepatocyte necrosis.³⁸

Although it has an accessory role as an EtOH-metabolizing enzyme in the liver, catalase, which resides in peroxisomes and normally detoxifies hydrogen peroxide (H₂O₂), also catalyzes oxidation of ethanol when it is present. H₂O₂ participates in this oxidation, which generates acetaldehyde and H₂O. While catalase is considered a minor ethanol oxidizing enzyme in the liver, it has a larger function in the brain, where the levels of ADH and CYP2E1 are significantly lower.³⁹

Acetaldehyde formed during catalysis by ADH, CYP2E1 and/or catalase is subsequently oxidized to acetate in a reaction catalyzed by ALDH, which also uses NAD⁺ as a cofactor. The reaction generates acetate and NADH. The acetate generated is believed to then enter the circulation. Major ALDH isoforms exist in the mitochondrial, microsomal, and cytosolic compartments of hepatocytes. In the mitochondrial matrix of liver cells, a low Km ALDH2 isozyme catalyzes the oxidation of the bulk of ethanol-derived acetaldehyde. Higher circulating acetaldehyde levels have been reported in problem drinkers because of enhanced acetaldehyde production and/or its slower removal, the latter owing to slower acetaldehyde clearance by the ALDH2 isozyme.⁴⁰ Higher acetaldehyde levels may also reflect impaired mitochondrial function that results from EtOH-induced mitochondrial depolarization, which reportedly results in the autophagic destruction of damaged mitochondria, also known as mitophagy.^41,42

Stages of liver injury

ALD pathologies include a spectrum of hepatic lesions, including steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), as shown schematically in Figure 2. These are stages that are not mutually exclusive, as they can coexist. Most patients with ALD have hepatic steatosis, which is usually asymptomatic and is a reversible condition if drinking ceases. However, with continued EtOH misuse, 20–35% of these patients progress to steatohepatitis (ASH), which is a more severe type of injury characterized by hepatocyte ballooning and degeneration, neutrophilic infiltration, and the development of Mallory–Denk (M-D) bodies within hepatocytes. M-D bodies are partially degraded, insoluble, misfolded, aggregated proteins that accumulate and form visible inclusions. There is also hepatic infiltration by leukocytes, including T cells and natural killer (NK) cells. A relatively small number of people with a history of prolonged, heavy alcohol misuse develop the clinical syndrome known as alcoholic hepatitis (AH), which manifests by the onset of jaundice, and is frequently accompanied by other features of liver failure, including hepatic encephalopathy, coagulopathy, and ascites. AH patients exhibit fibrosis and/or its terminal or late stage, cirrhosis, which is the deposition of high amounts of extracellular matrix proteins (e.g., collagen), secreted principally by activated hepatic stellate cells (HSCs). Initially, during AH, there is active pericellular fibrosis, which may progress to cirrhosis, the late stage of hepatic scarring. Hepatic fibrosis is a transient and reversible wound-healing response that can restore the liver architecture to normal in some patients who stop drinking. However, if drinking continues, chronic inflammation and sustained fibrogenesis progresses to substitution of liver parenchyma by scar tissue. The main pathological feature of cirrhosis is the formation of regenerative nodules of hepatic parenchyma surrounded by fibrous septa. Cirrhosis development progresses from a compensated phase, when the undamaged part of the liver functionally compensates for damaged tissue, to a decompensated phase, in which scar tissue fully envelops the organ. The latter is characterized by development of portal hypertension and/or liver failure. However, some degree of hepatitis is likely always present in cirrhotic patients, while hepatic fat is usually no longer prominent in these individuals. The World Health Organization 2018 Global status report on alcohol and health estimates that 50% of all deaths due to cirrhosis are attributed to alcohol abuse.²

Figure 2.

Stages of Liver Injury. Heavy alcohol consumption produces a wide spectrum of hepatic lesions. Steatosis (fatty liver) is the earliest, most common response that develops in more than 90 percent of problem drinkers who consume 4 to 5 standard drinks per day. With continued drinking, alcohol-associated liver disease (ALD) can progress to liver inflammation (i.e., steatohepatitis), fibrosis, cirrhosis, and liver cancer (hepatocellular carcinoma).

Methionine metabolic pathway and its relevance to ALD

Previous reports from many laboratories have demonstrated that ethanol consumption impairs several steps in methionine metabolism (reviewed in^43,44). Ethanol consumption predominantly inhibits the activity of a vital cellular enzyme, methionine synthase, that catalyzes the remethylation of homocysteine to methionine.^{43, 44, 45} By way of compensation, chronic ethanol administration increases the activity of betaine homocysteine methyltransferase (BHMT) in some species.^{43, 44, 45} This enzyme catalyzes an alternate pathway in methionine synthesis by utilizing hepatic betaine (trimethylglycine, a methyl group donor) to remethylate homocysteine⁴⁶ forming methionine, thereby maintaining adequate levels of S-adenosylmethionine (SAM), the key intracellular methylating agent.^{43, 44, 45} Under extended periods of ethanol feeding, however, this alternate pathway cannot be sustained because intrahepatic betaine is depleted.^{47, 48, 49} This results in lower hepatic levels of SAM and higher levels of S-adenosylhomocysteine (SAH) and homocysteine, both toxic metabolites. The consequent reduction in the hepatocellular SAM: SAH ratio impairs the activities of those SAM-dependent methyltransferases that have a lower K_i for SAH compared to the K_m for SAM.⁵⁰ Because methyltransferases have broad ranges of cellular functions,⁵⁰ there are several detrimental consequences that arise from inhibiting their activities (reviewed in.^43,44 These include increased hepatic fat deposition,⁴⁵ apoptosis,^51,52 accumulation of damaged proteins,^53,54 and altered signaling,⁵⁵ all of which are hallmark features of ALD. Studies also show the that the reduced SAM: SAH ratio impairs proteasome activity⁵⁶ and alters the mitochondrial respiratory chain proteome and function.⁵⁷ Both lysine- and protein arginine methyltransferases reportedly methylate residues on proteasome subunits, thereby decreasing proteasome activity.⁵⁶

Of all the therapeutic modalities presently being used to attenuate ethanol-induced liver injury, betaine appears to be most effective in a variety of experimental liver disease models. By donating a methyl group to remethylate homocysteine and SAH, betaine, detoxifies both these toxic metabolites, restores normal SAM levels, and reverses many indices of ALD, including steatosis, apoptosis, inflammatory changes, accumulation of altered proteins, and oxidant stress (reviewed in.⁵⁸

Alcohol-induced organelle dysfunction

The following are summaries that briefly describe EtOH-induced major organelle alterations that occur in liver cells:

• A.ATP is principally generated by mitochondria. EtOH consumption causes mitochondrial depolarization and inhibits oxidative phosphorylation by lowering the activities of respiratory chain enzymes to decrease hepatocellular ATP generation.⁵⁹ The ATP deficiency slows energy-requiring reactions and cellular processes, including secretion, transport, and endocytosis.^{60, 61, 62} Fatty acid oxidation is another key mitochondrial function that is blocked by EtOH oxidation and which contributes to steatosis due to decelerated breakdown of fatty acids.
• B.Studies reveal that chronic EtOH consumption reduces the proteolytic capacity and protease activities of hepatic lysosomes,^{63, 64, 65} as well as their biogenesis, which replenishes lysosome numbers.⁶⁶ Lysosomes degrade faulty organelles, accumulated lipid droplets, and all other macromolecular forms. Alcohol-induced hepatomegaly (liver enlargement) results in part from defective lysosomal degradation of long-lived proteins⁶⁷ and incomplete degradation of lipid droplets by lipophagy.⁶⁸
• C.Although it is not a true, membrane-bound organelle, the ubiquitin-proteasome system is a vital cytosolic proteolytic pathway that principally degrades short-lived native, as well as modified proteins. Chronic EtOH administration inhibits proteasome activity.⁶⁹ Causing accumulation of native and modified (e.g., adducted) proteins that contribute to EtOH-induced protein accumulation (also called proteopathy) and reduced generation of antigenic peptides.⁷⁰
• D.The endoplasmic reticulum (ER) is the intrahepatocyte center of plasma protein and lipid droplet biosynthesis. After chronic EtOH exposure, the hepatocyte ER becomes dilated, partially due to the accumulation of fatty acids and their accelerated esterification with glycerol. This is followed by enhanced packaging of triglycerides into lipid droplets. In addition, the movement of plasma proteins destined for secretion through the ER begin to accumulate, owing to slower ER transit. The latter events contribute to EtOH-induced ER stress which is marked by enhanced expression of stress markers, including the PERK-ATF4 pathway and the induction of N-nicotinamide methyltransferase, which is reportedly required for the onset of steatosis in EtOH-exposed hepatocytes.⁷¹
• E.The Golgi Apparatus is a membrane-bound organelle that is subdivided into cis, medial, and trans-Golgi compartments. It is downstream from and receives presecretory glycoprotein precursors from the ER. The Golgi further modifies each glycoprotein by catalyzing the removal and/or addition of monosaccharides and oligosaccharides and adding terminal sugars to the completed glycoprotein. The Golgi Apparatus is normally a tightly organized compartment. Ethanol oxidation in the liver cell causes Golgi fragmentation, which is clearly detected in hepatocytes under light microscopy⁷² This change is associated with abnormal glycosylation of certain glycoproteins, reductions in ADP ribosylation factor 1 and significant alterations in the proper localization of Golgi enzymes.⁷³ Such changes ultimately slow post-translational processing and secretion of glycosylated plasma proteins from the hepatocyte.

These alcohol-induced major organellar changes in the liver are schematically depicted in Figure 3.

Figure 3.

Alcohol-induced organelle dysfunction in the liver. Alcohol consumption causes depolarization of mitochondria, thereby affecting ATP generation and reducing other metabolic functions, including fatty acid oxidation. Liver lysosomes are damaged by EtOH misuse. Subsequent lysosome repair/replenishment (biogenesis) is impaired by alcohol consumption; this leads to steatosis and proteopathy due to decelerated lipid droplet and protein/organelle breakdown, respectively. Hepatic proteasome catalytic activity is diminished by chronic EtOH consumption, which leads to hepatocyte accumulation of modified, native, and senescent proteins, as well as dysfunctional antigen presentation. Hepatic endoplasmic reticulum (ER) membranes expand after heavy drinking as lipid droplet biogenesis in ER is enhanced, and secretory glycoprotein synthesis slows down. Both contribute to ER stress. EtOH exposure causes fragmentation of the tightly organized and compartmentalized Golgi Apparatus. This change disrupts Golgi organization and slows down glycosylation and packaging of glycoproteins destined for secretion.

Innate and adaptive immunity

Susceptibility or resistance to liver injury and development of end-stage liver disease after chronic ethanol consumption depends on innate and adaptive immunity statuses. There are many pathogen-associated or damage-associated molecular patterns (PAMPs/DAMPs), which are microbial products transferred from a leaky gastrointestinal tract or contents released from dying cells, respectively, which drive ALD progression. These PAMPs/DAMPs induce the production of proinflammatory cytokines, including TNFα, interleukin-1β (IL-1β), and monocyte chemoattractant protein 1 (MCP1). Proinflammatory cytokines cause liver cell dysfunction and attract circulating inflammatory cells to the liver.^74,75 Both liver parenchymal and nonparenchymal cells possess innate immunity activities, which provide a first line of defense against microbes, viruses, and danger signals by releasing cytokines, interferons, and chemokines. Nonimmune cells also have the protective properties, which may be induced by the activation of toll-like receptors (TLRs) on their surfaces.^{76, 77, 78} As an example, hepatocytes, which comprise 80% of liver cells, can generate interferon type 1 (IFN-1) response by activating TLR3/retinoic acid-inducible gene 1 (RIG1) signaling that induces phosphorylation of interferon regulatory factor 3 (IRF3) to activate IFNβ genes and the production of intracellular IFNβ, crucial for antiviral protection.^79,80 The released IFN type 1 binds to IFN receptors on the hepatocyte surface and induces signaling via the Janus kinase (JAK)-signal transduction and activator of transcription 1/2 (STAT1/2) pathway, causing downstream activation of IFN-stimulated genes (ISGs) with anti-viral activities. Ethanol exposure suppresses IRF3-mediated signaling^81,82 and decreases the phosphorylation and methylation of STAT1 while simultaneously upregulating inhibitors of this pathway, thereby increasing susceptibility to hepatotropic viruses, including hepatitis C virus (HCV).^83,84

Proinflammatory responses in ALD are often related to the impaired gut barrier function and increased release of gut luminal antigens, which activate innate immune cells.⁸⁵ These cells include macrophages, neutrophils, dendritic, NK, and NKT (natural killer T-lymphocyte) cells. Normally, innate immune cell response is well-balanced to both promote the protection and avoid excessive immune activation. However, alcohol misuse disrupts this balance to trigger inflammation.^86,87 Alcohol-induced hepatocyte cell death (apoptosis, pyroptosis, necrosis, ferroptosis) and the subsequent engulfment of dead hepatocytes by nonparenchymal cells cause the selection of certain liver innate immune cells populations, thus favoring inflammation.^{88, 89, 90} In addition, alcohol misuse directly affects the function of the immune cells, especially the macrophages (both resident Kupffer cells and circulating macrophages), important innate immune cells with diverse functions, including the clearance of infectious agents.⁹¹ The proinflammatory macrophage phenotype Ly6C^high switches to anti-inflammatory Ly6C^low upon prolonged alcohol exposure.⁹² Recent studies, using single-cell sequencing and lineage tracer techniques, reveal that within the first few days of alcohol exposure, approximately 30% of KCs undergo Foxo3-dependent apoptosis and are rapidly replaced by monocyte-derived neo-KCs. Studies show that after 16 weeks of alcohol exposure, mice develop steatohepatitis and pericellular fibrosis. The initial Kupffer cell population that dominated the liver prior to alcohol exposure was no longer present and was replaced by a population of highly protective KC forms, which are necessary to minimize liver injury and support hepatocyte function.⁹¹ Interestingly, engulfment of EtOH-exposed, pathogen-expressing (HCV, HIV) apoptotic hepatocytes by macrophages switches their profile to a proinflammatory phenotype.^84,93 Additionally, there are a variety of mediators that regulate macrophage polarization in response to ethanol, and which synergize with gut luminal antigens to induce macrophage activation in a disease stage-related manner, depending on the interplay in the cytokine network and the effects of different growth factors.⁹⁴

NK- and NKT-cells are also innate immuney cells that play an important role in host defense. As cytotoxic cells with a limited antigen-specificity, these cells are potent producers of IFNγ. However, chronic ethanol feeding to mice results in a significant reduction in NK cell activity.⁹⁵ At the alcohol-induced steatohepatitis stage, NK cells can also be downregulated by activation of a subset of NKT-cells that produce IL-10, which antagonizes the protective role of NK-cells in ALD.⁹⁶ However, supplementation with IL-15/IL15Rα successfully restores alcohol-induced NK cell deficiency.⁹⁷ IL-15 supplementation is not enough to overcome the EtOH-mediated suppression of downstream IFNγ-mediated antiproliferative effects on HSC and hepatocytes because IFNγ-triggered JAK-STAT1 signaling is dysregulated by oxidative stress.^98,99

Dendritic Cells (DC) provide the link between innate and adaptive immunity and are professional antigen-presenting cells that activate T-lymphocytes with helper functions (Th). Alcohol consumption suppresses the abilities of DC to initiate/amplify a cellular immune response.¹⁰⁰ In fact, co-culture of purified CD4+T-cells with CD11C + CD8αDC isolated from ethanol-fed mice results in reduced production of IL-6, IL-12, IL-17a, and IFNγ and increased IL-13 in response to ovalbumin stimulation.¹⁰¹ The alcohol-induced dysfunction of DCs leads to profound immunosuppression due to changes in expression of co-signaling molecules on these cells.¹⁰² While DC are mainly involved in MHC class II-restricted antigen presentation, which does not require the antigenic peptides processing by the proteasome, MHC class I-restricted antigen presentation on hepatocytes, the target cells for cytotoxic T-lymphocytes, can be altered by alcohol due to proteasome dysfunction^70,103 The importance of this mechanism for eliminating hepatocytes that express viral antigenic peptide-MHC class I complexes on their surfaces has been further demonstrated in HBV-infected ethanol-fed humanized mice¹⁰⁴ and in transgenic HCV mice.¹⁰⁵ Alcohol-induced impairment of antigenic peptide presentation in the context of both MHC class I and II illuminates the effects of EtOH on adaptive immunity.

Changes in adaptive immunity are strongly implicated in ALD pathogenesis. They include T- and B-lymphocyte-mediated immune responses.¹⁰⁶ Alcohol consumption causes robust apoptosis to decrease peripheral T-lymphocyte numbers.⁷⁵ EtOH misuse also suppresses activation of cytotoxic T-lymphocytes (CTLs), partially due to reduced production of co-stimulatory molecules, such as CD28 and to increased levels of soluble CD8+, which blocks CD8+ cell activation.^{107, 108, 109} The impaired CTL response results in poor elimination of HCV and HBV- infected hepatocytes, which likely causes chronic viral persistence. However, in many cases, ALD progression is associated with cellular immune responses to modified liver proteins adducted with malondialdehyde or malondialdehyde-acetaldehyde, and it reflects the switch to autoimmune reactions.¹¹⁰ Others report the induction of Th1 cellular immune response to alcohol dehydrogenase, which correlates with disease severity.¹¹¹

B-lymphocytes responsible for the humoral immune response are also decreased in ALD patients due to alcohol-induced suppression of progenitor B-lymphocyte differentiation.¹¹² An Increased circulating level of immunoglobulin A is typical for alcohol-associated liver cirrhosis, and it occurs by TLR9 priming of B-cells.¹¹³

The major suppressive effects of alcohol and alcohol metabolites on innate and adaptive immunity are summarized in Figure 4.

Figure 4.

Ethanol metabolism suppresses innate and adaptive immunity in liver cells. Alcohol metabolites (acetaldehyde and CYP2E1-generated reactive oxygen species) suppress both innate and adaptive immune responses in liver cells. Innate immunity dysfunctions are attributed to decreased interferon (IFN) production mainly by natural killers (NK) and dendritic cells (DC) and diminished secretion of cytokines with antiviral properties, as well as impaired IFN/cytokine signaling in hepatocytes. During viral infections, this leads to elevation of viral load in liver cells infected with hepatotropic HBV and HCV. Elimination of unwanted or infected liver cells or extracellular clearance of viral antigens by adaptive immunity is also downregulated by ethanol consumption. This is due to defective cytotoxic T-lymphocyte (CTL) responses and diminished production of antibodies (Abs) by B-lymphocytes. Unless drinking ceases, this unbalanced immunity promotes end-stage liver disease development as an outcome of ALD.

ALD modifiers

Among problem drinkers, about 35% develop an advanced liver disease because modifiers exist that exacerbate, slow down, or prevent ALD disease progression. These are depicted schematically in Figure 5 and are as follows.

• 1.Pattern of consumption and beverage type: This is the most important feature for determining the progression of liver disease. The beverage type, amount, and pattern of drinking (outside mealtime, binges) all influence disease progression. Daily intake of 40–80 g ethanol/day by males and of 20–40 g/day by females for 10–12 years is a predictor of more severe cases of ALD, including steatohepatitis, fibrosis, and cirrhosis.¹¹⁴
• 2.Gender: Epidemiologic data show that women are more susceptible to alcohol-related liver damage than men. This appears to be related to higher blood alcohol concentrations in women despite equal amounts of alcohol intake as men. This occurs because females have lower body water in proportion to their body weight than males.¹¹⁵ Additionally, lower first pass EtOH metabolism in women¹¹ allows higher EtOH concentrations to enter the portal circulation, thereby exposing their livers to higher ethanol levels than in men. Furthermore, gender-based differences in the sensitivity of KCs to endotoxins and hepatic inflammatory responses may also explain higher female susceptibility to ALD progression than that in males¹¹
• 3.Age: Most patients with serious ALD generally manifest signs and symptoms in the later decades of life¹¹⁶ to suggest that aging is an important contributor to liver disease progression. Age is also a predictor of ALD-related mortality.¹¹⁷ Despite epidemiological and other studies that implicate a relationship between aging and the development of advanced chronic liver diseases,^{118, 119, 120, 121, 122} very few studies have examined the underlying mechanism of how aging affects ALD progression.^123,124 Most of the earlier work that examined alcohol effects on the aging body demonstrated that older subjects exhibit greater sensitivity of the central nervous system to alcohol, which causes greater degrees of intoxication and loss of motor control in older, compared with younger subjects.^125,126 The higher “cerebral toxicity” in older adults is likely related to slower alcohol elimination rates, due to age-related alterations in alcohol absorption, hepatic EtOH metabolism and alcohol excretion.^{127, 128, 129} Indeed, significantly higher blood ethanol concentrations were seen in elderly people of both genders compared with those of their offspring after all subjects ingested 0.3 g ethanol/kg body weight.¹³⁰ As the global number of older adults is expected to double by 2050,¹³¹ it becomes important to gather comprehensive understanding of, not only age-related anatomical and physiological alterations in the liver (and other organs), but also the added effect of alcohol toxicity. This topic must be considered a high priority in future studies.
• 4.Race: Ethnicity is a major factor that affects the age and severity of presentation of different ALD subtypes.¹³² The reason(s) for these differences are not clear.
• 5.Genetics: Both genetic and epigenetic influences govern the initiation and progression of ALD. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms in alcohol-metabolizing enzymes, cytokine encoding genes, and antioxidant enzymes related to the progression of ALD.¹³³ Most recently, GWAS found significantly increased risk association of rs738409 in patatin-like phospholipase domain containing 3 (PNPLA3) and rs4607179 near 17-beta hydroxy steroid dehydrogenase 13 (HSD17B13) with alcohol-associated cirrhosis. Conditional analysis accounting for the PNPLA3 and HSD17B13 loci identified a protective association at rs374702773 in Fas-associated factor family member 2 (FAF2).¹³⁴ Two other previously known loci (SERPINA1 and SUGP1/TM6SF2).¹³⁵ Were also genome-wide significant in the meta-analysis.¹³⁴ Like the PNPLA3 and HSD17B13 gene products, the FAF2 product has been localized to lipid droplets in hepatocytes implicating this organelle and lipid regulating pathway(s) underlying alcohol-associated cirrhosis.
• 6.Nutritional Factors: Dietary fat is a macronutrient and a dietary modifier for ALD. In rodents, dietary saturated fat protects against, whereas dietary unsaturated fat, enriched in linoleic acid, reportedly promotes greater alcohol-induced liver damage.^136,137 Furthermore, superimposing alcohol on a high-fat or a western diet causes more severe liver injury than either alcohol or high-fat/western diet alone.^138,139
• 7.Drugs: Alcohol and other drugs, including prescription medications, over-the-counter agents, and illicit drugs, interact with each other to enhance hepatotoxicity. As described earlier, acetaminophen hepatotoxicity can be exacerbated by alcohol misuse.
• 8.Obesity: Population-based studies indicate a significantly higher degree and higher frequency of liver damage in heavy drinkers with high body mass indices (BMI).^140,141 Recent studies show that obese drinkers are more likely to develop cirrhosis and are at higher risk of liver-related mortality¹⁴² than those within a healthy weight range.^143,144
• 9.Hepatotropic viral infections: Alcohol consumption affects the pathogenesis of hepatotropic infections and treatment outcomes. While the mechanisms are different for various viruses, the progression of viral hepatitis depends on both the virus and ethanol metabolism, specifically, whether one or both components induce oxidative stress.¹⁴⁵

Figure 5.

Alcohol-associated liver disease modifiers. Among problem drinkers, about 35% develop an advanced liver disease because modifiers exist that accelerate or decelerate ALD progression, as schematically depicted.

HCV infection in alcohol misusers is a classic example of synergy between virus and alcohol. In this hepatotropic infection, the virus can only replicate in hepatocytes. EtOH exposure enhances not only viral replication¹⁴⁶ but also viral transmission to adjacent healthy hepatocytes via extracellular vesicles (EVs).¹⁴⁷ The major hepatotoxic mechanism is the combined oxidant stress produced by both the virus- and ethanol oxidation.^93,148,149 Elevated HCV protein content in hepatocytes arises from both increased viral replication¹⁵⁰ and the EtOH-induced impairment of the proteasome, which normally cleaves HCV proteins for antigen presentation.¹⁵¹ In contrast, human immunodeficiency virus (HIV) does not replicate in hepatocytes, but intracellular viral proteins still accumulate because of ethanol-induced proteasomal and lysosomal dysfunction.^93,152 However, alcohol-potentiated liver fibrosis in both types of viral infections is very similar through the activation of HSCs, following their uptake of viral components that contain apoptotic hepatocytes^93,153 regardless of whether hepatocytes are permissive cells for these viruses or not. Furthermore, alcohol suppresses innate immunity, as well as induction of IFN-stimulated genes (ISGs) in these infected hepatocytes.^93,154 In HBV infection, the mechanisms of potentiation of hepatitis pathogenesis by alcohol are a bit different from HCV and HIV infections since HBV replicating in hepatocytes does not induce apoptosis. However, ethanol metabolism blocks the presentation of HBV peptides in the context of MHC class I on hepatocyte surface, leading to decreased recognition of HBV-infected hepatocytes by cytotoxic T-lymphocytes^104,155 and the chronic persistence of HBV infection is due to cell immunity dysfunctions.¹⁵⁶ Thus, heavy drinking increases the susceptibility of liver cells to hepatotropic viral infections, which likely contributes to chronic hepatitis development that can progress to end-stage liver disease.

• 10.Adipose-Liver Axis: Chronic alcohol consumption profoundly disturbs adipose tissue (AT) function by inducing adipocyte lipolysis, reducing the secretion of adipokines, and enhancing the release of proinflammatory mediators, all of which promote ALD pathogenesis (reviewed in.^157,158 Particularly relevant to liver pathology is that EtOH-induced adipose lipolysis releases free fatty acids that are taken up by the liver, thereby exacerbating steatosis.^{159, 160, 161} Alcohol exposure also decreases adiponectin and leptin secretion from AT. These are key adipokines that reduce hepatic lipid content.^{162, 163, 164, 165} Further studies report that heavy drinking impairs AT methionine metabolism, characterized by higher SAH levels and a consequent decrease in the SAM: SAH ratio.^{165, 166, 167} This decline in methylation potential enhances activation of the adipose triglyceride lipase and hormone-sensitive lipase (HSL) to promote lipolysis^{165, 166, 167} and secretion of proinflammatory cytokines¹⁶⁷ while reducing the secretion of protective adipokines.^165,167
• 11.Liver Stiffness: Liver cirrhosis is associated with liver stiffness.¹⁶⁸ Recent studies have shown that, not only inflammation and activation of HSC to produce extracellular matrix affect the progression of ALD to end-stage liver diseases, but also pre-existing increases in liver stiffness promote pathological changes in liver cells. Studies conducted on hepatocytes plated on surfaces that mimic the stiffness of healthy (soft gels, 2 kDa) and cirrhotic (stiff gels, 55 kDa) livers revealed that cells grown on stiff surfaces exhibit lower albumin synthesis and cytochrome P450 enzyme activity¹⁶⁹ indicating that extracellular stiffness regulates hepatocyte function. Additionally, hepatocytes plated on stiff matrices and then exposed to second hits, including HCV-HIV coinfections, exhibited higher levels of apoptosis and viral infectivity.¹⁷⁰ As shown previously, engulfment of virus-infected apoptotic hepatocytes by macrophages and HSCs further promotes inflammation and fibrosis development.^93,153 These studies indicate that stiffness is an important variable that can promote ALD progression.

Potential new therapies and targets for ALD treatment

Recent research efforts have identified several new molecular drivers that promote ALD pathogenesis, some of which are described below:

Ghrelin: Ghrelin is an acylated polypeptide that is an endogenous ligand of the growth hormone secretagogue receptor type 1a (GHS-R1a). The stomach primarily produces ghrelin; lower amounts of ghrelin are detected in other organs.^{171, 172, 173} Circulating ghrelin levels rise during fasting and fall after a meal.¹⁷⁴ Ghrelin exerts a range of physiological functions. It stimulates growth hormone secretion and inhibits insulin secretion.^172,175 Ghrelin also modulates many intracellular signaling pathways, by promoting ghrelin-mediated GHS-R1a dimerization with other G-protein coupled receptors such as those for melanocortin, dopamine and serotonin, to reduce their functions.¹⁷⁶ Acute ethanol administration to healthy volunteers reduces plasma ghrelin levels.^{177, 178, 179} However, after chronic alcohol consumption higher serum ghrelin levels are reported in human drinkers and experimental animals.^{180, 181, 182, 183} Increased circulating ghrelin promotes ALD pathogenesis by slowing pancreatic insulin secretion, allowing adipose lipolysis to promote hepatic steatosis.^181,182 In addition to inhibiting insulin secretion, ghrelin directly promotes fat accumulation in hepatocytes by activating hepatic lipogenesis and increasing fatty acid transporters, while downregulating fatty acid oxidation.^181,184,185 Suppression of alcohol-induced steatosis was observed after ghrelin receptor antagonist [D-Lys-3] GHRP-6 treatment, which validated the role of ghrelin in steatosis development.¹⁸¹ In addition, preclinical and clinical investigations revealed that endogenous ghrelin levels are positively associated with alcohol craving.^{186, 187, 188, 189, 190} Thus, targeting ghrelin pathway represents a potential neuropharmacological target for AUD treatment to prevent alcohol abuse and liver injury.¹⁸⁶ Both are key therapeutic goals for patients with AUD and ALD.^1,191

CXC Chemokines: Chemokines attract infiltrating immune cells to the site(s) of organ damage. They also regulate cell differentiation, proliferation, fibrogenesis, vascular angiogenesis, and tumor metastasis.^192,193 Studies have shown that the levels of chemokines, such as CCL2, CXCL8, and CXCL5 in the liver positively correlate with higher neutrophil infiltration and greater mortality in patients with alcoholic hepatitis.^{193, 194, 195, 196} As a therapeutic intervention, targeting chemokine signaling pathways to neutralize their detrimental properties in patients with liver injury will likely ameliorate or prevent further damage.

Lipocalin 2: Lipocalin 2 (LCN2), also named neutrophil gelatinase-associated lipocalin (NGAL), is a 25-kDa glycoprotein mainly produced in neutrophils. However, upon stress or/and inflammatory stimuli, epithelial cells, macrophages, and hepatocytes also produce LCN2.^197,198 LCN2 has multiple functions in the regulation of metabolism, innate immunity, apoptosis, cell proliferation, and tumor metastasis.¹⁹⁷ In several studies, it has been reported that hepatic LCN2 expression and serum LCN2 level is markedly increased after chronic ethanol exposure¹⁹⁹ and LCN2 expression and serum LCN2 level were correlated with disease severity in AH patients.¹⁹⁷ The role of LCN2 in liver inflammation is inconsistent depending on the mode of liver injury. While mice lacking LCN2 show more liver injury after carbon tetrachloride, concanavalin A, or lipopolysaccharide exposure,^200,201 it protected against alcohol-induced liver injury by reducing neutrophil infiltration and hepatic steatosis.^199,202 In vitro studies demonstrated that LCN2 can act as a stimulus for HSCs activation, α-smooth muscle actin expression, and fibrogenesis. Further, in AH patients with portal hypertension, elevated hepatic LCN2 expression levels were positively correlated with the expression of a key fibrotic protein, collagen type I alpha 1, tissue inhibitor matrix metalloproteinase 1 (TIMP1), and endothelin 1 (EDN1).¹⁹⁷ Further evidence that LCN2 plays a key role in liver fibrosis was documented by the absence of the chronic alcohol-induced expression of TIMP1 and EDN1 in mice lacking LCN2.¹⁹⁷ Given a critical role for LCN2 in immunity and infection, pharmacological neutralization of LCN2 could be a therapeutic target in ALD patients.

Oxylipins: Oxylipins are fatty acid metabolites derived from polyunsaturated fatty acid (PUFA) via various pathways such as cycloxygenase (COX), lipoxygenase, and cytochrome P450 (CYP) pathways.²⁰³ Oxylipins play an important role in numerous biological and pathological process such as inflammatory response, cell adhesion, cell migration and proliferation, apoptosis, and oxidative stress. Clinical studies reported that serum oxylipin profiles were altered in patients with alcohol use disorder and AH compared to controls.²⁰³ Specifically, oxylipins derived predominantly from the n-6 PUFA arachidonic acid (AA) and n3 PUFAs eicosapentaenoic acid and docosahexaenoic acid were significantly altered in AH patients.²⁰⁴ Further, AA derived 20-hydroxyeicosatetraenoic acid levels in serum was positively correlated with steatosis and immune cell infiltration observed in liver biopsy and negatively correlated with 90-day survival in AH patients.²⁰⁴ Another clinical study documented that the oxylipin profile differs with the severity of liver injury.²⁰⁵ Since patients with ALD have different oxylipin profiles, and the oxylipins exert proinflammatory or dual anti-inflammatory and proresolution of inflammation activities,²⁰³ further studies are required to elucidate the regulators of oxylipin generating pathways (COX, lipoxygenase, and CYP), and their mechanistic role in ALD pathogenesis determine whether oxylipins can be used as disease biomarkers.

Fibroblast growth factor-inducible 14 (Fn14): Fibroblast growth factor–inducible 14 (Fn14) is a specific receptor for TNF-related weak inducer of apoptosis (TWEAK). TWEAK is secreted principally by macrophages/monocytes during tissue inflammation.²⁰⁶ Clinical analyses report increased Fn14 expression in ALD, chronic HCV, and HCC.^{207, 208, 209} In patients with ASH, Fn14 is overexpressed, compared with other liver diseases.²¹⁰ Increasing evidence indicates that the TWEAK/Fn14 signaling pathway has a pivotal role in the pathogenesis of liver fibrosis by activating HSC proliferation and aberrant ECM production.^{211, 212, 213} Thus, abrogation of Fn14 signaling is a potential therapeutic target for treating or preventing EtOH-induced liver fibrosis.

Osteopontin: Osteopontin (OPN) is a secreted extracellular matrix protein that has a multifunctional role. Under physiological conditions, OPN expression/secretion is restricted to kidneys and bone. Under pathological conditions OPN is expressed in immune cells, promoting inflammation, fibrosis, and carcinogenesis in various organs.^214,215 In the liver, OPN induces the infiltration of nonparenchymal cells into necrotic areas to activate liver fibrogenesis. Extensive evidence from clinical studies with ALD patients shows that plasma OPN levels are increased, and its expression correlates with disease severity in ALD patients.^216,217 Investigations into how/whether OPN induces liver fibrosis indicated that OPN activates HSCs by upregulating the hedgehog pathway²¹⁸ and Akt and Erk phosphorylations.²¹⁷ OPN also increases collagen type I expression by reducing miR-129–5p, an inhibitor of collagen expression.²¹⁹

90 kDa ribosomal S6 kinase (p90RSK): p90RSK is a serine/threonine kinase of the S6 ribosomal kinase (RSK) family, whose members are downstream effectors of the Ras/Raf/MEK/ERK signaling pathway, which controls cell growth, proliferation, survival, and apoptosis, cytokine production, and collagen synthesis.^220,221 Recently, clinical and experimental studies revealed that p90RSK is upregulated in livers of patients with chronic liver disease, as it enhances liver fibrogenesis.^{222, 223, 224} Based on evidence that p90RSK is involved in fibrosis, the p90RSK will likely be a suitable target to develop new treatments for patients with advanced liver fibrosis.

Stearoyl-CoA Desaturase (SCD1): Stearoyl-CoA desaturase 1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids, palmitoleate (POA, 16:1), and oleate (OA, 18:1) from palmitate (PA, 16:0) and stearate (SA, 18:0), respectively.²²⁵ POA and OA activate Wnt signaling pathways, which induce cell proliferation and survival and promote fibrogenesis and carcinogenesis.^{226, 227, 228} Recent studies report that SCD1 is overexpressed in activated HSC and HCC cells from fibrotic patients and that levels of SCD mRNA correlate positively with tumor state and inversely with patient survival time.²²⁹ Downregulation of SCD1 attenuates hepatic fibrosis in experimental animals indicating that SCD1 is a promising therapeutic target for treating advanced liver injury.^{229, 230, 231}

Macrophage migration inhibitory factor (MIF): This is a pluripotent cytokine/chemokine expressed in immune, endothelial and epithelial cells.²³² In the liver, hepatocytes and KCs produce MIF.²³³ It is an integral component of the host antimicrobial alarm system and stress response that induces the proinflammatory functions of immune cells.^{232,234, 235, 236} Recent studies report higher circulating and hepatic expression of MIF in ALD patients and EtOH-fed rodents.^{236, 237, 238, 239} Others report that MIF-deficient mice are more resistant to ALD development. These findings indicate that MIF-directed therapies may likely offer new treatment opportunities for ALD.

Gut microbiota: Bacteria, fungi, archaea, and viruses comprise the intestinal microbiome. Extensive evidence indicates that imbalances in these microflora populations (called dysbiosis) influence the development and progression of ALD.^{240, 241, 242, 243, 244} Microbial dysbiosis, causes altered bile acid metabolism and increased secondary bile acids production, which results in decreased activation of farnesoid x receptor.²⁴⁵ These receptors play a role in maintaining intestinal epithelial barrier function and regulating immunity.²⁴⁶ Further, dysbiosis is also associated with decreased level of short-chain fatty acids, which are important for maintaining gut barrier integrity, regulating inflammation, and lipid metabolism, improving satiety for alcohol use/drinking.²⁴⁵ Overall gut dysbiosis lead to a rise in systemic inflammatory mediators, as well as ammonia and endotoxemia, intestinal hyperpermeability, as well as alcohol craving.²⁴⁷ Given these findings, interventions to reverse the alcohol-induced gut microbiota changes are potentially important therapeutics to reduce alcohol cravings and decrease intestinal permeability in patients with AUDs. Clinical trials and several animal studies indicate that treatment with antibiotics, probiotics, synbiotics, or short-chain/long-chain fatty acids alleviates alcohol-induced liver injury and are effective strategies for preventing the ethanol-induced changes in the gut leakiness, intestinal microbiota, and liver pathology.^{241,248, 249, 250, 251, 252, 253, 254, 255, 256} Furthermore, patients with alcoholic hepatitis who received fecal microbiota transplant (FMT) showed higher survival rates than those who did not.^257,258 However, while FMT is effective in clinical settings, it can cause infection in patients with advanced liver disease. More investigations are needed regarding the interactions among the gut microbial inhabitants and their involvement in liver disease.

A majority of work on gut changes in ALD has been on bacterial microbiota until recently. However, in recent studies, researchers also focused on changes in the composition of the intestinal fungi (called the mycobiome) and viruses (virome) and their consequences in the development and progression of ALD. In experimental animal studies, chronic alcohol administration increases mycobiota populations, and administration of antifungal agents (amphotericin B) reduces intestinal fungal overgrowth and decreases β-1,3, glucan translocation, and ameliorates liver injury in a mouse model of ALD.²⁴³ Further, improved liver health in patients with AUD upon abstinence was associated with a reduction in mycobiome dysbiosis.²⁵⁹ Regarding viruses, the fecal virome (mainly composed of bacteriophages) profile is different in patients with alcohol use disorder (with or without liver disease) and patients with alcohol-associated hepatitis as compared with controls.²⁶⁰ However, future studies are needed to examine the role of altered virome profile in development and progression of ALD, or whether a cocktail of phages could be used to treat ALD.²⁴⁴

Hyaluronan (35 kDa): Hyaluronan (HA) is a high molecular mass (∼10⁷ Da) polysaccharide that is an abundant extracellular matrix component. During tissue injury and inflammation, HA deposition in the extracellular matrix rises.²⁶¹ Studies also report that HA fragments generated during enzymatic HA degradation can modulate inflammation during tissue injury.²⁶² With regard to ALD, clinical studies report that serum HA concentrations correlate positively with the severity of hepatic inflammation, hepatic fibrogenesis, and the degree of fibrosis in patients with ALD.^{263, 264, 265} However, related studies indicate that a 35 kDa HA fragment (HA-35) is a potent anti-inflammatory agent, which attenuates the EtOH-induced activation of KC by restoring the expression of microRNA, miRNA-181 b-3p, a negative regulator of TLR4 signaling in KCs.^266,267 Other investigations state that HA-35 treatment protects intestinal tight junctions from alcohol-induced barrier damage.²⁶⁸ Given these findings that HA-35 regulates KC activation and protects gut barrier function, this HA fragment may have therapeutic value for ALD treatment.

Fatty acid-binding protein 4 (FABP4) is a member of the fatty acid-binding protein family (FABPs). They are low molecular weight water-soluble proteins that bind long-chain fatty acids and other ligands to facilitate cellular ligand uptake and signal transduction.²⁶⁹ The FABP4 isoform is highly expressed in white and brown adipose tissue, monocytes, and macrophages.^269,270 Its expression is induced several-fold in livers of ALD patients and experimental animal models.²⁷¹ While many studies report that adipocyte-derived FABP4 delivers FAs to cancer cells to facilitate tumor growth/progression,^{272, 273, 274, 275} a recent study reported its role in stimulating hepatoma cell proliferation and migration via activation of JNK and/or ERK signaling pathways.²⁷¹ Further understanding of the role of liver FAPB4 in HCC progression and whether it is a potential target for ALD treatment is warranted.

Extracellular vesicles (EVs) as diagnostic tools

EVs are small membrane-bound vesicles that contain proteins, lipids, and/or nucleic acids as part of their cargo. These vesicles are subdivided into three groups: exosomes, micro-vesicles, and apoptotic bodies. Inside cells, intraluminal vesicles are formed by components of the endosomal-sorting-complex-required-for-transport machinery, lipids, and tetraspanins (e.g., CD63, CD81, etc.).²⁷⁶ When multivesicular bodies dock and fuse with the plasma membrane these intraluminal vesicles are also released as exosomes.²⁷⁶ The usual sizes of exosomes is 30–150 nm, while apoptotic bodies with the size of 1–5 μM are the largest. EVs establish the connection between the donor (EV-producing) and recipient (EV-capturing) cells. EVs’ half-life in the circulation is quite short (up to 6 h) since they are readily captured by target cells and are important intercellular messengers involved in organ-to-organ communications.²⁷⁷

Alcohol consumption substantially increases EV biogenesis and secretion from liver cells and also affects their contents.^278,279 The number of circulating EVs in plasma samples of acute alcoholic hepatitis patients was found to be higher compared with EV levels in healthy donors.²⁸⁰ EV content comprises DNA, protein, lipids cargo, metabolites, mRNA, and noncoding RNA. Since EV cargo is organ- and disease-specific, circulating EVs can be used as “barcodes” that display not only their tissue of origin but also allow differential diagnosis of alcohol misuse and disease state.²⁸¹ Biomarkers, including cytokeratin-18, vanin-1, asialoglycoprotein receptor- 1 and specific microRNAs (miRNAs) indicate a liver-specific origin.^{282, 283, 284}

Various studies have shown the importance of EV cargo-mediated ethanol-initiated crosstalk between hepatocytes and macrophages in inflammation development via their ability to transport macromolecules, including regulatory miRNAs, between cells of different organs.

To this end, hepatocyte-derived EVs express CD40L that activates macrophages in a caspase 3-dependent manner and induces a proinflammatory macrophage phenotype.²⁸⁵ Furthermore, EVs released from alcohol-exposed macrophages induce MCP-1 expression in naive hepatocytes to promote infiltration of circulating macrophages into the liver.²⁸⁶ Additional studies comparing the EVs generated by hepatocytes isolated from livers of mice fed control or ethanol diets, demonstrated that heat shock protein 90 (HSP 90) is expressed and transported with exosomes, which causes increased expression of the proinflammatory cytokines, TNFα and IL-1β, and suppression of anti-inflammatory markers, CD163 and CD206, on macrophages.²⁸⁷ The same authors demonstrated that these effects were reversed by HSP 90 inhibitor treatment.²⁸⁷

In addition, the transfer of nucleic acid cargo in EVs also plays a pathophysiological role in liver inflammation.²⁷⁸ The increase in the levels of miR122, miR192, and miR30a in EVs were observed in mice chronically fed ethanol diet and alcoholic hepatitis patients with the highest diagnostic accuracy for miR192.²⁸⁸ Further studies documented that hepatocytes exposed to alcohol secrete exosomes containing miR122 which sensitizes liver macrophages to the effects of LPS.²⁸⁸ Furthermore, the increased miR122, miR155 and miR-146a levels in exosomes correlated with elevated ALT levels in various mouse models of liver injury induced by alcohol, acetaminophen or TLR9 ligands.²⁸⁹

Importantly, EV levels and cargo allow differentiation of various stages of ALD, from fatty liver to mild alcoholic steatohepatitis.^290,291 Isolated hepatocytes and macrophages from mice at an early stage of disease release more EVs, with distinct miRNA profiles, such as let-7f, miR29a, and miR340^290,291. The progression of ALD from mild alcoholic hepatitis to severe injury with features of ballooning cell degeneration and liver fibrosis was associated with the release of EVs carrying profibrotic miR221, miR126, and miR27, which are unique for this disease stage and can be used as a barcode.²⁹¹ An increase of mitochondrial (mt) DNA in EVs was observed at this late stage of ALD, which by binding to TLR9 promotes proinflammatory activation.²⁹¹ As documented, TLR9 antagonist repressed the production of IL-17, but not IL-1β induced by EVs, which contributes to inflammation and liver injury.²⁹¹ The importance of EV-contained mtDNA and TLR9 signaling for ALD progression was also demonstrated in acute-on-chronic mouse ALD model, where mtDNA-enriched microparticles released from hepatocytes promoted neutrophilic inflammation via activation of TLR9, leading to hepatic damage.²⁹²

High levels of the EtOH-metabolizing enzyme, CYP2E1, in exosomes have also been characterized as barcodes for alcohol abuse.²⁹³ While this enzyme was found in circulating exosomes from nonalcohol consuming patients, its amount is higher in people with AUD, which was additionally confirmed using experimental animal models.²⁹³ These studies were corroborated by unpublished studies from our laboratory showing elevated levels of CYP2E1 in alcohol-exposed hepatocyte-derived exosomes, as well as enhanced amounts of ADH and the lipid peroxidation markers, MDA and MAA. Since hepatocytes are the main cells that metabolize ingested ethanol, the transfer of ethanol-metabolizing enzymes with exosomes from hepatocytes to other organs may not only promote alcohol toxicity but may also induce detrimental effects in ethanol non-metabolizing organs.

Furthermore, the EVs containing viral RNAs may contribute to the transmission of HCV infection to healthy hepatocytes.¹⁴⁷ Because HCV enhances exosome release from infected Huh 7.5.1 cells,²⁹⁴ and because alcohol increases EVs release from hepatocytes,²⁸⁸ the spread of HCV in the liver with EVs will likely be more efficient in people with AUD. This transmission of viral RNAs/DNAs with exosomes is not unique for HCV infection. This is also the case for HBV infection,^295,296 as well as for HIV infection in hepatocytes exposed to EtOH.²⁹⁷

Overall, EVs are becoming valuable tools for detecting cell-to-cell and interorgan communication after alcohol exposure.²⁹⁸ With further characterization, they will likely serve as essential diagnostic tools to characterize the varying pathologies that characterize the advancing stages in the spectrum of ALD. The scheme of EV-based interactions crucial for ALD development is presented in Figure 6.

Figure 6.

Alcohol-induced extracellular vesicles mediate cell-to-cell crosstalk in the liver. Alcohol exposure causes the release of extracellular vesicles (EVs) from parenchymal (hepatocytes) and nonparenchymal (Kupffer) liver cells. These EVs contain miRNAs, DNA, lipids, proteins, alcohol-metabolizing enzymes and viral RNAs/proteins (in virally infected livers). These cargoes are transmitted between hepatocytes and macrophages, which affect cell functions via EV-mediated crosstalk between donor and recipient cells.

ALD is a major cause of global disease burden and a leading cause of mortality. While many elements in ALD pathogenesis have been uncovered, there is still no FDA-approved therapy for this disease. Given the multifactorial nature of ALD development and progression, the multitude of disease modifiers, and the identification of potential new targets, it is conceivable that a multitherapeutic regimen may be needed to treat different stages in the spectrum of this disease.

Credit authorship contribution statement

All authors have equal and substantial contributions to the following: (1) the writing of the original draft, (2) reviewing and editing, and (3) final approval of the version submitted.

Conflicts of interest

None.

Funding

This review is the result of work supported by resources and the use of the facilities at the Omaha Veterans Affairs Medical Center. The original research cited in this review was supported by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Merit Review grants, BX004053 (KKK), NIH grants R01AA026723 (KKK), R01AA027189 (NAO), R01AA028504 (KR) and K01AA026864 (MG).