Targeting the gut barrier for the treatment of alcoholic liver disease

Abstract

Alcohol consumption remains one of the predominant causes of liver disease and liver-related death worldwide. Intriguingly, dysregulation of the gut barrier is a key factor promoting the pathogenesis of alcoholic liver disease (ALD). A functional gut barrier, which consists of a mucus layer, an intact epithelial monolayer and mucosal immune cells, supports nutrient absorption and prevents bacterial penetration. Compromised gut barrier function is associated with the progression of ALD. Indeed, alcohol consumption disrupts the gut barrier, increases gut permeability, and induces bacterial translocation both in ALD patients and in experimental models with ALD. Moreover, alcohol consumption also causes enteric dysbiosis with both numerical and proportional perturbations. Here, we review and discuss mechanisms of alcohol-induced gut barrier dysfunction to better understand the contribution of the gut-liver axis to the pathogenesis of ALD. Unfortunately, there is no effectual Food and Drug Administration-approved treatment for any stage of ALD. Therefore, we conclude with a discussion of potential strategies aimed at restoring the gut barrier in ALD. The principle behind antibiotics, prebiotics, probiotics and fecal microbiota transplants is to restore microbial symbiosis and subsequently gut barrier function. Nutrient-based treatments, such as dietary supplementation with zinc, niacin or fatty acids, have been shown to regulate tight junction expression, reduce intestinal inflammation, and prevent endotoxemia as well as liver injury caused by alcohol in experimental settings. Interestingly, saturated fatty acids may also directly control the gut microbiome. In summary, clinical and experimental studies highlight the significance and efficacy of the gut barrier in treating ALD.

1. Introduction

Long-term excessive alcohol consumption causes liver disease, namely alcoholic liver disease (ALD). ALD is one of the major causes of liver-related mortality worldwide. Alcohol-related deaths account for up to 48% of liver-related deaths in the United States,¹ whereas it has been estimated that 60%–80% of liver-related deaths in Europe are due to excessive alcohol consumption.² Unfortunately, effective therapies for ALD are currently unavailable. The major obstacle is the limited understanding of the pathogenesis of ALD. The spectrum of ALD involves three progressive stages: alcoholic steatosis (fatty liver), hepatitis and cirrhosis.^1,3 Alcoholic steatosis, characterized by macrovesicular and/or microvesicular lipid droplet accumulation in hepatocytes, is the earliest manifestation of ALD and is generally reversible with abstinence. Alcoholic hepatitis features inflammation and necrosis in the liver, which encompasses a spectrum of severity ranging from asymptomatic dysregulation of biochemistries to fulminant liver failure. Alcoholic cirrhosis, the most advanced form of ALD, refers to the replacement of functional liver tissue with nonfunctional fibrotic tissue and regenerative nodules, and may result in clinical manifestations of portal hypertension and liver failure. It is noteworthy that not all heavy drinkers develop alcoholic hepatitis, and the disease can occur in people who drink only moderately. Indeed, approximately 90%–95% of people who consume large quantities of alcohol develop steatosis, of which only 10%–40% eventually develop liver fibrosis.⁴ Despite the correlation between the per capita alcohol consumption and mortality rates from hepatic cirrhosis,⁵ there is little evidence suggesting that alcohol itself is able to cause permanent damage to the liver. Thus, factors other than alcohol intake influence the development and progression of ALD.

Early studies in ALD identified increased levels of lipopolysaccharide (LPS), a bacterial endotoxin, in the plasma of both ALD patients and experimental animal models of ALD (namely endotoxemia).^6–9 Experimentally-induced endotoxemia indicates that it is a key factor in the development of ALD.^10,11 In addition to LPS, increasing evidence from recent studies supports an emerging role for bacterial pathogen-associated molecular patterns (PAMPs) in ALD.^12–14 Clinical studies have shown that only alcoholics with gut leakiness develop liver injury,¹⁵ suggesting a pivotal role for gut-derived toxins and the gut barrier in ALD. This review focuses on the gut barrier and gut-derived PAMPs in the pathogenesis of ALD. Potential therapeutic approaches targeting the gut for the treatment of ALD will also be discussed.

2. Gut barrier

The primary function of the gastrointestinal tract is to digest food and absorb nutrients. It has the largest surface that the body exposes to the external environment, which puts the gastrointestinal tract at risk from exogenous pathogenic microorganisms such as bacteria, fungi and viruses.^16–18 Therefore, another essential function of the gastrointestinal tract is to act as a barrier preventing the invasion of the circulation by microorganisms. The gut barrier is a multi-layer system, and has both physical and immune defense functions. It consists of three major components, including mucus, epithelial cells and immune cells (illustrated in Fig. 1A).^19–21 While the mucus and epithelial cells act basically as physical barriers, all three layers contribute to the immune barrier function.

Fig. 1. Alcohol-induced gut barrier dysfunction.

(A) Schematic diagrams of the gut barrier under healthy and alcohol-intoxicated conditions. (B) Immunofluorescence of ileal tight junction protein ZO-1. Arrowheads indicate dissociated ZO-1. Abbreviations: AMP, antimicrobial peptide; LPS, lipopolysaccharide; PF, pair-fed; AF, alcohol-fed; ZO-1, zonula occludens-1; DAPI, 4′,6-diamidino-2-phenylindole.

2.1. Mucus layer

The first line of defense is the stratified mucus layer, which together with the glycocalyx of the epithelial cells, provides a protective spacer against physical and chemical injury caused by ingested food, microbes and microbial products.^20,21 The organization of the mucus system varies markedly along the gastrointestinal tract; the small intestine has a single unattached mucus layer which limits bacteria reaching the intestinal epithelium, whereas the stomach and colon has a two-layered mucus, with inner and outer layers. The inner colonic mucus layer is dense and firmly attached to the epithelial cells, and does not allow bacterial penetration. The outer colonic mucus layer is loose and unattached, and it is the natural habitat of commensal bacteria.²²

Mucus is mainly produced and secreted by goblet cells.²³ The major structural component of the intestinal mucus is mucins, which are large highly glycosylated glycoproteins. The two major types of mucins can be functionally distinguished; transmembrane mucins and secreted mucins.^24,25 Transmembrane mucins, including MUC1, MUC3, MUC4, MUC11–13, MUC15–17, MUC20 and MUC21, have a single membrane-spanning domain and are essential components of the glycocalyx of the mucosal surface, and are involved in intracellular signaling.²⁴ Secreted mucins, especially gel-forming mucins, make up the skeleton of the mucus layer. In the small intestine and colon, the mucus is built of the gel-forming MUC2 mucin, compared with MUC5AC mucin in the stomach.^26,27 Mucins can be considered a two-edged sword, as their normal function protects against unwanted substances and microorganism penetration while malfunction of mucins may be a causal factor in disease.²⁸ In addition to MUC2 secretion, the intestinal goblet cells also secrete a number of other mucus components, including trefoil factor peptide 3 (TFF3), resistin-like molecule β (RELMβ), Fc-γ binding protein (FCGBP), zymogen granule protein 16 (ZG16) and calcium-activated chloride channel regulator 1 (CLCA1),^20,23 all of which contribute to a highly viscous extracellular layer.

To maintain intestinal homeostasis between the host and microorganisms, a variety of biomolecules are produced and released into the mucus layer by cells other than goblet cells. A subset of these biomolecules, antibacterial peptides (AMPs), are particularly important. Intestinal AMPs are secreted by both Paneth cells and enterocytes. Paneth cells are exocrine cells located at the base of crypts of Lieberkühn, which secrete various AMPs, including lysozyme, C-type lectins, secretory phospholipase A2 (sPLA2), angiogenin 4 (Ang4) and α-defensins (HD5 and HD6 in humans and cryptdins in mice).^29–31 The enterocytes do not merely provide a passive barrier function but also contribute actively by secreting AMPs.³² The major AMPs secreted by villus enterocytes include β-defensins (hBD-1, 2, 3, 4), cathelicidins (LL-37 in humans and cathelin-related antimicrobial peptide in mice) and regenerating islet-derived protein 3 β (Reg3β) and Reg3γ. These AMPs generate an antibacterial gradient in the mucus layer, and prevent microorganisms from penetrating to the epithelial cell surface.

2.2. Intestinal epithelial cells

Epithelial cells provide both a physical and immune defense barrier in the intestine. This selectively permeable barrier prohibits passage of microorganisms and toxins while permitting transport of nutrients and water.^33,34 The paracellular permeability of the intestinal epithelium is controlled by an apical junction complex, composed of tight junctions, adherens junctions and desmosomes, in an apical to basal orientation. The tight junction is located at the most apical position of epithelial cells, and forms the actual seal between adjacent cells.^34,35 It is the principal determinant of intestinal epithelial permeability. Adherens junctions and desmosomes provide the adhesive forces necessary for maintenance of cell-cell interactions, and prevent mechanical disruption of the epithelium.^36,37 The tight junction is composed of transmembrane proteins, including occludin, claudins, junctional adhesion molecule and tricellulin, and cytoplasmic scaffold proteins, such as zonula occludens (ZO-1, ZO-2 and ZO-3).^34,38 The most crucial transmembrane proteins are claudins, which define tight junction permeability. Claudins are categorized into barrier forming (claudin-1, -3, -4, -5 -8, -9, -11 and -14) and channel pore forming (claudin-2, -7, -12 and -15) subtypes. The barrier forming claudins decrease, whereas the channel pore forming claudins increase, the paracellular permeability.³⁹ The function of occludin has not been fully elucidated thus far. Knockdown of intestinal occludin in mice increased the gut permeability to macromolecules.⁴⁰ Furthermore, mice deficient in occludin develop chronic inflammation and hyperplasia, which suggests that the function of occludin is more complex than originally thought.⁴¹ ZO proteins regulate tight junction assembly and maintenance through anchoring occludin or claudins to the cytoskeleton. Disruption of the integrity of the tight junction results in dysfunction of the gut barrier and the diffusion of macromolecules from the intestinal lumen into the blood.

In addition to the barrier functions of intestinal epithelial cells and the substances they secrete as described above, the intestinal epithelial cells also perform immune surveillance and send signals to the mucosal immune system by producing cytokines, such as interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor (TNF)-α, and chemokines, including CXC-motif chemokine ligand (CXCL) 8, CXCL10, CC-motif chemokine ligand (CCL) 2, CCL6, CCL20 and CCL25.^19,42,43 The primary role of the cytokines/chemokines produced is to induce immune cell migration and promote innate and adaptive immunity. Notably, a subset of chemokines, including intestinal epithelium-derived CCL6 (human homologs CCL14 and CCL15) display antimicrobial properties.^44–47

2.3. Mucosal immune cells

The intestine possesses an integrated mucosal immune system. Gut-associated lymphoid tissue (GALT) is a prominent part of the mucosal-associated lymphoid tissue, and contains up to 70% of the immune cells of the whole human body.⁴⁸ The GALT harbors diverse immune cells, including dendritic cells, T and B lymphocytes, plasma cells, innate lymphoid cells (ILCs), macrophages and neutrophils.^49–51 While residential macrophages are responsible for phagocytosing bacteria diffused into the lamina propria,⁴⁹ ILCs protect the mucosa against bacterial invasion by secreting cytokines.^52–54 Of special importance are dendritic cells, which play a key role in shaping the intestinal immune response through their ability to orchestrate protective immunity and immune tolerance.⁵⁵ T and B lymphocytes which reside in the lamina propria are major effector cells in adaptive immune responses that are induced and directed by dendritic cells. T cells respond to signals from the gut lumen and initiate immune responses.^56,57 B cells, especially IgA-producing plasma cells, contribute to the protection of the gut barrier.^58,59 Of note, immune cells also play a role in regulation of the intestinal barrier function by secreting cytokines. For example, IL-22 produced by type 3 ILCs and CD4⁺ T cells has been shown to stimulate AMP secretion from the intestinal epithelial cells and upregulate epithelial tight junction proteins.^60,61 IL-10 secreted by regulatory T cells (Tregs) and macrophages promotes mucosal wound healing and enhances gut barrier function.^62–64

3. Gut barrier dysfunction and bacterial translocation in ALD

3.1. Alcohol-induced gut hyperpermeability

Gut permeability is a term describing the control of the passage of macromolecules through the epithelium into systemic circulation.⁶⁵ Macromolecules in the gut lumen are not able to penetrate into the blood when the gut barrier functions normally. However, under disease conditions, the gut barrier function is impaired or disrupted, which leads to uncontrolled passage of macromolecules. Multiple methods have been used to assess gut permeability, including in vivo assay of macromolecules penetrating from the gut to the blood, ex vivo assay of macromolecules passing through the mucosa of intestinal explants, and morphological and biochemical analysis of tight junction structure and/or proteins.^66–68 The in vivo gut permeability assay in humans is usually conducted by oral administration of a pair of molecules, an indigestible large molecule (i.e., lactulose) that penetrates through the mucosa only when the gut barrier is impaired and a small molecule (i.e., mannitol) that crosses the mucosa freely regardless of gut barrier function. The degree of gut permeability is reflected by the ratio of urinary excretion of these two molecules.

Alcohol-induced gut hyperpermeability has been well documented in both clinical and experimental studies.^69–73 In fact, the gut appears to be the first site of injury upon alcohol intoxication.^11,73 Patients with ALD showed a significant increase in gut permeability to a variety of permeability markers, such as lactulose/mannitol, polyethylene glycol (PEG) and ⁵¹Cr-ethylenediaminetetraacetic acid (⁵¹Cr-EDTA). In a clinical study, alcoholics with chronic liver disease demonstrated a marked increase in lactulose absorption as well as in urinary lactulose/mannitol ratio compared with alcoholics with no liver disease and nonalcoholics with liver disease.¹⁵ Another clinical study tested gut permeability to PEGs in patients with ALD, and found that urinary levels of PEGs and plasma endotoxins are significantly higher in patients with ALD than those in healthy controls.⁷⁴ Moreover, alcohol-induced gut hyperpermeability seems to be a persistent effect in ALD patients; elevated plasma ⁵¹Cr-EDTA could be detected in alcoholics with liver cirrhosis even after 2 weeks of abstinence, whereas it was more transient in healthy subjects and in alcoholics without cirrhosis.⁷⁵

As summarized in Fig. 1A, mechanistic studies suggest that alcohol breaches intestinal integrity at multiple levels. Acute alcohol intoxication may cause histopathological alterations, such as loss of the epithelial cells at the top of intestinal villi, both in vivo and in vitro.^76–78 Chronic alcohol exposure has been reported to reduce the distribution of tight junction proteins without significantly affecting intestinal histopathology in mice.⁷⁰ Colon biopsies from patients with ALD exhibited a reduction of ZO-1 protein levels in comparison with normal controls.⁷⁹ Animal studies demonstrated that ileal tight junction proteins, such as occludin and ZO-1 (Fig. 1B), are reduced in mice chronically fed alcohol.⁷⁰ A recent study showed that deficiency of occludin exacerbates alcohol-induced gut barrier dysfunction and liver damage in mice,⁸⁰ which provides direct evidence that disassembly/depletion of tight junction proteins is likely an important mechanism underlying alcohol-induced gut permeability increase. Alcohol promotes the disruption of intestinal tight junctions through multiple pathways, including induction of oxidative stress,⁸¹ elevation of microRNAs,^15,79 perturbation of circadian rhythm,⁸² and malnutrition.^70,83,84 Our group has reported that chronic alcohol exposure induces intestinal oxidative stress, which leads to zinc deficiency;⁷⁰ and that zinc deficiency may sensitize alcohol-induced disassembly of tight junctions through inactivating hepatocyte nuclear factor 4α.⁸⁵ Of note, alcohol metabolites, rather than alcohol itself, are believed to be more responsible for the deleterious effects caused by alcohol. Acetaldehyde, a major toxic metabolite of alcohol, is accumulated in the intestine after alcohol exposure,^83,84 and has been shown to reduce tight junctions and promote leakiness of Caco-2 cells.^86,87

A growing body of evidence has revealed that alcohol also affects the gut mucus layer and immune cells in addition to the epithelial junctions. The expression of MUC2 was decreased in the ileum of mice fed-alcohol for 8 weeks.⁸⁸ Recovery of the alcohol-reduced Akkermansia muciniphila population, a mucin-degrading bacterium that resides in the mucus layer, enhanced mucus thickness, and ameliorated experimental ALD.⁸⁹ However, another study showed that knockout of MUC2 ameliorates ALD in mice through adaptively upregulated Reg3β and Reg3γ.⁹⁰ This controversy necessitates further investigation into the function of MUC2 and its role in the pathogenesis of ALD. Several studies have reported that alcoholics have increased levels of IgA.⁹¹ A recent study further showed that alcohol causes increased IgA levels in tissue homogenates and decreased IgA levels in the intestinal contents, which suggests impaired secretion of IgA.⁹² Loss of IgA is not sufficient to promote the development of ALD in mice due to various compensations, such as increased levels of intestinal and plasma IgM.⁹³ Antimicrobial peptides, Reg3β and Reg3γ, are suppressed in the small intestine of mice after alcohol exposure.^94,95 Moreover, alcohol damages intestinal stem cells.⁹⁶ As stem cells are pivotal in intestinal cell proliferation and differentiation, dysfunction of intestinal stem cells may represent a key mechanism for alcohol-induced long-lasting damage.

3.2. Bacterial translocation in the pathogenesis of ALD

Bacterial translocation is the invasion of viable intestinal bacteria or microbial products through the gut mucosa to extraintestinal sites, such as the mesenteric lymph nodes, liver, spleen, and bloodstream. Translocation of these pathogens to the liver elicits an inflammatory cascade, oxidative stress, and consequently liver damage. One of the most well studied phenomena is endotoxemia in ALD. Elevated blood LPS levels are found both in ALD patients and animal models of ALD.^6–9 It has been reported that endotoxemia and gut barrier dysfunction are early events prior to the development of ALD,⁷³ and persist through to advanced stages of alcoholic cirrhosis.⁹⁷ The blood endotoxin levels correlate well with the severity of ALD and TNF-α levels,⁹⁸ and are higher in alcoholic cirrhosis compared with other stages of ALD.⁹⁷ Our previous work showed that orally administered LPS can be detected in the plasma of alcohol-intoxicated mice but not in the control mice,⁷⁶ which provides direct evidence indicating that alcohol increases gut permeability to endotoxin. Gut permeability assays have shown that alcohol intoxication increases permeability of the duodenum,⁹⁹ ileum,⁷⁰ proximal colon^100,101 and distal colon¹⁰¹ to macromolecules.

LPS in the systemic circulation activates hepatic Kupffer cells via Toll-like receptor 4 (TLR4) to produce inflammatory cytokines and chemokines which, in turn, attract neutrophils and monocytes to the liver.¹⁰² Engagement of TLR4 signaling in the liver has been repeatedly reported in both clinical and experimental studies of ALD. TLR4 is essential for the progression of alcohol-induced hepatic steatosis, inflammation and fibrosis.¹⁰³ Mice deficient in TLR4, CD14 or LPS binding protein that have a perturbed LPS-receptor complex are protected from alcohol-induced liver injury.^104–107 Moreover, the plasma endotoxin levels are comparable between TLR4 knockout and wild type mice,¹⁰⁵ which suggests that TLR4 signaling is not involved in the modulation of gut permeability. Fig. 2 summarizes the process of alcohol-induced bacterial translocation in the pathogenesis of ALD at the gut-liver axis.

Fig. 2. Gut-liver axis in the development of alcoholic liver disease.

(A) Schematic diagram of pathological alterations at the gut-liver axis after alcohol consumption. (B) Hematoxylin and eosin staining of the liver. Alcohol causes hepatic lipid accumulation (arrowheads) and inflammatory cell infiltration (arrows). Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; ROS, reactive oxygen species; PAMP, pathogen-associated molecular pattern.; PF, pair-fed; AF, alcohol-fed.

In addition to LPS, multiple microbial products can also translocate from the intestine to other organs after alcohol intoxication, and play a critical role in ALD progression. Bacterial DNA was elevated in the plasma of ALD patients.¹⁰⁸ Acute alcohol binge drinking resulted in increased bacterial 16S ribosomal DNA in correlation with serum LPS levels in healthy human volunteers.¹⁰⁹ Bacterial DNA is recognized by TLR9 and sensitizes to LPS-induced liver injury.¹¹⁰ Peptidoglycan, a component of gram-positive bacteria, is detected in human ALD patients,¹¹¹ and injected peptidoglycan deteriorated liver injury and inflammation in alcohol-fed mice.¹³

3.3. Intestinal dysbiosis and ALD

The intestinal commensal bacteria plays a major role in regulating the host immune response and maintaining the integrity of the gut mucosa.¹¹² Alcohol decreases short chain fatty acids (SCFAs) and branched chain amino acids, which are a source of nutrition for microbes, in the gastrointestinal tract.⁸⁴ Thus alcohol may directly and/or indirectly alter the composition of gut microbiota. Indeed, quantitative (bacterial overgrowth) and qualitative changes of the gut microbiome have been reported in ALD.^94,113 This review will only focus on the pathological relationship between intestinal dysbiosis and the onset of ALD. Detailed information regarding the alcohol-perturbed microbiome has been discussed in-depth in other reviews.^114–116 First, microbial-derived acetaldehyde may represent a mechanism of how microbiota participate in the development of ALD. As mentioned, acetaldehyde is known to disrupt the gut barrier through disassembling tight junctions.^86,87 Overgrowth of bacteria affects intestinal acetaldehyde levels which, in turn, elevates intestinal permeability.¹¹⁷ Oral administration of metronidazole to rats led to high levels of acetaldehyde in the intestinal lumen by increasing aerobic bacteria and reducing anaerobic bacteria in the intestine.¹¹⁸ Treatment with the antibiotic ciprofloxacin reduced colonic microbiota and prevented acetaldehyde accumulation.¹¹⁹ Second, alcohol induces the expansion of bacteria that may augment bacterial translocation under disease conditions. The Proteobacteria phylum includes Gram-negative bacteria, most of which are regarded as opportunistic pathogens, and is a major source of LPS. A proportional increase of Proteobacteria has been reported in ALD patients with liver cirrhosis as well as in mice chronically fed alcohol,^120,121 which suggests a causal link between alcoholic dysbiosis and endotoxemia as well as hepatic inflammation. Third, intestinal microbiota may directly mediate the development of ALD. A recent study showed that mice harboring intestinal microbiota from alcoholic hepatitis patients develop more severe liver inflammation, greater gut permeability, and higher bacterial translocation.¹²² Another study reported that alcohol increased intestinal Enterococcus spp. and translocation of Enterococcus spp. led to hepatic inflammation and hepatocyte death.¹²³ In addition to the aspects mentioned above, fungi, another part of the intestinal microbiome, may also mediate the pathogenesis of ALD. It has recently been reported that chronic alcohol consumption increased gut fungal populations in mice, and the subsequent translocation of fungal β-glucan induced liver inflammation.¹²⁴ These studies demonstrate that dysbiosis contributes to the development of alcoholic hepatitis and necessitates further mechanistic investigation.

4. Targeting the leaking gut to treat ALD

Efforts exploring potential ALD therapies have ongoing for decades, and one of the major focuses with promising findings is on sealing the leaky gut. As emphasized above, the pathophysiology of ALD is clearly linked with alcohol-induced gut barrier dysfunction. Indeed, animal studies demonstrated that neutralizing circulating endotoxin abrogates the endotoxin signaling cascade, and thereby attenuates alcohol-induced hepatic cytokine production, inflammatory cell infiltration and liver damage.¹²⁵ In contrast, knockout of either Reg3β or Reg3γ enhances bacterial translocation and promotes ALD progression in mice.⁹⁵ Therefore, further discussion on potential ALD therapies targeting alcohol-induced gut barrier dysfunction is merited. The discussion will focus on microbiota-based and nutrient-based treatments.

4.1. Microbiota-based treatments

4.1.1. Antibiotics

Experimental and pre-clinical studies indicate that treatment with antibiotics reduces Gram-negative bacteria and prevents ALD.^126–128 In a preliminary study involving a small number of ALD patients, administration of antibiotics (norfloxacin and neomycin) led to an improvement of the Child-Pugh score after three and six months of treatment.¹²⁹ However, due to the fear of antibiotic resistance and possible hepatic side effects, further studies investigating antibiotic treatment in patients with ALD are lacking. Rifaximin, a nonabsorbable antibiotic with broad spectrum antimicrobial activity, represents an alternative in treating ALD. Indeed, rifaximin is found to reduce endotoxemia resulting from intestinal decontamination and improves not only the prognosis of patients but also cirrhosis-related thrombocytopenia.^130–132

4.1.2. Prebiotics

In contrast to antibiotics, which kill or inhibit the growth of bacteria, the concept of using prebiotics and/or probiotics is to restore gut microbiota symbiosis, and warrants study as a potential treatment for ALD. Prebiotics are indigestible dietary polysaccharides that can only be digested by commensal microbiota, such as Bifidobacteria and Lactobacilli, to promote the growth of a subset of gut microbiota.¹³³ Dietary supplementation with oats mitigated alcohol-induced liver damage by improving gut permeability and reducing endotoxemia in rats.¹³⁴ In a separate study utilizing mice, administration of prebiotic fructooligosaccharides restored the host antimicrobial peptide Reg3γ, reduced bacterial overgrowth, and ameliorated steatohepatitis caused by alcohol.⁹⁴ Intake of lactulose by cirrhotic patients was reported to be effective in treating subclinical hepatic encephalopathy.¹³⁵ However, studies that explore the efficacy of prebiotics in ALD patients are somewhat limited.

4.1.3. Probiotics

Probiotics are live bacteria that are benign for the host, especially in regard to gut homeostasis. Studies of probiotic administration to ALD patients or rodents with ALD suggest that probiotics may improve the prognosis of ALD (summarized in Table 1). The first report was the study by Nanji et al.,¹³⁶ which showed that Lactobacillus GG supplementation reduced alcohol-induced endotoxemia and liver injury in rats. Since then, a number of probiotics, mainly Lactobacillus spp. and Bifidobacteria spp., have been tested in the context of ALD. A short-term 5-day therapy with Bifidobacterium bifidum and Lactobacillus plantarum 8PA3 to ALD patients lowered plasma alanine aminotransferase and aspartate aminotransferase levels, restored the gut microbiota, and improved ALD compared with patients treated with standard therapy (abstinence plus vitamins) alone.¹³⁷ Intake of Lactobacillus subtilis and Streptococcus faecium for 7 days reduced LPS levels and the severity of liver damage in alcoholic hepatitis patients.¹³⁸ In another study, Lactobacillus casei Shirota administration three times daily for 4 weeks reestablished microbiota balance and restored neutrophil phagocytic capacity in alcoholic cirrhotic patients.¹³⁹ In addition, a long-term study administering the probiotic VSL#3 for up to three months showed significantly reduced oxidative stress and cytokine production and improved liver function in patients with ALD.¹⁴⁰

Table 1.

Studies exploring the protective effects of probiotics/prebiotics against alcoholic liver disease.

Probiotic/Prebiotic	Subjects	Duration of treatment	Outcome	Reference	Year
Probiotics
Lactobacillus rhamnosus GG	Male Wistar rats	1 month	Probiotic feeding reduced alcohol-induced endotoxemia and liver injury.	Nanji et al.136	1994
A mixture containing 450 billion bacteria (VSL #3)	Alcoholic cirrhosis patients	3 months	Treatment of probiotic lowered plasma levels of cytokines and oxidative stress parameters.	Loguercio et al.140	2005
L. casei Shirota	Alcoholic cirrhosis patients	4 weeks	Probiotic supplementation restored neutrophil phagocytic capacity.	Stadlbauer et al.139	2008
Heat-killed L. brevis SBC8803	C57BL/6N mice	35 days	L. brevis SBC8803 ameliorated alcohol- induced liver injury and fatty liver.	Segawa et al.182	2008
Bifidobacterium bifidum and L. plantarum 8PA3	Male Russian adults	5 days	Patients treated with probiotics had significantly lower ALT and AST activity, and restored gut microbiota compared with patients treated with standard therapy alone.	Kirpich et al.137	2008
L. rhamnosus GG	Male Sprague-Dawley rats	10 weeks	L. rhamnosus GG reduced alcohol-induced gut leakiness and attenuated alcohol-induced oxidative stress and inflammation both in the intestine and liver.	Forsyth et al.72	2009
L. rhamnosus GG	Male C57BL/6N mice	Last 2 weeks of the 8-week feeding	L. rhamnosus GG supplementation reduced alcohol-induced endotoxemia and hepatic steatosis.	Wang et al.141,183	2011, 2013
L. paracasei	Male Fischer 344 rats	10 weeks	L. paracasei altered the fatty acid composition of the plasma and liver.	Komatsuzaki et al.184	2012
L. rhamnosus GG culture supernatant	Male C57BL/6N mice	5 days	Bacteria-free L. rhamnosus GG culture supernatant ameliorated acute alcohol-induced gut leakiness and liver injury.	Wang et al.185	2012
	Male Sprague-Dawley rats	Up to 8 weeks	Probiotic administration reduced plasma elevated-endotoxin levels caused by alcohol and altered gut microbiota.	Zhang et al.186	2012
Live or heat-killed VSL #3	Male rats	Up to 12 hours	VSL #3 administration reduced plasma endotoxin levels and cytokine production caused by alcohol exposure.	Chang et al.144	2013
Heat-killed L. casei MYL01	HepG2 cells	20 hours	L. casei MYL01 modulated proinflammatory cytokine production.	Chiu et al.187	2014
Escherichia coli Nissle 1917 secreting pyrroloquinoline quinone	Male Foster rats	10 weeks	Probiotic treatment ameliorated alcohol- induced oxidative damage and hyperlipidemia in rats	Singh et al.188	2014
Lactobacillus subtilis/Streptococcus faecium	Patients with alcoholic hepatitis	7 days	Oral supplementation with L. subtilis/S. faecium reduced E. coli in stool, and decreased the levels of LPS and TNF-α.	Han et al.138	2015
L. rhamnosus GG	C57BL/6 mice	11 days	L. rhamnosus GG upregulated ileal tight junction proteins, decreased E. coli protein in the liver, and balanced Treg and Th17 cells in peripheral blood.	Chen et al.143	2016
Prebiotics
L. rhamnosus GG or oats	Male Sprague-Dawley rats	10 weeks	Supplementation with L. rhamnosus GG or oats prevented alcohol-induced altered colonic mucosa-associated microbiota composition in rats.	Mutlu et al.189	2009
Fructooligosaccharides	Male C57BL/6J mice	3 weeks	Administration of fructooligosaccharides to alcohol-fed mice reduced bacterial overgrowth and ameliorated alcoholic steatohepatitis through partially restoring the host antimicrobial protein Reg3γ.	Yan et al.94	2011

Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; LPS, lipopolysaccharide; TNF-α, tumour necrosis factor-alpha; Treg cell, regulatory T cell; Th 17 cell, T-helper 17 cell.

Several potential mechanisms through which probiotics improve ALD symptoms have been proposed. Administration of probiotics can enhance liver function by reducing endotoxin levels, decreasing oxidative damage,¹⁴⁰ and improving immune response to enteric pathogens.¹³⁹ Lactobacillus GG supplementation in mice has been shown to increase intestinal tight junction expression, prevent gut leakiness, balance Treg and T-helper (Th) 17 cells, and attenuate hepatic TNF-α production as well as oxidative stress.^72,141–143 Notably, the beneficial effects of probiotics are achieved not only by live bacteria, but also by heat-inactivated bacteria or bacteria culture supernatant.¹⁴⁴ Importantly, a study employing 16S ribosome RNA sequencing revealed that Lactobacillus GG not only reduced bacterial overgrowth in alcohol-fed mice, but also prevented alcohol-induced expansion of the Proteobacteria and Actinobacteria phyla, which implicates the capacity of Lactobacillus GG in orchestrating gut microbiota symbiosis.¹²¹ A recent study reported that Akkermansia muciniphila, a Gram-negative commensal bacterium, is diminished both in mice and humans with ALD, and oral supplementation of A. muciniphila promotes intestinal barrier integrity and ameliorates experimental ALD.⁸⁹

Synbiotics, combinations containing both probiotics and prebiotics, represent another promising microbiota-based treatment for ALD. A recent study reported that Lactobacillus plantarum and a prebiotic for Lactobacillus plantarum, epigallocatechin gallate, synergistically reversed alcohol-induced endotoxemia and liver injury in rats.¹⁴⁵ Further pre-clinical and mechanistic investigations are needed to confirm the potency of synbiotics in ALD treatment.

4.1.4. Fecal microbiota transplant (FMT)

FMT is the process of transplantation of fecal material, which contains bacteria from a healthy individual, to a recipient.¹⁴⁶ It is an effective therapeutic approach addressing various conditions characterized by microbiota dysbiosis, such as ulcerative colitis.¹⁴⁷ The mechanism of FMT may involve the establishment of beneficial bacterial strains and production of antimicrobial components.¹⁴⁶ The first attempt utilizing FMT in treating ALD has just been published recently. In the study, feces from alcohol-resistant donor mice were transplanted to alcohol-sensitive recipient mice three times a week for three weeks, which resulted in prevention of alcohol-induced gut dyshomeostasis and hepatic steatohepatitis.¹⁴⁸

4.2. Nutrient-based treatments

Alcohol abuse is often associated with malnutrition.^149,150 Dietary supplementation is therefore a potent strategy for treating ALD. Related studies have shown that nutrient-based treatments not only improve the function of the liver per se, but also impact on the gut-liver axis through regulating the gut barrier function.

4.2.1. Zinc

Zinc is the second most abundant trace element in the body.¹⁵¹ It plays a vital role in maintaining physiological processes, such as metabolism, signaling transduction, and cell growth and differentiation.¹⁵² Hypozincemia (low serum zinc levels) and reduction of hepatic zinc levels have long been observed in patients with ALD, and serum zinc levels correlate positively with the severity of liver damage.^153,154 In a mouse model of ALD, acquired zinc deficiency occurs as early as after two weeks of alcohol feeding.¹⁵⁵ Our group has reported that in addition to serum and hepatic zinc decreases, alcohol-induced zinc deficiency could also be detected in the intestine, especially in the distal small intestine or ileum, which exacerbates gut barrier dysfunction caused by alcohol.⁷⁰ We also found that dietary zinc deficiency exaggerates alcohol-induced endotoxemia and liver damage.⁶⁹ These observations suggest an important role of zinc in regulating gut barrier function, and the potency and necessity of zinc supplementation in treating ALD.

Zinc supplementation has been shown to tighten the leaky gut under a variety of disease conditions. Patients with Crohn’s disease showed improved gut permeability after oral zinc sulfate supplementation.¹⁵⁶ Evaluation of the tight junction ultrastucture by electron microscopy demonstrated that dietary zinc supplementation in rats with colitis reduced the number of opened tight junction complexes in the colon epithelium.¹⁵⁷ The effect of zinc treatment on alcohol-induced leaky gut has been tested in both acute and chronic rodent models of ALD. In an acute model of alcohol intoxication, mice were treated with three doses of ZnSO₄ at 5 mg elemental zinc/kg in a 12-h interval prior to an oral dose of alcohol. Alcohol intoxication elevated plasma endotoxin levels and caused pathological liver changes, which were abrogated by zinc pretreatment.⁷⁶ Dietary zinc supplementation also attenuated alcohol-increased ileal permeability, restored the distribution of tight junction proteins, and mitigated hepatic endotoxin signaling in rats.¹⁵⁸ Therefore, zinc prevention of increased intestinal permeability contributes to the beneficial effects of zinc on alcohol-induced liver damage.

4.2.2. Niacin

Niacin, also known as nicotinic acid, is a naturally occurring B3 vitamin. A well-established role for niacin is as a broad-spectrum hypolipidemic drug.^159,160 It also exhibits potent antioxidant and anti-inflammatory properties.^161,162 Niacin is the precursor of nicotinamide adenine dinucleotide (NAD⁺), which plays a crucial role in energy metabolism, including ethanol/acetaldehyde clearance. Chronic alcohol abuse causes niacin deficiency in human, termed pellagra,¹⁶³ which may further worsen alcohol metabolism-perturbed redox imbalance. Supplementation with niacin has been reported to attenuate hepatic oxidative stress and reverse indices of ALD in mice.¹⁶¹ Studies by our group showed that niacin increases hepatic fatty acid oxidation and decreases de novo lipogenesis in rats.¹⁶⁴ Dietary supplementation with niacin also reversed alcoholic endotoxemia, and upregulated the expression of tight junction proteins, especially claudins.⁸³ Interestingly, niacin significantly lowered endotoxin and acetaldehyde levels in the intestinal lumen, which indicates a direct impact of niacin on gut microbiota.⁸³

An intimate interrelationship between niacin and zinc has been suggested for decades. Zinc participates in the regulation of niacin metabolism,¹⁶⁵ and zinc deficiency is associated with increased histidine oxidation and consequently pellagra,¹⁶⁶ whereas zinc supplementation is capable of increasing NAD⁺ concentrations.¹⁶⁵ In contrast, niacin may enhance zinc absorption and utilization.¹⁶⁷ Therefore, a possible synergistic effect of niacin and zinc should be taken into account when interpreting data generated by supplementation of either alone, and a combination of niacin and zinc may represent another possible therapy in ALD.

4.2.3. Fatty acids

Fatty acids differ in length of carbon chain (e.g., short, medium and long chain fatty acids) and degree of saturation (e.g., saturated, monounsaturated and polyunsaturated fatty acids). There are several lines of evidence suggesting that different types of dietary fatty acids may mitigate alcohol-induced intestinal barrier dysfunction, endotoxemia and liver injury.

Short-chain fatty acids (SCFAs) are fatty acids with fewer than six carbon atoms, and are produced by bacterial fermentation.¹⁶⁸ They are the main source of energy for colonic cells. Our group has reported that alcohol exposure dramatically decreased intestinal luminal levels of all SCFAs, except for acetic acid, in rats fed alcohol for eight weeks.⁸⁴ Supplementation with butyrate (tributyrin) mitigated alcohol-induced gut barrier disruption and liver injury both in acute and chronic alcohol-intoxicated mice.^169,170

The fatty acids in medium chain triglycerides (MCTs) are mainly saturated with carbon chain lengths ranging from 6–12. Studies have demonstrated that dietary MCTs improved alcohol-induced liver histological changes through multiple mechanisms involving both the liver and the intestine.^88,171–173 Kirpich and colleagues examined the therapeutic intervention against ALD using a diet enriched in saturated fats (MCTs: beef tallow) in mice. They reported that the saturated fat-enriched diet improves intestinal tight junction expression,¹⁷³ alleviates intestinal inflammation,⁸⁸ enhances MUC2 expression,⁸⁸ and modulates gut microbiome and metabolome in mice intoxicated by alcohol.¹⁷² In a separate study using rats, a MCT-enriched diet normalized alcohol-reduced intestinal tight junction proteins, occludin and ZO-1, prevented endotoxemia, and alleviated hepatic LPS signaling.¹⁷⁴

In humans and mice, alcohol abuse decreases the capacity of the microbiome to synthesize saturated long chain fatty acids (LCFAs), and reduces the proportion of Lactobacillus, a strain of bacteria known to metabolize saturated LCFAs.¹⁷⁵ Administration of saturated LCFAs to alcohol-fed mice increased Lactobacillus spp., enhanced gut barrier function, and reduced intestinal inflammation and liver injury. These findings indicate that alterations in bacterial metabolism contribute to the pathogenesis of ALD.¹⁷⁵ The protective roles of saturated LCFAs in the intestine may also involve stimulating release of gut hormones, including glucagon-like peptide (GLP)-1 and GLP-2,¹⁷⁶ regulating the production of MUC2 by goblet cells,¹⁷⁷ and enhancing antimicrobial activity.¹⁷⁸ Future studies are required to explore whether these mechanisms are involved in saturated LCFAs-mediated protection against ALD.

Although dietary interventions are the focus of the present review, other methods of targeting the leaky gut in ALD should not be ignored. For example, several reports have described the protective role of IL-22 in treating ALD in rodents.^179–181 In a mouse model of alcohol plus burn injury, alcohol augmented burn-induced IL-22 reduction and gut permeability increase; administration of IL-22 prevented these deleterious effects.¹⁷⁹ The same group further demonstrated that the IL-22 induced protection is through signal transducer and transcription activator 3-mediated upregulation of AMPs expression and reduction of gut Enterobacteriaceae.¹⁸¹

5. Conclusion

The significance of the gut-liver axis in the pathogenesis of ALD has been widely accepted, although the mechanisms remain largely unknown. Based on the observation that increased gut permeability is a leading cause of alcohol-induced endotoxemia and liver damage, modulation of the gut barrier seems to be a promising strategy. Investigations targeting the gut barrier have been conducted in pre-clinical and clinical settings to understand the relationships between alcohol, the gut barrier and liver damage. The long-term clinical benefits and safety of these treatments should be evaluated in a clinical setting involving ALD patients. Recent studies suggest that gut commensal bacteria play a pivotal role in shaping both the host immune response and the integrity of the gut barrier. Exploration of specific microbial strains that contribute or even control the progression of alcohol-disrupted barrier function as well as the underlying mechanism is challenging, but could provide potential new avenues of research for a better understanding of ALD progression.