The Sugar-Coated Truth of Alcohol-Associated Liver Disease

Abstract

Alcohol-associated diver disease is a major driver of end-stage liver diseases globally. Alcohol functions as a hepatotoxin by overwhelming cell stress response pathways and deregulating hepatocellular protein, amino acid, and lipid metabolism. In addition, alcohol alters innate and adaptive inflammatory immune responses and acts on extrahepatic organs to flood the liver with pro-inflammatory stimuli. Here we examine how galectins, a class of highly conserved carbohydrate-binding proteins, regulate liver homeostasis and pathology. Next, we define how galectins affect key pathways that drive alcohol-induced liver disease, including hepatocyte cell biology (eg, altered lipid metabolism, endoplasmic reticulum and lysosomal stress, mitochondrial dysfunction), innate and immune response, intestinal dysfunction, and liver fibrosis. We then document the roles of galectins in the setting of alcohol-associated liver disease. Finally, we discuss galectins as theragnostic markers and therapeutic targets for alcohol-associated liver disease and address key open questions in the field.

Alcohol-associated liver disease (ALD) remains a major public health burden and is the most common cause of cirrhosis globally, responsible for the majority of end-stage liver disease cases in Europe, Latin America, and North America.¹ Although epidemiologic studies have linked excessive alcohol consumption to liver disease for centuries, expansive efforts over the past 2 decades have elucidated broad understanding into the cellular and molecular mechanisms underlying the ALD pathophysiology (extensively reviewed elsewhere^2,3). Briefly, alcohol is an established hepatotoxin; it can subvert numerous cellular programs through altered cellular redox, increased endoplasmic reticulum (ER) stress, and mitochondrial dysfunction, among others. Although metabolism of ethanol can occur in the lung and kidney (approximately 10%), it is principally metabolized in hepatocytes by cytosolic alcohol dehydrogenase, with additional metabolism occurring in the microsomal system (mainly by cytochrome P450 2E1) or in peroxisomes via catalase enzymes. The oxidation of ethanol by these pathways results in accumulation of acetaldehyde and hydrogen ions, which can directly disrupt intermediate lipid, carbohydrate, and fatty acid metabolism and cause aberrant lipid accumulation (eg, steatosis) through various mechanisms including altered peroxisome proliferator–activated receptor-⍺ (PPAR-⍺), AMP-activated protein kinase (AMPK), and sterol regulatory element-binding protein signaling.³ In addition, increased reactive oxygen species (ROS) and homocysteine loads within cells can promote ER stress, which can drive apoptotic programs, inflammatory cytokine secretion, and metabolic reprogramming. Finally, increased oxidant and acetaldehyde loads within hepatocytes cause aberrant lipid peroxidation, including on mitochondrial membranes. These effects, coupled with increased mitochondrial DNA mutational burdens, promote mitochondrial membrane permeabilization and dysfunction, which act on apoptotic pathways and induce hepatocyte necrosis causing damage-associated molecular pattern secretion, inflammasome induction, and activation of proinflammatory cytokines (eg, IL-1b).²

In addition to these effects within hepatocytes, innate and adaptive immune responses can act on the liver to potentiate disease. Notably, alcohol-induced permeability defects in the small intestine can drive increased endotoxin exposure in the liver⁴; excessive endotoxin (eg, lipopolysaccharide) activates Toll-like receptor 4 (TLR4) signaling on Kupffer cells,⁵ which in turns leads to cytokine production [eg, tumor necrosis factor (TNF)]⁶ and ROS generation.⁷ Endotoxin-driven TLR4 signaling also exerts effects on hepatic stellate cells (HSCs) to drive fibrosis via down-regulation of the transforming growth factor beta (TGF-β) pseudoreceptor bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), leading to TGF-β sensitization.⁸ Endotoxins can also act on hepatocytes to induce intercellular adhesion molecule 1 expression and promote neutrophil recruitment (enhancing so-called “satellitosis”).⁹ In addition, altered IgA metabolism caused by chronic alcohol exposure leads to sinusoidal IgA deposition and monocyte activation, increasing TNF secretion.^10,11 Coupled with these effects, adaptive immune responses are also believed to drive pro-inflammatory responses in ALD (reviewed previously¹²), in which neo-antigens produced by protein adducts generated by increased ROS and acetaldehyde trigger production of liver-specific autoantibodies. Chronic alcohol exposure can enhance T-cell activation and turnover, alter T-cell patterning (with a shift toward more memory T cells), and reduce the numbers of total circulating T and B cells in patients.¹² Furthermore, increased T helper 17 cells seen in the setting of chronic alcohol exposure increase neutrophil infiltration and stimulate HSC activation. Finally, effector T cells display enhanced programmed death 1 and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) expression secondary to endotoxin-derived TLR4 activation on monocytes, indicating a component of T-cell exhaustion in ALD. However, the extent to which these immune dysfunctions drive versus enhance disease progression remains unclear and is the current focus of intense investigation.

Although hepatocyte injury remains a hallmark of ALD, extensive parenchymal fibrosis and tissue remodeling that occurs with chronic exposure underlies much of the morbidity and mortality experienced in patients, as reviewed previously by Lackner and Tiniakos.¹³ In both compensated and decompensated ALD, the degree of liver fibrosis is a reliable predictor of poor clinical outcome, including the development of hepatocellular carcinoma (HCC) and liver transplantation.¹⁴ In the setting of ALD, centrizonal fibrosis is often predominant, owing to the metabolism of ethanol by cytochrome P450 2E1, which is enriched around the central veins. Secretion of hedgehog ligands, which are released from ballooning hepatocytes, reactive ductules, and myofibroblasts, drives secretion of the extracellular matrix from stromal cells within the hepatic parenchyma.¹⁵ Moreover, increased levels of damage-associated molecular patterns, pathogen-associated molecular patterns (eg, lipopolysaccharide), and extracellular ROS seen in the setting of alcohol consumption promote cytokine secretion from Kupffer cells, which act on specialized cells within the space of Disse, HSCs, leading to their activation and trans-differentiation into activated myofibroblasts.^15,16 The presence of activated HSCs seems to be a key contributor to the advancement of liver fibrosis across all liver diseases, as their attenuation can abrogate advanced fibrosis in animal models.^17,18 Activated HSCs contribute to fibrinogenesis through a variety of mechanisms, including increased fibrillar collagen secretion, TGF-β signaling, and extracellular matrix remodeling through altered matrix metalloproteinase activity, among others.18, 19, 20, 21 Although the maturation of fibrosis in ALD has not been described in detail, alcohol seems to directly amplify fibrogenesis through the alteration of innate immune responses, notably inhibition of natural killer (NK) cell–mediated death of activated HSCs²²; in addition, emerging evidence indicates that alcohol may directly promote epigenetic changes in activated HSCs to enhance extracellular matrix secretion and alter their differentiation through effects on retinoic acid metabolism.^23,24

Galectins as Multifaceted Regulators of Cell and Developmental Biology

Galectins represent a broad class of evolutionarily conserved intracellular and secreted glycan-binding proteins (or lectins) unified by their ability to bind β-galactoside epitopes on cognate glycoproteins and glycolipids. Since their original identification in the electric eel in 1975, studies have shown that galectins are conserved across metazoans, where they exert broad effects on cell and developmental programs through regulation of intracellular and extracellular signaling pathways, stress responses, and endomembrane trafficking^25,26 (Figure 1). In total, up to 15 galectins are now recognized in vertebrates, which are typically placed into one of three families: prototypical, tandem repeat, and chimeric galectins. Prototypical galectins (Gal-1, Gal-2, Gal-5, Gal-7, Gal-10, Gal-11, Gal-14, and Gal-15) consist of globular proteins with one carbohydrate recognition domain, which can form homodimers to crosslink proteins. Tandem repeat galectins (Gal-4, Gal-6, Gal-8, Gal-9, and Gal-12) represent a class of galectins that contain N- and C-terminal carbohydrate recognition domains separated by a variable linker domain. Chimeric galectins (Gal-3) are defined by their hybrid characteristics, containing one C-terminal carbohydrate recognition domain and a long N-terminal tail that allows for additional protein–protein interactions, including oligomerization.

Figure 1.

Overview of galectins and their cell biology. Top: Galectins are a class of glycan-binding proteins defined by their highly conserved carbohydrate-binding regions [carbohydrate recognition domain (CRD)] that recognize β-galactoside epitopes on glycoproteins and glycolipids. Three broad families of galectins are classified based on the arrangement and number of their CRDs: prototypical, tandem repeat, and chimeric (galectins in bold red are associated with liver homeostasis/pathology). Bottom: Galectins of each family participate in both intracellular and extracellular interactions. Intracellular interactions include both carbohydrate-mediated interactions with organelles and direct interactions with proteins that influence cell survival, organelle repair, cell metabolism, and cell signaling. Extracellular interactions include lattice formation, cell-to-cell, and cell-to-matrix interfaces that regulate cell activation, cytokine secretion, and cell migration. Gal, galectin. Figure created with BioRender.com (Toronto, ON, Canada).

In addition to sharing carbohydrate recognition domain sequence homology allowing for β-galactoside binding, galectins share additional biochemical characteristics that distinguish them from other carbohydrate-binding proteins; notably, galectins are sensitive to protein oxidation, are expressed in the cytosol on free ribosomes, and are secreted in a calcium-independent manner through a poorly described nonclassical secretory pathway. Once outside the cells, galectins are thought to engage extracellular glycans in homotypic and heterotypic interactions, where they can stabilize cognate protein/lipid in a galectin-glycan lattice, engage signaling modalities, promote cell migration, and engage on ligands across numerous cell types and membranes (Figure 1). Galectins can also affect cell behavior through carbohydrate-dependent and carbohydrate-independent interactions with intracellular binding partners. Galectins are broadly expressed across cell types in metazoans, although individual galectins exhibit enrichment at certain sites and cell types over others. Notably, Gal-7 appears enriched in keratinized squamous epithelium (skin), whereas adipose tissue is enriched for Gal-12. Similarly, mucosal surfaces appear enriched for Gal-2, Gal-3, Gal-4, Gal-6, Gal-8, and Gal-9, with Gal-4 exhibiting robust expression in the intestine.²⁷ Importantly, although galectins exhibit diverse expression across immune populations and subtypes, as reviewed by Liu and Stowell,²⁸ Gal-1, Gal-3, and Gal-9 are enriched in the liver in homeostatic and diseased states (see Galectins as Mediators of ALD).

Galectins as Regulators of Hepatic Homeostasis and Pathology

Given the ability of galectins to broadly affect cellular processes and tissue patterning,²⁶ they have been extensively studied to define their role in human pathologies, including hepatic diseases (reviewed by Mimura et al²⁹). Galectins are fundamental regulators of injury and repair in the liver, where they regulate processes involving hepatocyte cell biology, innate and adaptive immune responses, and liver fibrosis, among others (Figure 2). Following is a brief description of the roles of galectins implicated in each of those processes.

Figure 2.

Overview of galectins as regulators of liver disease. Galectins are multifaceted regulators of liver homeostasis and pathology, including aspects directly related to alcohol-associated liver disease (ALD). Depicted are the various interactions that galectins affect in processes promoting liver pathology and influence (ALD). This includes galectins from each of the three structural families of galectins (ie, prototypical, tandem repeat, and chimeric). Bottom right inset: Key regulators of transcription/translation of the three most well-studied galectins in the liver: galectin-1 (Gal-1), galectin-3 (Gal-3), and galectin-9 (Gal-9). BRG-1, Brahma-related gene 1; ER, endoplasmic reticulum; IFN, interferon; TGF-β, transforming growth factor beta. Figure created with BioRender.com (Toronto, ON, Canada).

Galectins as Regulators of Epithelial Cell Biology in the Liver

Since the discovery of galectins, studies have reported altered epithelial expression of individual galectins in liver disease, including for Gal-1, Gal-3, Gal-4, and Gal-9 (reviewed elsewhere³⁰). Gal-1 expression is tightly regulated in hepatocytes by multiple upstream factors, including miRNA (eg, miR-22), CpG methylation, and by TGF-β signaling,31, 32, 33 whereas Gal-3 is regulated by Brahma-related gene 1, NF-κB, and AP-1³⁴ (Figure 2). Emerging data on Gal-9 show control by the transcriptional repressor NFX1, in which lysosomal degradation of NFX1 leads to increased expression of Gal-9, thereby modulating CD8⁺ T cell–mediated cancer cell immunity and tumor progression.³⁵ Although definitive characterization of transcriptional regulators of Gal-9 within hepatocytes remains unclear, interferon (IFN) signaling promotes Gal-9 expression through a histone deacetylase 3–dependent mechanism and likely contributes to Gal-9 expression in the setting of ALD³⁶ (Figure 2).

Within hepatocytes, studies have highlighted diverse roles of galectins in regulating lipid composition and metabolic pathways that drive steatosis (Figure 2); studies confirming these findings in the context of ALD are lacking, however. Murine studies show that Gal-1 and Gal-3 are required for the transition or reversion of steatosis in hepatocytes³⁷; mechanistically, although these effects remain poorly understood, recent studies found that Gal-3 can activate PPAR-γ signaling and up-regulate the fatty acid transporter CD36.³⁸ Intriguingly, Gal-1^–/– mice fed a high-fat diet also exhibit reduced hepatic steatosis that is secondary to decreased PPAR-γ activity in the liver and CD36 expression; this potentially indicates that galectins share conserved pathways to regulate lipid accumulation through PPAR-γ signaling.^39,40 In parallel, galectins are direct regulators of AMPK signaling in hepatocytes, and recent studies show that Gal-3 can interact with CD133 in liver progenitor cells to drive AMPK and focal adhesion kinase activation in the setting of regeneration and injury.⁴¹ Furthermore, Gal-9 can be recruited to damaged lysosomes and, through interactions with TGF-β–activated kinase 1, promote AMPK phosphorylation and activation.⁴² Finally, in addition to regulating PPAR signaling and AMPK, galectins augment sterol regulatory element-binding protein signaling, another significant driver of ALD pathophysiology. Gal-1 can bind to isoforms of sterol regulatory element-binding protein 1 and alter its localization in cultured cells⁴³; moreover, sterol regulatory element-binding protein 1 up-regulates Gal-3 expression in smooth muscle cells.⁴⁴ Whether these pathways are similarly activated in hepatocytes, including in the context of ALD, remains unknown.

Galectins are also critical regulators of cellular stress response pathways, which, when overwhelmed, can act as amplifiers of ALD through damage-associated molecular pattern secretion, ROS release, and inflammatory cytokine expression (see discussion above). Early studies defining intracellular roles of Gal-3 found that exogenous Gal-3 could attenuate cell death in the setting of increased apoptotic stimuli. Subsequent studies revealed that Gal-3 prevents mitochondrial damage and cytochrome c release through regulating Bad/BCL-2 interactions upon enrichment at mitochondrial membranes via interactions with synexin⁴⁵ (Figure 2). More recently, studies show that Gal-3 regulates inflammasome formation in hepatocytes, via binding to TLR4 on hepatocytes and oligomerization of NOD-like receptors such as NOD-, LRR-, and pyrin domain–containing 3 in hepatic macrophages.⁴⁶

Galectins are also critical regulators of endosomes and membrane-bound organelles, through their regulation of autophagy and lysosomal stress responses (Figure 2) (reviewed in Liu et al⁴⁷). Autophagic and lysosomal stress can in turn augment ER stress and redox control in cells.⁴⁸ Consistent with this, in an in vitro cell culture model of alcohol exposure and viral infection, exposure to both an acetaldehyde-generating system and HIV promotes lysosomal damage, impaired lysosomal biogenesis, and altered transcription factor EB activity, thereby sensitizing cells to intrinsic apoptotic stimuli.⁴⁹ Future studies defining the role of these pathways in ALD, and the requirement for individual galectins in initiating and promoting hepatic dysfunction, will be an intense area of focus in the coming years as an understanding of ALD pathophysiology increases.

Galectins as Regulators of Innate and Adaptive Immune Responses in the Liver

In 1983, a homolog of Gal-1 (then known as electrolectin) was shown to regulate adaptive immunity by binding to lymphocytes.⁵⁰ Subsequently, galectins emerged as broad regulators of innate and adaptive immune cell signaling across metazoans.²⁸ In the context of the liver, Gal-1, Gal-3, and Gal-9 have prominent roles regulating the immune response to injury implicating their potential impact on ALD (Figure 2).

Immunomodulatory roles of Gal-1 within the liver are well documented, particularly in regulating leukocyte biology. Early studies looking at concanavalin A–induced liver injury found that exogenous Gal-1 could ameliorate cell injury by increasing the turnover of concanavalin A–activated T cells and attenuating proinflammatory macrophages.⁵¹ Studies have also revealed an immunosuppressive role of Gal-1 through control of T helper 1 and T helper 17 cells and their cytokines in promoting allograft tolerance.⁵² Similarly, in ischemia-reperfusion injury, recombinant Gal-1 treatment reduces proinflammatory cytokine secretion (eg, TNF, IL-6, IFN-γ) while increasing IL-10 levels in hepatic macrophages.⁵³ Thus, Gal-1 has primarily been associated with attenuating inflammatory response in mouse models of liver injury.

Like Gal-1, Gal-9 has emerged as a clear regulator of immune responses during liver injury through coordination of T-cell responses, as reviewed by Golden-Mason and Rosen.⁵⁴ In a murine model of acute hepatitis, exogenous Gal-9 ameliorated concanavalin A–induced hepatitis by blunting the T helper 1 responses, increasing the regulatory T-cell populations, and decreasing proinflammatory cytokines (including TNF and IL-6) through interactions with its receptor TIM-3. Similarly, in the setting of ischemia-reperfusion injury and allograft models, Gal-9 exhibits protective effects through attenuating T-cell activation and promoting CD4⁺ T-cell apoptosis, while decreasing local infiltration of innate and adaptive immune cells, reducing proinflammatory cytokines, and suppressing hepatocyte apoptosis. Gal-9 also shapes T-cell responses in viral hepatitis, in which increased expression on inflammatory leukocytes, Kupffer cells, and hepatocytes from liver biopsy specimens drive regulatory T-cell expansion, loss of IL-21–producing CD4⁺ T cells, and progression toward chronic infection. In the context of toxic/metabolic disease, additional immunoregulatory roles for Gal-9 in shaping immune response include controlling NK T cells. Gal-9 can bind TIM-3 on NK T cells to drive apoptosis, but Gal-9 can also act on Kupffer cells expressing TIM-3 to drive IL-15 expression, which expands NK T cells and overall ameliorates diet-induced steatosis.

Immune-related functions of Gal-3 have also been extensively examined in the liver. In murine models, Gal-3 seems to promote an influx of TNF, IFN-γ, IL-17, and IL-4 producing proinflammatory CD4⁺ T cells, while limiting the numbers of anti-inflammatory IL-10–producing CD4⁺ T cells.⁵⁵ Gal-3–deficient mice were also associated with reduced NK cells, NK T cells, and activated dendritic cells, with skewed polarization of macrophages toward the immunosuppressive M2 phenotypes.⁵⁵ Similarly, in a model of drug-induced liver injury, Gal-3–deficient mice also exhibited decreased injury, reduced inflammatory cytokine production, and increased M2 macrophage polarization.⁵⁶ In contrast to Gal-9, regulatory T cells did not appear altered. Gal-3 derived from activated Kupffer cells also appears to regulate immune responses in metabolic dysfunction, where it can act as a ligand for TLR4 and promote inflammasome activation.⁵⁷

However, the degree to which these immunomodulatory interactions exist in and contribute to ALD remains relatively unknown (as discussed in Galectins as Mediators of ALD). Moreover, whether known well-characterized interactions of galectins with other immune populations, notably neutrophils and eosinophils, contribute to liver homeostasis is still unexplored.⁵⁸

Galectins as Regulators of Liver Fibrosis

As inflammatory mediators and cellular alarmins, galectins have long been implicated in tissue fibrosis associated with immune-mediated injury.⁵⁹ Although their effects on epithelial and innate and adaptive immune populations undoubtedly contribute to tissue fibrosis, direct roles for galectins regulating mesenchymal populations are well characterized (Figure 2). Within the context of the liver, extensive focus has centered on Gal-1 and Gal-3. In cell culture models, both Gal-1 and Gal-3 are up-regulated during the HSC trans-differentiation to myofibroblasts, in which Gal-1 and Gal-3 stimulate the proliferation and migration of cultured HSCs via mitogen-activated protein kinase kinase 1/2–extracellular signal–regulated kinase 1/2 signaling.⁶⁰ In murine models of liver fibrosis, Gal-3 is required for TGF-β–dependent activation of HSCs and myofibroblast activation through both autocrine and paracrine pathways, which was confirmed in patients with cirrhosis.^61,62 GR-MD-02 (belapectin), a Gal-3 inhibitor, reduces nonalcoholic steatohepatitis and thioacetamide-driven liver fibrosis^63,64 and inhibits the expression of fibrogenic mediators, including inducible nitric oxide synthase CD36, and α-smooth muscle actin.⁶³ More recently, studies in mouse models showed that Gal-1 helps regulate HSC activation and migration through a complex with N-glycans on neuropilin-1 to activate platelet-derived growth factor and TGF signals.⁶⁵ Gal-1 also positively regulates TGF-β, connective tissue growth factor, and α-smooth muscle actin levels, and correlates with intrinsic regulation of HSC mitochondrial function, proliferation, and migration.⁶⁶ Taken together, these findings suggest that Gal-1 and Gal-3 are drivers of hepatic fibrosis. Not surprisingly, these two galectins are at the center of clinical trials investigating their clinical utility in liver fibrosis.

Gal-7, Gal-9, and Gal-12 have also been associated with liver fibrosis, although their roles are less fully characterized (Figure 2). In cultured HSCs, Gal-7 is required for hepatocyte growth factor–driven suppression of TGF-β–responsive gene expression after carbon tetrachloride injury.⁶⁷ Gal-9 serum levels correlate with levels of liver fibrosis and fibrosis progression in patients with chronic liver disease.⁶⁸ Although it is not yet known what cell types are responsible for Gal-9 serum levels, HSCs, macrophages, and Kupffer cells all produce Gal-9.^69,70 Gal-12 deficiency enhanced liver fibrosis in an end-stage mouse model of nonalcoholic fatty liver disease by promoting M2 polarization of macrophages, thereby promoting production of TFG-β and stimulation of HSCs.⁷¹ These studies reveal the potential for studying galectins in the context of liver fibrosis relevant to ALD.

Extrahepatic Functions of Galectins

Although the aforementioned functions highlight roles of galectins within the liver in regulating hepatic homeostasis, galectins also have well-described extrahepatic functions that are drivers of ALD. Notably, galectins have roles in regulating barrier function and microbial populations, which in turn can act as potentiators of liver damage during acute and chronic alcohol use (Figure 2). Galectins have varied roles in regulating cell barrier function. In epithelial wound healing models, Gal-3 can drive expression of matrix metalloproteinase 9, likely through cell surface interaction with CD147, thereby inducing tight junction disassembly.⁷² In the intestine, Gal-9 is also required for epithelial restitution, although mechanistic insight is lacking.^73,74 Gal-3 can also reduce the stability of adherens junctions by increasing N-cadherin turnover.⁷² Similarly, Gal-1 can decrease endothelial barrier function by regulating vascular endothelial cadherin.⁷³ However, Gal-3 can also promote desmosome stability in the intestine by inhibiting desmoglein-2 proteasomal turnover by stabilizing desmoglein-2 on the plasma membrane.⁷² In parallel, galectins (including Gal-1, Gal-3, Gal-8, and Gal-9) are regulators of luminal microbes through glycan-specific and glycan-independent interactions, as reviewed by Vasta,⁷⁵ and emerging evidence highlights galectins as central regulators of the microbiome through regulation of bacterial agglutination.⁷⁶ In sum, these findings emphasize a role for galectins in regulating intestinal epithelial cell functions and microbial populations, which, when dysregulated, can lead to excessive bacterial translocation and hepatic lipopolysaccharide/damage-associated molecular pattern exposure.

Galectins as Mediators of Hepatocellular Carcinoma

Finally, extensive alcohol exposure and fibrosis can drive neoplastic transformation, including the development of HCC. Although the current mini-review is focused on non-neoplastic roles of galectins in regulating liver disease and alcohol-related liver injury, the role of galectins in the development and progression of HCC has been extensively examined and reviewed elsewhere.^77,78 Briefly, galectins serve as intrinsic regulators of cell growth and apoptotic pathways to regulate tumor growth while also acting outside the cells to engage in interactions with the extracellular matrix to drive invasion and metastasis. Moreover, Gal-1, Gal-3, and Gal-9, which are all dysregulated in hepatocytes in HCC, can regulate innate and adaptive immune responses (see discussion above) (Figure 2), which can further augment HCC development and progression. Considering these observations, it is not surprising that galectins are linked to clinical outcomes in the setting of HCC; notably, tumors with high levels of Gal-1 are associated with worse clinical outcomes (vascular invasion, incomplete encapsulation, poor differentiation, multiple number, and large tumor size) and reduced survival,⁷⁹ while increased circulating Gal-3 and Gal-9 levels predict reduced patient survival and recurrence in patients with HCC.79, 80, 81

Galectins as Mediators of ALD

Not surprisingly given the multifaceted roles of galectins in regulating hepatic responses to injury, they are emerging as direct regulators of ALD, especially Gal-1, Gal-3, and Gal-9 (Table 1). Gal-1 expression is strongly up-regulated in mouse models of alcohol intake, particularly in the setting of HCV infection.⁸² Gal-3 levels are elevated in patients with alcohol-related liver cirrhosis and negatively correlate with liver function in this setting.⁸⁶ In addition, in a mouse model of alcohol-related liver cirrhosis, the development of cirrhosis was associated with increased expression of Gal-3 from macrophages and migration of bone marrow cells to the liver. Furthermore, bone marrow cell transplantation in this model reduced Gal-3 expression and ameliorated liver cirrhosis.⁸³ Gal-3 serum concentrations are also significantly higher in patients with alcohol-related/nonalcohol-related cirrhosis and toxic hepatitis, with serum Gal-3 concentrations higher in subjects exposed to alcohol than in patients with toxic hepatitis and yielding a significant correlation between serum Gal-3 levels and stage of disease.⁸⁵ Moreover, microbial exacerbation of alcohol-induced gut barrier dysfunction increases TGF-β1 and Gal-3 production as well as HSC activation through a pathway that involves TLR4-dependent activation of IL-6 and TNF.⁸⁴ This action implicates Gal-3 as a mediator of liver damage in the oral-gut-liver axis during ALD.

Table 1.

Studies Directly Relating Galectins to ALD

Protein	Preclinical/clinical	Result	Reference
Gal-1	Preclinical (mouse)	Gal-1 up-regulated after alcohol ingestion in ethanol-fed core-transgenic mice	Tsutsumi et al82
Gal-3	Preclinical (mouse)	Levels of Gal-3 expression correlated with liver fibrosis in mice treated with CCL4 and EtOH	de Oliveira et al83
	Preclinical (mouse)	High levels of Gal-3 associated with worse outcomes in mice fed a Lieber-DeCarli liquid diet and administered Porphyromonas gingivalis	Gao et al84
	Clinical	Serum concentrations of Gal-3 significantly higher in ALD and associated with worse clinical outcomes	Gudowska et al85
	Clinical	Systemic, portal, and hepatic levels of Gal-3 negatively associated with liver function in patients with alcoholic liver cirrhosis	Wanninger et al86
Gal-9	Preclinical (human)	scRNA-seq reveals higher Gal-9 expression in Kupffer cells in patients with ALD compared with those with other inflammatory pathologies	Zhang et al87
	Clinical	CD4 and CD8 T cells from patients with acute alcohol-related hepatitis expressed higher levels of Gal-9 and TIM-3. Elevated levels of plasma Gal-9 in patients with AAH compared with healthy control subjects	Markwick et al88
	Clinical	Soluble TIM-3 ligands Gal-9 and CEACAM-1 were elevated in plasma of patients with ALD	Riva et al89
	Clinical	Polymorphisms in Lgals9 are linked to ALD development in patients with excessive alcohol exposure	Rosen et al90

AAH, acute alcoholic hepatitis; ALD, alcohol-associated liver disease; CCL4, carbon tetrachloride; CEACAM-1, carcinoembryonic antigen-related cell adhesion molecule 1; EtOH, ethanol; Gal-1, galectin-1; Gal-3, galectin-3; Gal-9, galectin-9; scRNA-seq, single-cell RNA-sequencing; TIM-3, T-cell immunoglobulin and mucin domain-containing protein 3.

Gal-9 has also been extensively studied in advanced ALD, particularly through regulating T-cell exhaustion through the receptor TIM-3. In patients with acute alcohol-related hepatitis, T cells express higher levels of programmed death 1, programmed death-ligand 1, TIM-3, and Gal-9, and this increased expression correlates with increased T-cell exhaustion; furthermore, blockade of TIM-3 restores T-cell IFN-γ production and reduces IL-10–producing T cells.⁸⁸ In parallel studies, TIM-3 and Gal-9 plasma levels are higher in patients with ALD and closely correlate with inflammatory state, increased bacterial translocation, and clinical indices of disease severity in ALD.⁸⁹ Furthermore, in bacteria-challenged peripheral blood mononuclear cell cultures, recombinant soluble TIM-3 plus ⍺-lactose (a galectin antagonist) was sufficient to activate bacterial immune response (IFN-γ and IL-17) and restore anti-inflammatory IL-10 production in patients with alcohol-related hepatitis. Finally, a recent single-cell RNA-sequencing analysis from patients with alcohol-related liver cirrhosis found that immune dysfunction, including T-cell exhaustion, is a key feature in patients with alcohol-related liver cirrhosis and was correlated with high Gal-9 expression from Kupffer cells in these patients.⁸⁷ Taken together, these studies indicate that by driving T-cell exhaustion and immune dysfunction, Gal-9 can promote ALD pathophysiology, likely by impairing antimicrobial immune response and sepsis.⁸⁸ Consistent with a direct role in ALD, genetic analysis of heavy alcohol consumers found that two polymorphisms in LGALS9 (Gal-9) are associated with development of ALD.⁹⁰ These polymorphisms (rs4239242 and rs4794976) are associated with higher transcription and protein expression of Gal-9 after stimulation of monocytes with IFN-γ and ethanol.

Future Perspectives

As secreted alarmins with broad biologic effects that can be easily assessed in patient plasma and on tissue sections, galectins show promise across a myriad of disease for their theragnostic value. Plasma levels of Gal-3 are a clinically actionable biomarker for heart, kidney,⁹¹ autoimmune,⁹² and neoplastic diseases, among others.⁹³ Similarly, groups propose Gal-9 as a biomarker of disease severity across broad classes of diseases, including autoimmune disorders, infections, cancer, chronic kidney disease, type 2 diabetes, coronary artery disease, atherosclerosis, and benign infertility-related gynecologic disorders.⁹⁴ In parallel, evidence examining prototypical galectins shows similar promise in their utility as clinically actionable biomarkers⁹⁵; preliminary studies indicate that Gal-3 shows comparable sensitivity and specificity with the hyaluronic acid and prothrombin time index and other current biomarkers.⁸⁵ Within the context of the liver, elevated levels of Gal-3 and Gal-9 in the plasma predicts stage of liver disease and outcomes in inflammatory pathologies, as reviewed by An et al.⁷⁸ However, extensive analysis on whether these individual galectins, not to mention combinations of galectins, can predict clinical outcome or response to therapy within the context of ALD remains unknown. Yet, given the numerous links outlined earlier between Gal-1, Gal-3, and Gal-9 and ALD pathophysiology, efforts to clarify this open question likely will prove fruitful in the years to come.

As discussed earlier, individual galectins display broad-reaching impacts on ALD pathophysiology. These include interactions within the liver, regulating diverse aspects of epithelial cell biology, innate and adaptive immune responses, and HSC activation, as well as outside the liver where galectins can affect intestinal barrier function and the microbiome (Figure 2). Thus, future studies using genetic model organisms, in which the requirement for individual galectins within individual cell populations can be assessed, are needed to define the cell- and tissue-specific requirements of individual galectins within ALD pathophysiology. Moreover, integration of galectins and glycomic profiles into large-scale spatial omics (eg, transcriptomics, proteomics, multiplex immunofluorescence) will provide context for the role of individual galectins in real time within ALD research. Together, these findings will inform not only a better understanding into the role of galectins in liver disease but an understanding into ALD pathophysiology in general.

Finally, given their diverse clinical significance, accessible binding pocket, and well-characterized structural characteristics, galectins remain an intense focus of the pharmacologic industry for therapeutic manipulation.⁹⁶ To date, two Gal-3 inhibitors, GR-MD-02 and GB1211, are actively under investigation for the treatment of liver fibrosis and cirrhosis in the setting of metabolic dysfunction–associated steatohepatitis/nonalcoholic fatty liver disease, with other Gal-3 modulators under development and showing promise in preclinical models.⁹⁷ Similarly, monoclonal antibodies targeting Gal-9 are being investigated for their utility in cancer treatment, where they have shown promise in phase 1 clinical trials.⁹⁸ Whether these inhibitors, or others, can exhibit similar effects or utility in ALD remains unknown and should be the focus of intense scrutiny in the years to come.

Disclosure Statement

None declared.