The integrity of the gut barrier plays a key role in prohibiting harmful intestinal materials being translocated into the bloodstream (Figure 1). All patients with various degrees of ALD have disruption of the gut barrier (9, 10). The oxidative and nonoxidative metabolites of ethanol produced by gut bacteria and intestinal epithelial cells disrupt the gut barrier by degrading the tight junction proteins or destroying the interaction of claudin-1 and ZO-1 (11–13). In chronic alcohol feeding mice, enteric dysbiosis-related intestinal inflammatory response causes intestinal barrier injury, and the restoration of eubiosis using non-absorbable antibiotics inhibits intestinal inflammation and barrier dysfunction (14). However, researchers find that mouse colonized with harmful C. albicans and cytolytic E. faecalis has no effect on intestinal permeability when exposed to alcohol (6, 7). To sum up, the relationship between intestinal dysbiosis and barrier function needs further studies.

The gut vascular barrier (GVB) is another gatekeeper that prevent bacteria translocation across the gut to reach the portal vein. Salmonella typhimurium broke the GVB in a manner that is dependent on the declined Wnt/β-catenin signaling in gut vascular endothelial cells (15). In experimental cirrhosis and non-alcoholic fatty liver disease (NAFLD), farnesoid X receptor (FXR) agonists modulate the GVB to reduce bacterial translocation through driving β-catenin activation in endothelial cells (16, 17). Leakage of intestinal vascular endothelia has been observed in experimental ALD mice and patients with ALD (18, 19).

The intestinal bacteria are significantly altered in patients with ALD and experimental animals. At the phylum level, mice fed with alcohol diet have relatively higher abundances of Bacteroidetes and Verrucomicrobia when compared to mice fed with a control diet, whereas the mice fed with a control diet have a relative predominance of Firmicutes (20). However, alcoholics, with decreased communication of the microbial network in the colon (21) (Table 1), have the lower median abundances of Bacteroidetes and the higher median abundances of Proteobacteria than the healthy subjects (22). The commensal microbiota is exhausted in ALD patients with and without cirrhosis (23). At the species level, when compared with the healthy group, the numbers of Bifidobacteria, Lactobacilli, and Enterococci are significantly reduced in the alcoholics (24). Patient with alcoholic cirrhosis have 27 times more Enterobactericaea in their feces than healthy volunteers, and Enterobactericaea is the most common liver translocated bacterium in patients with cirrhosis (25).

The severity of ALD closely relates to the degree of intestinal flora alternations (26). Alcohol dependence is negatively correlated with levels of butyric-producing clostridium species (23). Severe patients with alcoholic hepatitis have increased Bacilli, Lactobacillales, Veillonella, and decreased Eubacterium_g23, Oscillibacter and Clostridiales in the fecal compared with the healthy controls (27). The reduced Akkermansia and increased Bacteroides are used to identify alcohol use disorder patients with an accuracy of 93.4% (28). Cytolysin-positive E. faecalis is correlated with the severity of liver disease and mortality in patients with alcoholic hepatitis (34). However, a research from Arun J Sanyal el at. points out that when compared with heavy drinkers, alcoholic hepatitis have more obvious microbiome characteristics, while the bacterial signature between moderate and severe alcoholic hepatitis is not differential (35).

Except for bacteria, gut microorganisms consist of fungi, archaea, and viruses. The role of intestinal fungi in ALD has caught researchers' attention recently. Compared with the control subjects, alcoholic patients have lower fungal species richness and diversity (31). An increased systemic immune response to fungi and their products is associated with increased mortality in patients with alcoholic hepatitis (32). The overgrowth of Candida, especially C. albicans, is observed in patients with ALD compared with non-alcoholic controls, where Penicillium is dominant in the gut of non-alcoholic controls (7, 31, 32). Moreover, the species of C. albicans are significantly decreased in alcohol abusers after 2 weeks of abstinence (33). In chronic ethanol diet feeding mice, the commensal fungus Meyerozyma guilliermondii is significantly increased compared with mice fed with control diet (36).

In addition to the direct effects of alcohol, several molecules have been identified as important factors that contribute greatly to alcohol-related intestinal barrier dysfunction. Hif1a knockout in mice intestinal epithelial cells results in a significantly decrease of the Firmicutes/Bacteroidetes ratio and the Lactobacillus level, exacerbating gut leakiness (38). Mucin 2, secreted by goblet cells, protects against pathogens penetrating the inner mucus layer. However, Muc2 knockout protects mice from alcohol-induced liver injury, prevents the intestinal bacterial overgrowth, and enhances the expression and activity of Reg3b and Reg3g (39). Mmp7^−/− mouse with a deficiency in active α-defensins of intestinal paneth cells promotes PAMP translocation and worsens the liver damage of alcohol-exposed mice (40).

Immune dysfunction is another important cause of alcohol-related intestinal barrier dysfunction. Intestinal flora is essential for maintaining the immune homeostasis of the gut-liver axis (41). On one hand, the innate and adaptive immune systems influence the component and diversity of intestinal microorganisms (42). On the other hand, gut microbiota has the potential to model intestinal immune responses in healthy and disease states (43). Alcohol feeding significantly upregulates the expression of proinflammatory cytokines interleukin-1 (IL-1) beta, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), high mobility group protein box-1 (HMGB-1), interleukin-17 (IL-17), interleukin-23 (IL-23), and inducible nitric oxide synthase (iNOS) in the intestine (44, 45). High TNF-α ruptures the tight junctions by phosphorylating the myosin light chain kinase (MLCK) of intestinal epithelial cells through TNF-receptor I (14). Mice fed with a chronic ethanol diet have decreased the level of fecal immunoglobulin A (IgA) but increased the systemic level of IgA increases compared with the control mice (46). Binge-on-chronic alcohol reduces the number and maturation of mucosa-associated invariant T cells in mice intestines (47). Gut high permeability and dysbiosis result in the highly compromised antibacterial defense of mucosa-associated invariant T cells in patients with ALD (48). Ethanol-induced dysbiosis lower type 3 innate lymphoid cells' production of interleukin-22 (IL-22), and restore IL-22 produced by engineered bacteria in the intestine protecting mice from ethanol-induced steatohepatitis (49). In addition, IL-22 activates the hepatic signal transducer and activator of transcription 3 (STAT3), decreases the hepatic expression of fatty acid transport protein, and ameliorates alcohol related liver inflammatory injury and hepatic oxidative stress (50).

The translocation of intestinal bacteria and their constituent parts, such as peptidoglycan, LPS, flagellin, and CpG DNA, play an important role in ALD progression. Most studies have reported a change in LPS. LPS translocation through the leaky intestine results in circulating endotoxemia and aggravates alcohol-induced liver inflammation through TLR4 signaling in liver (51). Activation of the LPS-TLR4 signaling pathway promotes the release of pro-inflammatory factors, such as TNF-α and IL-6. Hepatocyte TLR4 deficiency decreases lipogenic genes expression, enhances fatty acid oxidation, and reduces inflammatory genes expression of white adipose tissue, then prevents mice from alcohol-induced liver injury (29). Chronic ethanol exposure sensitizes hepatic macrophages to LPS and enhances liver inflammatory damage through TLR4 signaling (52). The absence of MyD88, one of TLR4 signaling adaptor, prevents the progression of hepatic steatosis and inflammation in chronic ethanol-exposed mice (53). However, TLR7-mediated signaling suppresses hepatic injury, steatosis, and inflammation of chronic binge ethanol fed mice (54). In addition, TLR9 signaling protects chronic alcohol exposure mice from hepatic oxidative stress but worsens hepatic inflammation.

Increased exposure to bacterial exotoxins and reduced toxin clearance in the liver also aggravate alcohol-related liver injury and inflammation. Cytolysin from E. faecalis, which caused hepatocyte death, are correlated with the severity of liver disease and mortality in patients with alcoholic hepatitis (34). The lack of α1-2-fucosylation in the intestine might contribute to increase the cytolytic E. faecalis in chronic ethanol exposed mice (55). Chronic ethanol feeding impairs the hepatic clearance of translocated pathobionts due to the reduced complement receptor of immunoglobulin (CRIg) expression in Kupffer cell (56).

Fatty acid metabolism is significantly altered in ALD. Short-chain fatty acids (SCFA), the fermented product of dietary fiber, are the energy source for intestinal epithelial cells and for maintaining barrier integrity (57). The 16S rRNA gene and whole genome shotgun metagenomic analysis have showed the damage of acetyl-coenzyme A (CoA) butyrate synthesizing pathway in butyrate-producing bacterial genera which caused a decreased intestinal level of butyrate by chronic ethanol feeding (58). Tributyrin can inhibit ethanol-induced intestinal barrier and liver injury (59, 60). Saturated long-chain fatty acids are decreased in ethanol intragastric mice, and maintaining the levels of saturated fatty acids in the intestine promote commensal Lactobacillus growth and stabilize gut barrier (30). The n3-polyunsaturated fatty acids (PUFAs) can attenuate experimental ALD through decreasing neutrophil chemoattract (61).

Bile acid homeostasis is disturbed in ALD. Gut bacteria modulate bile acid metabolism as bile acid diversity is lower and the proportion of taurine-conjugated bile acids is increased in germ-free rats compared with conventional subjects (62). Chronic alcohol administration induces the high expression of choloylglycine hydrolase that is responsible for the deconjugation of bile acid in mice bacteria and the increased levels of unconjugated bile acids, such as cholic acid and muricholic acid in the ileum and plasma, compared with control mice (63). Persons with chronic alcohol consumption have increased bile acid synthesis and bile acid pool in the liver (64). The bile-acid receptor TGR5 maintains biliary homeostasis, as TGR5 deficiency mice have intestinal microbiota dysbiosis, higher plasma and liver levels of secondary bile acids, and greater liver steatosis and inflammation when fed with ethanol diet than WT mice (65). The apical sodium-dependent bile salt transporter (ASBT) inhibitor GSK2330672 attenuates the liver injury of chronic plus binge alcohol mouse by decreasing intestinal bile acid accumulation, and increasing hepatic CYP7A1 expression (66). Restoring bile acid homeostasis by a proliferator-activated receptor-delta agonist seladelpar (MBX-8025) could reduce chronic ethanol-induced liver damage and improve ethanol-associated dysbiosis (67).

The FXR plays an important role in energy metabolism and bile acid synthesis, and thus are extensively studied in ALD. The importance of FXR is different in the gut and the liver in ALD. Intestine-specific FXR knockout mice are more susceptible to ethanol-induced liver steatosis and inflammation compared with WT mice (68, 69). The intestine-restricted FXR agonist fexaramine mitigates hepatic injury and inflammation of chronic ethanol-fed mouse (63). However, FXR deletion in hepatocytes has no effect on the severity of steatosis, inflammation, or liver fibrosis in chronic plus binge alcohol feeding mice, although slight liver lipid deposition and collagen accumulation are increased (70). Nevertheless, an administration of ursodeoxycholic acid (UDCA) attenuates NF-kB activation and inflammatory infiltrations in the liver of FXR knockout mice exposed to ethanol (71). These demonstrate the significance of FXR interaction with gut microbiota in the pathophysiology of ALD.

Aromatic amino acids are important factors to maintain the homeostasis of intestinal flora, among which tryptophan is fully studied in ALD. Tryptophan and tryptophan-derived metabolites are reduced in the fecal and serum of alcoholic hepatitis patients with cirrhosis when compared with nonalcoholic controls, despite that the ability of microorganisms to synthesize tryptophan has improved (72). In ALD animal experiment, Lactobacillus rhamnosus GG derived exosome enriched with bacterial metabolites of tryptophan can improve intestinal barrier function through AhR signaling that promotes IL22 production by intestinal immune cells (73). Alcohol-induced intestinal dysbiosis reduces intestinal IL-22 production and indoce-3-acetic acid (IAA) levels. IAA supplementation, as a microbial-derived ligand of AhR, protects mice from ethanol-induced liver steatohepatitis by preventing bacterial translocation to liver (49). Intestinal epithelial cell-specific Ahr knockout exacerbates ethanol-induced liver injury through promoting the translocation of Helicobacter hepaticus and Helicobacter ganmani to liver (74). Ficz (6-formyllindolo (3, 2-B) carbazole), an AhR agonist, reduces ALD liver injury with similar effects to prebiotics. Moreover, alcohol-fed Ahr knockout mice eliminates the beneficial effects of prebiotics (75).

The 1,3-β-glucan from the overgrowth of fungi in chronic alcohol-fed mice binds to the C-type lectin domain family 7 member A (CLEC7A) of Kupffer cells and promotes liver inflammation (31). Patients with alcoholic hepatitis have an intense immune response to fungus, as serum anti–Saccharomyces cerevisiae antibodies are significantly high compared to patients with alcohol use disorder and nonalcoholic controls (32). Candidalysin positive C. albicans exacerbate ethanol-induced liver injury not dependent on the further impairment of intestinal barrier function through Candidalysin, but dependent on directly cytotoxic to hepatocytes (7). The commensal fungus M. guilliermondii induced alcoholic hepatic steatosis is probably due to translocated β-glucan increasing PGE₂ production in the liver (36).

Comparing with patients treated with the standard of care, fecal microbiota transplantation from family members attenuates the disease severity and improves the survival rate of severe patients with alcoholic hepatitis (76). In mice models, fecal microbiota transplantation from alcohol-tolerant donor mice to alcohol-sensitive recipient mice can correct alcohol-induced dysbiosis and prevent alcohol-induced liver injury (77).

Many probiotic therapies have been carried out in human patients with ALD and experimental ALD mice where they have received inspiring results. Patients with alcoholic hepatitis who receive 7 days of Lactobacillus subtilis/Streptococcus faecium have reduced gut-derived microbial LPS and TNF-α level (78). Different species of Lactobacillus, such as Lactobacillus plantarum (79), Lactobacillus acidophilus (80), Lactobacillus fermentum (81), and L. rhamnosus GG (82) are all reported to protect against alcohol-induced liver injury through improving intestinal barrier function, modulating gut bacteria, and balancing T_reg and T_H17 cells in peripheral blood of mice. Akkermansia muciniphila supplementation decreases ethanol-induced gut leakiness and hepatic injury (83). L. plantarum LC27 and Bifidobacterium longum LC67 inhibit the activation of NF-κB mediated by LPS, restore the disturbed intestinal flora, and ultimately reduce alcoholic steatosis in mice (84). Bacillus subtilis relives alcohol-induced liver damage by reducing bacterial endotoxin translocation and liver inflammation (34). A new strain of Pediococcus pentosaceus alleviates ethanol-induced liver injury by increasing the abundance of bacteria that produce SCFAs and strengthening tight junctions of intestinal epithelial cells (85). Faecalibacterium prausnitzii and potato starch supplementation attenuate chronic-binge ethanol-induced liver injury by increasing propionate abundance in mice cecum and mitigating the losses of SCFA transporter in the proximal colon (86). VSL#3 treatment prevents intestinal bacteria and their products from spreading to portal circulation and downregulates liver inflammation mediated by TNF-α (87).

Saccharomyces cerevisiae var. boulardii which is anti-carcinogenic, antibacterial antiviral, antioxidant, and able to reduce serum cholesterol level, has been used for treating various gut-related diseases (88). Saccharomyces boulardii administration attenuates acute liver injury (89). Besides, S. boulardii administration attenuates hepatic steatosis, low-grade inflammation, and changes the gut microbiome (90). Hanseniaspora osmophila, Lachancea thermotolerans, and S. cerevisiae strains are proved to have the most potential as health-promoting probiotics (91). However, it has also been reported that Clostridium difficile colitis and neutropenic patients have S. cerevisiae fungemia after treatment with S. boulardii as probiotic (92, 93). For future treatments, doctors should concern about the potential risk when prescribing fungal probiotics, especially to immunocompromised patients.

Some amino acids, fatty acids, and probiotic fermentation are found to alleviate alcoholic liver disease. Ethanol feeding exhausts protein thiols, raises oxidized protein thiols in mice gut, while glutamine complement can attenuate the protein thiol oxidation of distal colonic mucosa (94). What's more, glutamine prevents the ethanol-induced disruption of the tight junction by EGFR-dependent mechanism (95, 96). L-cysteine attenuates acetaldehyde-induced transepithelial electrical resistance (TEER), and inhibits the ROS injury of Caco-2 cells (11). Tributyrin supplementation has been found to attenuate both acute and chronic-binge ethanol induced intestinal leakage and liver damage (60, 97, 98). For chronic alcohol intragastric mice, supplementation with saturated fatty acids can enhance the intestinal barrier and reduce alcohol-induced liver injury (30). The fermentation broth of the mixture of Pueraria lobata, Lonicera japonica, and Crataegus pinnatifida by L. rhamnosus 217-1 is reported to alleviate alcohol-induced intestinal microbiome disorders, and reduce oxidative stress and inflammatory signals in the liver (99).