MICROBIOTA REPROGRAMMING FOR TREATMENT OF ALCOHOL-RELATED LIVER DISEASE

Abstract

In the past decade knowledge has expanded regarding the importance of the gut microbiota in maintaining intestinal homeostasis and overall health. During this same time, we have also gained appreciation for the role of the gut-liver axis in the development of liver diseases. Alcohol overconsumption is one of the leading causes of liver failure globally. However, not all people with alcohol use disorder progress to advanced stages of liver disease. With advances in technology to investigate the gut microbiome and metabolome, we are now beginning to delineate alcohol’s effects on the gut microbiome in relation to liver disease. This review presents our current understanding on the role of the gut microbiota during alcohol exposure, and various therapeutic attempts that have been made to reprogram the gut microbiota with the goal of alleviating alcoholic-related liver disease.

INTRODUCTION

Burden of Alcohol-related liver disease

Alcohol is the seventh leading risk factor for death and disability adjusted life years (DALY) world-wide.¹ Alcohol use is very common globally with 38% of the adult population reporting alcohol intake within the preceding 12 months of this survey, while in the United States 70% of the population reported consuming alcohol.^2–4 Approximately 1 in 12 adults report heavy alcohol consumption, which is associated with more than 200 disease states.^3,5

Alcohol ingestion can cause both direct and indirect liver toxicity through multiple metabolic and biochemical derangements.⁶ Overconsumption of alcohol causes a broad range of histopathologic changes in the liver, ranging from simple hepatic steatosis, alcoholic steatohepatitis, and varying degrees of fibrotic changes, to alcoholic liver cirrhosis and hepatocellular carcinoma.^7,8 Alcoholic hepatitis is associated with high morbidity and mortality, and liver cirrhosis is identified as the 12^th leading cause of death in the United States, of which 48% of cases are alcohol related.^9–11 The economic burden of alcohol-related liver diseases is substantial, estimated to be several billion dollars annually.^12–14 Despite being a huge economic burden, therapeutic options for the management of alcohol-related liver disease (ARLD) is limited to alcohol abstinence and supportive care, and liver transplantation is the only definitive curative therapy for liver failure.^15,16 Ethical dilemma surrounding liver transplantation for ARLD highlights the need for continued exploration of novel therapeutic options for this patient population.¹⁶

PATHOPHYSIOLOGY OF ALCOHOL-RELATED LIVER DISEASE

Alcohol-induced pathologic changes in liver are categorized mainly as alcoholic fatty liver (hepatic steatosis), alcoholic steatohepatitis, alcoholic fibrosis and cirrhosis, and hepatocellular carcinoma.^6,7 Steatosis is the earliest manifestation of alcohol-induced liver injury. Both through direct and indirect effects, alcohol not only stimulates lipogenesis in the liver, but simultaneously it also inhibits fatty acid metabolism.^6,17–20 Alcohol consumption upregulates sterol-regulatory element-binding protein-1c (SREBP-1c), a transcription factor involved in fatty acid synthesis in the liver.²¹ While this direct effect is mediated by the first alcohol metabolite acetaldehyde, alcohol also stimulates SREBP-1c indirectly via activating other processes which upregulate SREBP-1c, such as lipopolysaccharide (LPS) signaling through toll-like receptor 4 (TLR4) and tumor necrosis factor (TNF)-alpha.^19,21–29 Mice deficient in SREBP-1c showed decreased fatty changes in the liver with significantly lower triglyceride accumulation.³⁰ Alcohol also inhibits hepatic metabolism and oxidation of fatty acids by inhibiting the activity of peroxisome proliferator-activated receptor (PPAR)-α, a major hormone receptor involved in the transportation and oxidation of fatty acids in the liver.³¹ Alcohol also inhibits autophagy, a process which plays a key role in removal of lipids from hepatocytes.^32–34

Alcoholic steatosis can further advance to alcoholic hepatitis which is characterized by active inflammatory infiltration of hepatic parenchyma resulting in hepatocellular injury.⁶ Acetaldehyde induces generation of reactive oxygen species, in combination with depletion of mitochondrial glutathione and S-adenosylmethionine, induces hepatocyte injury and hepatocyte apoptosis.^6,35 Alcohol-induced gut dysbiosis and increased intestinal permeability results in increased LPS – TLR4 activity in the liver, leading to the generation of proinflammatory cytokines, such as TNF-α, further increasing oxidative stress. This is accompanied by alcohol-mediated complement system and Kupffer cell activation, and the resulting hepatic immune system dysregulation ultimately results in hepatocellular injury.²² Alcoholic hepatitis is characterized by neutrophil and T lymphocyte infiltration.⁶ Acetaldehyde, LPS, and TNF-α promote chemokine production, activate hepatic stellate cells (HSCs), and increase IL-8 and CXCL1 production which stimulates neutrophil infiltration.³⁶ With ongoing hepatic injury, activated HSCs increase the production of extracellular matrix, including portal fibroblasts and myofibroblasts.¹⁵ These proinflammatory triggers can lead to HSC and myofibroblast producing collagens, non-collagenous glycoproteins, proteoglycans, and glycosaminoglycans up to 10-fold compared to normal liver tissue, which results in the development of fibrosis in patients with chronic hepatic injury and inflammation.¹⁵ Interestingly, only a subset of people with alcoholic use disorder progress beyond hepatic steatosis to the more severe stages of ARLD, suggesting other contributing factors to ARLD development.

ROLE OF GUT MICROBIOTA IN PATHOPHSIOLOGY OF LIVER DISEASE

Global research efforts over the past decade indicate the importance of the gut microbiota in supporting health.^37–40 The gastrointestinal (GI) tract harbors the majority of the body’s microbiota, and its disruption is associated with many disease states including liver disorders.^39,41–43 Gut microbial alterations are linked with alcoholic and non-alcoholic liver disorders, both early and advanced stages of liver disease.^44–46 Recent compelling evidence indicates a potential role of the gut microbiota in the pathogenesis of Non Alcoholic Fatty Liver Disease (NAFLD) in several animal models and human clinical studies.^47–49 Chiu et al. studied the importance of gut microbial composition by inoculating feces from healthy controls and patients with Non-alcoholic steatohepatitis (NASH) into germ-free mice.⁵⁰ They found that gut microbiota from patients with NASH aggravated hepatic steatosis and inflammatory responses when these mice were fed a high-fat diet.⁵⁰ Similarly, restoration of a healthy gut microbiota normalized portal hypertension in a rat model of NASH.⁴⁷ Although more studies are needed to establish a pathophysiological pathway, it is apparent that the gut microbiota plays a complex intermediary role, impacting the interaction between environmental factors and the host, possessing the capability of modulating pathological processes in liver diseases.

RELATIONSHIP BETWEEN ALCOHOL AND GUT MICROBIOTA IN ALCOHOL-RELATED LIVER DISEASE

Alcohol-induced gut dysbiosis dates back several decades with one of the first studies by Bode et al. who utilized bacterial culture methods to explore this phenomenon.⁵¹ Noting that plasma endotoxin increased after alcohol ingestion, their team laid a foundation for the hypothesis that gut-derived endotoxins might play a role in the pathophysiology of alcohol-induced liver disease.⁵¹ With the development of advanced methods for microbiota characterizations such as RNA gene sequencing and DNA-PCR (polymerase chain reaction), newer studies confirmed that alcohol induced gut dysbiosis and this was associated with alcohol-induced endotoxemia.^52–55 Increased endotoxemia essentially reflects increased circulating LPS, the components of the cell wall of gram-negative bacteria. Hepatocytes play a major role in clearing LPS from systemic circulation with its eventual excretion in the bile.⁵⁶ TLR4-deficient mice were found to have lower degrees of alcohol-induced inflammatory changes in the liver, supporting the notion that LPS binding to TLR4, expressed by hepatocytes, Kupffer cells, and hepatic stellate cells, mediates proinflammatory pathways in the liver. Alcohol-induced disruption of the intestinal epithelial barrier is believed to be mediated in part by effect of alcohol metabolites on the gut microbiota.^22,56,57

Several animal studies have reported characteristic changes in the gut microbiota in response to alcohol.^52,53,58,59 Yan et al. investigated a mouse model of intragastric feeding and compared the impact of alcohol feeding in contrast with an isocaloric diet on the gut microbiota, and reported that 3-weeks of alcohol feeding induced intestinal bacterial overgrowth.⁵² An overall decrease in Firmicutes and an increase in Bacteroidetes abundance was noted, with depletion of Lactobacillus, Pediococcus, Leuconostoc, and Lactococcus abundances.⁵²

In one of the first human studies, Mutlu et al. used non-culture methodologies to analyze the changes in the gut microbiota via mucosal biopsy in chronic alcoholics with and without liver dysfunction, and compared it with healthy controls.⁵⁴ Alcoholics with dysbiosis had decreased Bacteroidetes and higher Proteobacteria abundances.⁵⁴ Despite being a robust study, their inability to detect clearer differences were attributed to a relatively lower sample size and a specific focus on mucosa associated bacterial composition which does not take into account the overall fecal microbiota composition.⁵⁴

DRINKING PATTERN, GUT MICROBIOTA AND EFFECTS ON LIVER

Several preclinical studies highlight the interaction between the gut microbiota and liver.^60–62 Chen et al. created an acute-binge alcohol exposure model and utilized germ-free and conventional C57BL/6 mice to compare its impact on the liver.⁶⁰ Evaluating changes in the hepatic parenchyma 9 hours after the binge ethanol episode (3 g/kg), they reported that healthy gut microbiota in the conventional mice was hepatoprotective against alcohol-induced acute liver injury compared to germ-free mice, which exhibited increased hepatic steatosis and upregulation in genes involved in fatty acid and triglyceride synthesis.⁶⁰ Canesso et al. studied the impact on liver after 7 days of alcohol administration (10% v/v) in drinking water with an oral alcohol gavage (5 mg/kg) on day 7 in germ-free and conventionalized germ-free mice. Germ-free mice had a lesser degree of liver injury and neutrophil infiltration, and did not exhibit intestinal permeability compared to conventionalized mice.⁶¹ The conventional mice treated with alcohol exhibited intestinal bacterial overgrowth and dysbiosis, particularly enterobacteria, compared to the conventional control mice.⁶¹ These studies highlight the dynamic role played by the gut microbiota in development of alcohol-induced liver injury.⁶² While normal gut microbiota have protective effects against acute alcohol ingestion, liver injury resulting from frequent serial ingestion of alcohol was likely mediated by alcohol-induced gut microbial dysbiosis.

DIFFERENT PATTERNS OF GUT DYSBIOSIS IN THE SPECTRUM OF ALCOHOL-RELATED LIVER DISEASES

Alcoholic Steatohepatitis

Several studies have identified characteristic dysbiotic gut microbial patterns in severe alcoholic steatohepatitis.^63–65 Patients with severe alcoholic steatohepatitis reportedly exhibit a gut microbial composition with higher abundance of Streptococci, Enterobacteria and Bifidobacteria, and a lower abundance of Clostridia taxa and Faecalibacterium prausnitzii.^44,63,66 Ciocan et al. noted differences in the distinct pattern of dysbiosis between healthy alcoholic controls, patients with severe alcoholic steatohepatitis and chronic alcoholic pancreatitis.⁶⁵ While severe alcoholic steatohepatitis and chronic alcoholic pancreatitis groups showed a higher degree of dysbiosis compared to healthy alcoholic controls, patients with severe alcoholic steatohepatitis particularly showed a higher abundance of Haemophilus parainfluenzae (Pasteurellaceae).⁶⁵ Chen et al. noted previously that higher abundance of Pasteurellaceae was associated with higher mortality from acute-on-chronic liver failure.⁶⁷ Smirnova et al characterized the gut microbial ecology in patients with alcoholic hepatitis (mild and moderate) and compared it with heavy drinkers without liver disease and healthy controls.⁶⁸ Microbial taxa identified by 16S pyrosequencing was distinct between heavy drinkers and those with alcoholic steatohepatitis, but no significant differences were noted between the alcoholic hepatitis severities.⁶⁸

Grander et al. reported that Akkermansia muciniphila abundance reduced with increasing severity of alcohol-induced liver injury and was noted to be lowest in patients with alcoholic steatohepatitis.⁶⁹ Llopis et al. studied hepatic inflammation in germ-free mice after they received transplanted stool from alcoholic patients with and without hepatitis.⁶³ After a 5-week period of similar food and alcohol intake, hepatic inflammation and necrosis was more severe in the mice receiving fecal transplant from the patient with severe alcoholic steatohepatitis.⁶³ These mice showed a higher infiltration of CD45+ lymphocytes which was associated with a higher percentage of CD3+, CD4+, CD8+ and natural killer T cells.⁶³ Llopis et al. noted that individual susceptibility to alcoholic steatohepatitis was also driven by intestinal microbiota.⁶³ Transplantation of fecal matter from alcoholic patients without hepatitis into mice that had received fecal matter from patients with severe alcoholic steatohepatitis improved plasma ALT levels and decreased liver regeneration, steatosis and overall inflammation scores.⁶³ These findings suggest that normalization of gut microbiota can potentially counteract alcohol-induced liver lesions, and this effect is independent of frequency and amount of alcohol intake.⁶³

Alcohol-related liver disease without Cirrhosis

Leclareq et al. investigated the gut microbiota in patients with alcohol-related liver disease who did not have significant fibrosis (e.g., F0 and F1 on fibroscan).⁷⁰ They found that 26 out of 60 (43%) patients had elevated intestinal permeability when measured by radioactive probe ⁵¹Cr-EDTA method, while the intestinal permeability was within normal range for remaining 57% (n=34) of the patients. Interestingly, patients with alcohol-related liver disease and increased gut permeability had drastically decreased abundance of Faecalibacterium prausnitzii. Increased intestinal permeability was also associated with a lower abundance of Ruminococcaceae taxa, which reversed after a 3-week period of abstinence and detoxification, but abstinence had no impact on the abundance of F. prausnitzii.⁷⁰ Decreased microbial diversity and higher intestinal permeability was also associated with a higher degree of depression, anxiety and alcohol craving.⁷⁰

Alcohol-related liver disease with Cirrhosis

Several studies have analyzed the gut microbiota of cirrhotic patients and identified specific microbial patterns.^66,71,72 Differences in the degree of dysbiosis is noted for different etiologies and disease severity of cirrhosis.^71,72 Qin et al. reported that in both healthy controls and patients with cirrhosis, that while the most dominant phylotype was Bacteroides, its abundance was significantly decreased and Streptococcus and Veillonella species were more abundant in patients with cirrhosis compared to healthy controls.⁷¹ Bajaj et al. reported that patients with alcoholic-related cirrhosis have a higher degree of dysbiosis compared to patients with cirrhosis for other indications, and this dysbiosis seems to persist despite abstinence.⁷² Dubinkina et al. compared the metagenomic composition of the gut microbiota of alcohol dependent patients without cirrhosis with actively drinking patients with alcoholic cirrhosis.⁷³ While both groups had decreased gut microbial diversity, a distinctively higher dysbiosis (greater number of genera and species changes) was noted in patients with alcoholic cirrhosis. They also noted a significant depletion of major commensals from the Bacteroidales order and a simultaneous rise of taxa normally inhabiting the oral cavity, including Lactobacillus salivarius, Veillonella parvula, and Streptococcus salivarius.⁷³

Bajaj et al. developed the Cirrhosis Dysbiosis Ratio (CDR) which is calculated based on the ratio of autochthonous to non-autochthonous taxa.⁷² Beneficial autochthonous bacteria included Lachnospiraceae, Ruminococcaceae and the Clostridiales Family XIV Incertae Sedis, while potentially pathogenic taxa were Enterobacteriaceae and Bacteroidaceae.⁷² In this study of 244 subjects with cirrhosis and age-matched healthy controls, healthy controls had the highest CDR. Patients with a lower CDR, indicating a degree of dysbiosis, had a stronger association with organ failure and 30-day mortality.⁷² The CDR was lowest in patients with alcoholic cirrhosis, which correlated with a higher abundance of gram-negative Enterobacteriaceae and higher degree of endotoxemia.^72,74

GUT MICROBIOTA REPROGRAMMING

Co-evolving with their host, gut microbes are vital for the development of a healthy gut. A healthy gut microbiome is involved with normal GI tract functions including digestion, absorption, immune function, and pathogen exclusion. Beneficial gut microbes also synthesize vital compounds for the host such as vitamins, enzymes, and short-chain fatty acids, the fermentation byproducts of indigestible dietary polysaccharides. Crosstalk between the gut microbiota and the host supports a mutualistic relationship and functional stability of the complex gut ecosystem.

The basis for treatment of ARLD is total alcohol abstinence, yet despite achieving this goal, a subset of ARLD patients (5–15%) will progress to advanced stages of liver disease, fibrosis and cirrhosis. Aside from genetic factors, environmental factors are thought to contribute to ARLD progression. As the gut microbiota is important in maintaining homeostasis, microbiota reprogramming as a means to correct gut dysbiosis and restore defects in the functions of the gut microbiota as a potential therapy for ARLD is appealing. Here we review current efforts to date using antibiotics, prebiotics, probiotics, synbiotics and fecal microbiota transfer as a means to reprogram the gut microbiome and mitigate ARLD.

Antibiotics

Alcohol use disorder is associated with overgrowth of gram-negative bacteria in the gut and increased systemic endotoxin.^{51,53,54,75,76} As gut microbial-derived endotoxin is a known driver of liver Kupffer cell activation and ARLD pathophysiology, an approach to decrease translocation of microbial components is to decrease the microbial burden in the gut with non-absorbable antibiotics.^77,78 A preclinical model tested whether two nonabsorbable antibiotics (neomycin and polymyxin B sulfate) would be hepatoprotective during chronic alcohol feeding in rats.⁷⁸ While alcohol induced overgrowth of gram negative bacteria in stool, serum aspartate aminotransferase and plasma endotoxin levels, and hepatic lipid accumulation responses in rats co-treated with the antibiotics were dampened and similar to those in the controls. The same antibiotics were also tested in the last 4 weeks of an 8-week alcohol feeding mouse model.⁷⁹ Antibiotic treatment protected mice from alcohol-induced intestinal inflammation and permeability. Few studies have investigated provision of antibiotics as a means to treat ARLD. With bacterial overgrowth noted in patients with alcoholic cirrhosis, provision of non-absorbable antibiotics has been particularly targeted at this patient population. A small study tested the effects of six months of antibiotics (neomycin and norfloxacin) on improving intestinal motility, small intestinal bacterial overgrowth (SIBO) and liver function in patients with alcoholic cirrhosis.⁸⁰ Compared to the placebo-controlled patients, the patients receiving antibiotics demonstrated improved small intestinal contractility and decreased SIBO incidence at 6 months, and improved liver function per Child-Pugh scores at 3 and 6 months. Rifaximin is a broad-spectrum non-absorbable antibiotic which reduces endotoxin and inflammation, but does not appear to grossly alter gut microbial composition.⁸¹ A study tested the efficacy of rifaximin on thrombocytopenia and blood endotoxin levels in patients with biopsy-proven alcoholic cirrhosis.⁸² Compared to patients randomized to receive no treatment, increased platelet counts were significantly correlated with decreased endotoxin levels in those receiving rifaximin for 4-weeks, and this coincided with decreased serum IL-1, IL-6, and TNF-α levels. Rifaximin, both water soluble and water-insoluble forms, are currently being tested in clinical trials for multiple indications of liver failure.⁸³

Alcoholic hepatitis is an inflammatory liver injury that carries a 30% mortality rate in those that do not respond to corticosteroids.⁸⁴ A current randomized prospective Phase I clinical study is evaluating the additional role of an oral antibiotic (ciprofloxacin 500 mg twice daily) to prednisolone therapy on patient mortality (28-day, 3- and 6-month).⁸⁴ A randomized double-blinded controlled Phase III clinical study is testing the effect of 30-day prednisolone treatment with or without antibiotics, amoxicillin and clavulanic acid, on 2 month survival in patients with alcoholic hepatitis.⁸⁵

While antibiotics can dampen deleterious bacterial overgrowth, its therapy is not without consequences. Antibiotic treatment decreases gut microbial community taxonomic richness, diversity, and evenness, producing a shift to an alternative state that is different from the baseline.⁸⁶ While in most humans, gut microbial communities partially but not fully, recover following antibiotic cessation. Rather, it seems a new configuration similar to the original one results, and that these responses are highly variable amongst individuals. How the effects of antibiotics on gut microbial community structure alters gut microbial functions in humans remains uncertain. Concern for complications related to long-term antibiotic therapy, such as antibiotic resistance and hepatic side-effects, dampen the use of this means to reprogram the gut microbiota.

Prebiotics

A prebiotic is commonly a natural or synthetic polysaccharide that escapes host digestion. Through its metabolization by microorganisms in the gut, a prebiotic modulates the composition and/or activity of the gut microbiota, thus conferring a beneficial physiologic effect on the host.⁸⁷ A prebiotic is selectively utilized by a restricted group of microbes (e.g., Lactobacillus, Bifidobacteria) rather than substantial portions of the gut microbiota.⁸⁸ The most commonly studied prebiotics are soluble fibers, inulin, fructooligosaccharides (FOS) and galatooligosaccharides, human milk oligosaccharides, and resistant starches.

In a mouse model of continuous intragastric chronic alcohol feeding, mice developed liver injury with steatosis and early stages of fibrosis, which coincided with an increased abundance of intestinal Bacteroidetes and Verrucomicrobia compared with control mice that had a relative abundance of Firmicutes bacteria.⁵² Gene expression of regenerating islet-derived 3 beta (Reg3b) and gamma (Reg3g) were reduced in the small intestine of mice fed alcohol for 1- or 3 weeks as compared to control mice. Similarly, duodenal biopsies of patients with alcohol use disorder still actively drinking also had decreased Reg3g levels compared to healthy controls. When mice were supplemented with FOS during alcohol-feeding, the antimicrobial peptide Reg3b protein, but not Reg3g protein, was maintained to near control levels, and this was linked with decreased intestinal bacterial overgrowth and liver injury.

A rat model in which alcohol was gavaged twice daily for 10 weeks, tested whether intestinal and liver injury induced by alcohol could be mitigated by daily supplementation with oats (10 g/kg).⁸⁹ Oats contain many nutrients including vitamins, minerals and essential fatty acids, as well as beta-glucan, a fermentable fiber with prebiotic effects.⁹⁰ Supplementation with oats did reduce liver injury, gut permeability and endotoxemia. A follow-up study used a similar rat alcohol feeding model with oat supplementation provided for 12-weeks to evaluate intestinal oxidative tissue damage.⁹¹ Oat supplementation prevented alcohol-induced upregulation of inducible nitric-oxide synthase (iNOS), nitric oxide overproduction in the colonic mucosa, and increases in protein carbonyl and nitrotyrosine levels. This protection was linked with preservation of intestinal actin cytoskeleton and tight junction protein assembly.

Lactulose, a non-digestible and non-absorbable synthetic disaccharide consisting of galactose and fructose, is a prebiotic substrate found to increase Bifidobacterium, Lactobacillus and bacterial metabolites (e.g., short-chain fatty acids).⁹² Low doses are shown to have prebiotic effects in healthy people.⁹² Lactulose has long been a treatment for hepatic encephalopathy at doses of 60–100g which induces malabsorption with excessive diarrhea and decreases blood ammonia levels, however, the prebiotic effects of this dose are unknown. Nonetheless, lactulose treatment combined with a low protein diet has been shown to be an effective treatment in patients with subclinical hepatic encephalopathy with respect to psychometric tests.⁹³ Studies exploring efficacy of prebiotics in ARLD patients are limited and warrant further investigation.

Probiotics

A probiotic is a live, nonpathogenic microorganism that when consumed in adequate amounts, provides a health benefit on the host.³⁸ The most commonly studied probiotics contain Lactobacillus and bifidobacteria, and they are the species most commercially available. Mechanisms of action of probiotics are strain specific. Proposed mechanisms include enhancement of the intestinal epithelial barrier, increased probiotic adhesion to the intestinal mucosa, enrichment of the mucus layer, inhibition and exclusion of pathogen colonization, production of antimicrobial substances, enhancement of the immune system, and suppression of intestinal inflammation.⁹⁴

Animal Models

Investigations into the role of probiotics in protecting against ARLD in animal models dates back to 1994 with a study that showed Lactobacillus rhamnosus GG (LGG) supplementation reduced alcohol-induced endotoxemia and liver injury in rats.⁹⁵ LGG supplementation also attenuated ARLD in mouse models of chronic-alcohol exposure.^96,97 Protective effects of LGG included preservation of protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), attenuation of TLR4 and TLR5 mRNA levels, and reduced TNFα expression in the liver.⁹⁷ LGG supplementation also prevented alcohol-induced reductions of hypoxia-inducible factor 2α (HIF-2α), vascular endothelial growth factor (VEGF), and intestinal trefoil factor protein levels in the ileum, which was associated with higher tight junctional protein level expression of zonulen occludins-1 (ZO1), occludin, and claudin-1 mRNA levels, and occludin protein levels in the ileum.⁹⁶ Two soluble proteins, p75 and p40, were shown to be released from LGG and to promote intestinal epithelial homeostasis through specific signaling pathways.⁹⁸ Thus, in addition to viable LGG, heat-inactivated LGG and LGG supernatant have been tested in mouse models of chronic alcohol exposure and shown to improve intestinal barrier, balance Treg and T-helper (Th) 17 cells, reduce TNFα and oxidative species production, and mitigate liver injury.^97,99–101 By utilizing 16S ribosomal RNA (16S rRNA) sequencing, LGG has been shown to reduce chronic alcohol-induced gut dysbiosis in mice, and prevent expansion of Proteobacteria and Actinobacteria phyla, which coincide with lower plasma endotoxin, fecal pH, hepatic inflammation and injury.⁵⁸ This demonstrates the capacity of LGG to reprogram the gut microbiome skewed by chronic-alcohol in mice. Rather than testing a single strain probiotic, the multi-strain probiotic cocktail, VSL#3®, was tested in a rat model of short-term (3 days) high dose alcohol (5 g/kg) gastric feeding.¹⁰² VSL#3® contains 8 strains of live lyophilized microbes (Bifidobacterium breve, B. longum, B. infantis, L. acidophilus, L. plantarum, L. paraasei, L. bulgaricus, and Streptococcus thermophilus). Provision of VSL#3® (viable or heat-killed) 30 minutes prior to alcohol exposure prevented elevations in plasma endotoxin and TNFα, and preserved mRNA and protein expression of tight junctional proteins, ZO-1 and occludin.

Human Studies

Hospitalized patients with alcoholic hepatitis were randomized to receive both Lactobacillus subtilis and Streptococcus faecium (n=60) versus placebo (n=57) for 7 days immediately following 7 days of standard alcohol detoxification treatment.¹⁰³ While both patient groups showed improved liver function tests following 7 days of detoxification, the probiotics group showed significant differences (day 1 to 7) in serum albumin and TNFα, and stool Escherichia coli compared to the control group. Patients with alcoholic psychosis admitted to a psychiatric hospital were randomized to receive both B. bifidum and L. plantarum 8PA3 or standard care (abstinence plus vitamins) for 5 days.¹⁰⁴ Following treatment, the probiotic supplemented group had higher numbers of bifidobacteria and lactobacilli compared to the control group, and this coincided with lower liver enzyme activities. L. casei Shirota provision 3 times daily for 4 weeks was studied in patients with alcoholic cirrhosis (n=12) and compared to placebo (n=8) and in healthy controls (n=13) to determine the effect of supplementation on neutrophil oxidative burst, phagocytosis, TLR expression, and plasma cytokines.¹⁰⁵ At the end of the study period, cirrhotic patients receiving probiotic supplementation had restored plasma neutrophil phagocytic capacity compared to those receiving the placebo. Long-term treatment (3 months) with VSL#3® was tested in patients with alcoholic liver cirrhosis and was shown to significantly improve oxidative stress, cytokine production, and liver enzymes.¹⁰⁶

Postbiotics

Primary and secondary metabolites that are formed due to the direct or indirect metabolism of prebiotics have been correlated with many health benefits in humans. Short-chain fatty acids (SCFA) have ≤ 6 carbons and are produced by the gut microbiota due to the fermentation of carbohydrates, amino acids, and other nutrients that escape digestion and absorption in the proximal small intestine. The predominant SCFAs produced are acetate, propionate and butyrate, in a nearly constant molar ratio of 60:25:15, acetate:propionate:butyrate.¹⁰⁷ Although the least abundant SCFA, butyrate appears the most dynamic. Butyrate provides energy for the colonocyte, improves gut barrier function, aids in water and electrolyte absorption, inhibits activation of the transcription factor NF-kB which decreases expression of inflammatory cytokines and provides a consequent anti-inflammatory effect, and as an inhibitor of histone deacetylase, butyrate also affects gene expression.¹⁰⁸ Absence of butyrate in the intestinal tissue is associated with apoptosis, inflammation, and mucosal atrophy.^107,109 Higher acetate:butyrate ratios are associated with colonic pathology including colon polyps and tumors.¹¹⁰

Alcohol metabolism occurs primarily in the liver, but may also be metabolized in the intestine and by the gut microbiota. Through the alcohol dehydrogenase (ADH) and Cytochrome P450 2E1 (CYP2E1) pathways, alcohol metabolism generates acetaldehyde, which is further metabolized to acetate by aldehyde dehydrogenase. In humans, 70–80% of oxidized ethanol appears as free acetate in the hepatic vein.¹¹¹ Acetate redistribution from hepatic to peripheral tissues occurs during ethanol metabolism when saturating conditions are reached for acetate metabolism.^112,113 Rats fed chronic alcohol exhibited depleted intestinal levels of SCFA, except for acetate.¹¹⁴ Recently shown was that in addition to reduced SCFA producing bacteria (Lachnospiraceae and Ruminococcaceae), fecal SCFA were decreased in patients with alcoholic hepatitis.⁶⁸

Tributyrin is a structured lipid with butyrate esterified to glycerol at the 1, 2, and 3 positions. It is neutral, chemically stable, and rapidly hydrolyzed by pancreatic and gastric lipases to glycerol and butyrate. Cresci, et al investigated the role of tributyrin supplementation in multiple mouse animal models of alcohol exposure.^115,116 During chronic alcohol exposure (25 days, 32% of kcal), tributyrin supplementation provided daily in a liquid diet protected against alcohol’s effects on the disorganization of tight junctional proteins in the proximal colon, however it did not prevent liver injury assessed by elevations in the liver enzyme, alanine aminotransferase (ALT), and liver triglyceride accumulation.¹¹⁵ However, when tributyrin was supplemented daily as an oral gavage, mice exposed to alcohol for short-term (2 days, 32% of kcal), binge (single dose of 5 g/kg), and chronic-binge (27% total kcal for 10 days plus 5 g/kg on day 11) demonstrated preserved assembly of intestinal tight junctional proteins as well as decreased liver injury and inflammation.^115,116 The direct effect of butyrate in protecting the intestinal epithelial barrier during alcohol exposure was further demonstrated in vitro.¹¹⁶ Further investigation found tributyrin also preserved intestinal immune responses, mitigated oxidative stress, and maintained the integrity of the vasculature within the colonic villi disrupted by chronic-binge alcohol exposure in mice.¹¹⁷ In an effort to determine epigenetic effects of butyrate on hepatic protection during alcohol exposure, Donde, et al utilized a mouse model of chronic alcohol exposure (4 weeks, 27% kcal) and also supplemented a group of mice daily with tributyrin (2 g/kg, 5 days/week) via oral gavage; and treated primary rat hepatocytes with alcohol (50 mM) and butyrate (2 mM).¹¹⁸ They found alcohol exposure inhibited hepatic carnitine palmitoyltransferase-1 (CPT-1) expression by interfering with the interactions of key regulatory proteins on DNA via epigenetic mechanisms. Tributyrin supplementation prevented these alcohol-mediated changes and reduced fat accumulation and liver injury. Taken together, these data testing the role of tributyrin/butyrate during alcohol exposure support the role of the gut microbiota and its fermentation byproducts in protecting gut-liver injury during alcohol exposure. Further study into whether supplementation of SCFA, particularly butyrate, could be an effective therapy for ARLD is warranted.

Synbiotics

A synbiotic is a physical combination of prebiotics and probiotics. When attempting to reprogram the gut microbiome, a synbiotic is an appealing therapeutic approach. Individuals in need of gut microbiome reprogramming may not be consuming the adequate food substrates to support the growth and viability of a probiotic supplement; and likewise, if a prebiotic is provided, the targeted gut microbes may not be represented in a skewed gut microbiota. Thus, providing a synbiotic may overcome these physiologic and clinical situations.

A microstructured synbiotic consisting of Lactobacillus plantarum MTCC 2621 and epigallocatechin gallate, a phenolic compound and L. plantarum prebiotic, was tested in a chronic alcohol-exposure study in rats.¹¹⁹ Synbiotic-supplemented rats had significantly lower levels of blood alcohol, endotoxin, and liver enzymes and protected hepatoarchitecture compared to the controls and rats supplemented with the individual synbiotic components. Due to the protective effects of tributyrin supplementation on protecting the gut-liver axis disrupted by alcohol^115–117, a synbiotic designed to physiologically target butyrate was developed and tested in a mouse model of chronic-binge alcohol exposure.^120,121 Butyrate-producing bacteria are a part of the Firmicutes phyla, which contains butyrate-producing bacteria and whose abundance is diminished with alcohol exposure. A targeted synbiotic consisting of Faecalibacterium prausnitzii, a gram-positive, butyrate-producing and anti-inflammatory microbe, and a resistant starch (potato starch), which upon fermentation yields butyrate⁸⁸ was tested in a mouse model of chronic-binge alcohol exposure.^120,122 Daily supplementation with the butyrate-targeting synbiotic protected tight junctional protein expression and butyrate transporter expression in the proximal colon, as well as diminished hepatic oxidative stress and inflammation.¹²⁰ Upon further analysis, synbiotic supplementation during alcohol exposure prevented diminished adherens junctional protein losses in hepatocytes and reduced expression of endothelial barrier proteins in the liver.¹²²

A synbiotic consisting of 7 probiotics (Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus salivarius, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium longum) and 3 prebiotics (inulin, FOS, GOS) was provided daily for 8 weeks in 20 male subjects that were classified as high risk drinkers using the alcohol use disorders identification test (AUDIT).¹²² At the end of the study period, subjects demonstrated significant improvements in total AUDIT score, as well as levels of gamma glutamyl transferase, lipopolysaccharide, and immunoglobulin A from baseline levels.

Taken together, these data highlight a role for synbiotic supplementation as a therapeutic approach to reprogram the gut microbiome disrupted by alcohol as a means to mitigate ARLD. Further studies elucidating the ideal synbiotic composition and mechanistic targets for ARLD treatment are warranted.

Challenges in utilization of Pre- and Probiotics:

Despite extensive literature supporting the health benefits of prebiotics and probiotics, their utilization has been rather very limited for ARLD. Several barriers preclude their large-scale use into clinical practice.¹²³ Many studies are of low quality and vary with formulation, dosage, and therapy duration. For the probiotics demonstrating efficacy in the literature, the majority are not commercially available. Of importance, is that probiotic mechanisms of action are strain specific, not species specific. Most commercially available probiotics do not list the designated strain, but only the species, which could lead to clinician over-generalization of the product and the lack of clinical efficacy. In most countries, including the United States, these products are mainly marketed as food, dairy or dietary supplements-type of products, and labeling of the probiotic strain is not required by labeling laws and is left to the manufacturer discretion to include.¹²⁴ In the United States nutritional supplements, including pre-and probiotics, are not regulated by the Food and Drug Administration (FDA) and therefore are not intended for therapeutic treatment as drugs.¹²⁵ They are currently regulated by Center for Food Safety and Applied Nutrition, and therefore, no mention of disease or illness is allowed when marketing of these products.^124,126 However, due to recent advances in this area, the U.S. FDA has started taking initiatives to develop regulations for probiotics which show potential of being utilized as therapeutic drugs.¹²⁷ While such an initiative by FDA would address the clinician’s concerns regarding the safety and quality of pre-and probiotics, it might also increase their cost. In addition to that, insurance coverage of these products may continue to be a barrier.

Fecal Microbiota Transplantation

Fecal microbiota transplantation (FMT) involves transplanting fecal material containing the full array of components in the gut luminal content (e.g., microbes, bacteriophage, antimicrobial substances, postbiotics) from a healthy donor into a recipient.¹²⁸ FMT represents the most comprehensive and effective means of therapeutically reprogramming the gut microbiome. FMT has consistently achieved cure rates of >90% for refractory Clostridioides difficile infection. FMT is also being tested in other gastrointestinal and non-gastrointestinal conditions, showing promising results.¹²⁸

A preclinical study of alcohol exposure (27% kcal for 10 days) tested the effectiveness of fecal transfer from mice resistant to alcohol-induced liver injury into mice that were sensitive.⁶⁴ The FMT mitigated liver steatosis and inflammation, and restored gut microbial homeostasis similarly to prebiotic supplementation (pectin) in mice sensitive to alcohol-induced liver injury. Current treatment guidelines for patients with severe alcoholic hepatitis is corticosteroid therapy, however many patients do not respond to this treatment and their mortality rate is high. An open-label study of FMT in patients with steroid-resistant alcoholic hepatitis (n=8) was performed.¹²⁹ Stool from several donors was transferred through a nasojejunal tube daily for 7 days; and patients were followed up for 1 year. Compared to a patient cohort that did not receive stool transfer, FMT resulted in reduced Proteobacteria and increased Firmicutes levels one year after transfer. This was associated with improved overall survival (87.5% versus 33.3%). Another open-label study evaluated the effects of corticosteroids, nutritional support, pentoxyfylline, or FMT on male patients with severe alcoholic hepatitis on the gut microbiota, as well as the incidence of infections, inflammation, oxidative stress, and their clinical outcomes.¹³⁰ Patient survival at the end of 1 and 3 months in the steroids, nutrition, pentoxifylline, and FMT group were 63%, 47%, 40% and 75% (p=0.179) and 38%, 29%, 30%, and 75% (p=0.036), respectively. In the FMT group, improved gut microbial patterns, infections, inflammation and oxidative stress coincided with improved clinical outcomes. These data support the notion that gut microbial reprogramming with FMT may be a potential therapeutic approach for patients with severe non-responding alcoholic hepatitis. However, FMT is not without risk, and further blinded, targeted clinical trials are needed.

FUTURE DIRECTIONS

Bacterial engineering

Rationale engineering of probiotics with the aim to directly alter the functional readout of the microbiome to achieve a benefit to the host is gaining interest. This approach may introduce non-native pathways in probiotics through heterologous expression of enzymes. Rather than yield general health benefits on the host, this approach aims to deliver targeted benefits.¹³¹ This concept has been applied to various probiotic strains to yield various byproducts and host benefits to include: Esscherichia coli Nissle 1971 to delivery glucagon-like-peptide 1 in rats to restore insulin sensitivity¹³², and Lactococcus lactis to produce anti-inflammatory cytokines for low grade colonic inflammation.¹³³ In the context of ARLD, the probiotic L. reuteri was engineered to produce IL-22, and supplemented in mice during chronic-binge alcohol feeding.¹³⁴ Overexpression of the antimicrobial peptide, regenerating islet-derived 3 gamma (REG3γ), protects mice against alcohol-induced liver injury, and REG3γ is regulated by IL-22, but alcohol feeding reduces IL-22 production in mice. In this study, L. reuteri/IL-22 supplemented mice had reduced inflammation, bacterial translocation to the liver, and liver damage which coincided with upregulated intestinal expression of REG3γ.

Bacteriophages

A bacteriophage is a type of virus that kills bacteria by injecting its own DNA into bacterial cells. The recent discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated nuclease 9 (Cas9) system combined with bacteriophages has introduced strategies to target the microbiome with precision. Bacteriophage are much less harmless than antibiotics, and have been tested in terminally ill patients with multidrug resistant infections of Pseudomonas aeruginosa and Acinetobacter baumannii.^135,136 Multiple biotech companies are currently conducting FDA-regulated clinical trials in an effort to potentially bring this modality into mainstream medicine.¹³⁷ As specific pathogenic gut bacteria have been shown to infect and penetrate the intestinal epithelium and translocate to the liver and trigger ARLD, phage therapy is being evaluated for patients with primary sclerosing cholangitis.¹³⁸ Another milestone study by Duan et al. investigated the therapeutic effectiveness of bacteriophages in humanized mice colonized with bacteria from the feces of alcoholic hepatitis patients.¹³⁹ Their specific target was cytolytic E. faecalis because in their multiple stage study the presence of this bacteria was shown to be correlating with severity of liver disease and mortality in patients with alcoholic hepatitis.¹³⁹ This effect was mediated by cytolysin which is an exotoxin produced by these bacteremia which hepatotoxic properties. They found that the precise reprogramming of intestinal microbiota was possible with phage treatment targeting cytolysin-positive E. faecalis which was able to abolish alcohol induced liver injury and steatosis.¹³⁹ Mice treated with bacteriophages had lower levels of ALT and hepatic triglycerides, lower percentage of transferase uridyl nick end labelling (TUNEL)-positive hepatic cells, and lower oil red O-staining.¹³⁹ Although further human clinical studies are needed to test the effectiveness of phage treatment for ARLD, in the near future we will see more studies employing “phage cocktails” designed to specifically target particular bacterial strains, but leaving the healthy gut microbiome intact.

SUMMARY

Here we focused on strategies used to reprogram the gut microbiota disrupted by alcohol exposure as a preventative or therapeutic strategy for ARLD. Earlier studies did not have access to the “omic” technology currently available which is needed to identify specific targets for reprogramming. Thus, as successful manipulation and disease mitigation relies on extensive knowledge about the gut microbiota, the multiple metabolic pathways and metabolites, and the cross-talk with the host, gut microbial reprogramming is in its infancy. Past and current efforts show great promise for gut microbiota reprogramming in ARLD. Future research should build upon this fundamental understanding of the gut microbiota and host interactions in order to harness this evolving field as a therapeutic option for ARLD.