The importance of microbes for xenobiotics metabolism was underscored by a study that demonstrated an increase in hepatic expression of ethanol metabolizing genes in germ-free mice, and exacerbation in hepatic steatosis . Non-alcoholic and alcoholic liver diseases are characterized by increased luminal and circulating levels of ethanol and its metabolites, acetaldehyde and acetate . These metabolites have independently been associated with liver damage . Acetaldehyde has been implicated in weakening the intestinal tight junctions, compromising the gut barrier and enabling translocation of microbial products . It has also been associated with down regulating the expression of antimicrobial peptides in the intestine , and eliciting inflammatory and adaptive host immune responses . Additionally, alcoholic liver disease is marked by reduced levels of intestinal butyrate , which is linked to weakening of intestinal tight junctions and, hence, permeability .NAFLD refers to a spectrum of liver disease that can be broadly classified into two categories: nonalcoholic fatty liver , the non-progressive form of NAFLD, and NASH, the progressive form of NAFLD . NASH is generally linked to type 2 diabetes mellitus, cardiovascular risk factors and obesity , although NAFLD has also been reported in lean individuals, emphasizing that genetic and environmental factors also contribute to disease development . Patients with NAFLD have a higher prevalence of SIBO and microbial dysbiosis . Using 16S amplicon sequencing, Boursier et al. found that the bacterial genera, Bacteroides and Ruminococcus were substantially increased, and Prevotella was reduced in patients with NASH compared to those without NASH. Loomba et al. utilized whole-genome metagenomics to characterize the gut microbiota in patients with NAFLD with and without advanced fibrosis and showed an increased abundance of Escherichia coli and Bacteroides vulgatus in patients with advanced fibrosis. An enrichment of Escherichiawas also seen in paediatric patients with NASH compared with children with obesity but without NASH .

Consistent with preclinical studies,growing blueberries in pots these studies indicate an association between Gram-negative bacteria and progression of liver fibrosis . Genetically modified mouse models have been used to study NAFLD-associated gut dysbiosis and permeability for mechanistic insights in liver disease progression. Rahman et al. used JAM-A -knockout mice to demonstrate that deficiency in this tight junction protein with a diet rich in saturated fats, fructose and cholesterol leads to increased intestinal permeability and liver inflammation. This inflammation could be alleviated by administering antibiotics, underscoring the importance of microbial translocation in promoting immune response in the liver. Another group used mice deficient in Muc2and found that there was a compensatory increase in intestinal levels of antimicrobial protein-coding genes, Reg3b and leading to an overall protective response against NAFLD . The contribution of liver-damaging inflammation in response to translocation of microbes and MAMPs has been elucidated . Using inflammasome-deficient mouse models , Henao-Mejia et al. conclude that there is an accumulation of MAMPs in portal circulation, which enhanced the expression of hepatic TNF, thereby promoting liver inflammation and NASH progression. Furthermore, cohousing inflammasome-deficient mice with wild-type controls exacerbated diet-induced hepatic steatosis and obesity in healthy cage mates, suggesting transferability of disease via the microbiota. Increasing links between NAFLD and the gut microbiome at both the observational and mechanistic levels make the gut microbiota an attractive source of biomarkers for early diagnosis of NAFLD. In a comparison between children with obesity with and without NASH, Zhu and colleagues observed markedly elevated gut microbial production of ethanol in those with NASH. Adults with NAFLD also show increased serum TMAO and hepatic bile acid synthesis , and decreased production of phosphatidylcholine . Furthermore, Loomba et al. observed differences in carbon and amino acid metabolism in gut microbiome of patients with NAFLDassociated advanced fibrosis . This proof-of-concept study provides preliminary evidence to support the utility of a microbiome-derived metagenomics signature to detect advanced fibrosis as well as candidacy for anti-fibrotic treatment trials in NAFLD.

The manifestation of ALD in patients who chronically abuse alcohol is a consequence of multifactorial interactions involving genetics, immune system, gut microbiome and environmental factors . Like NAFLD, the non-progressive form of ALD is characterized by accumulation of fat inside the liver , whereas the progressive form is marked by inflammation and liver injury . Our understanding of the compositional and mechanistic contributions of the gut microbiota in ALD is improving. As in NAFLD, SIBO has been demonstrated as an important hallmark of alcohol-associated liver disease in humans and mouse models . Intestinal dysbiosis in individuals who abuse alcohol is characterized by marked enrichment of Enterobactericaeae and reduction in abundances of Bacteroidetes and Lactobacillus. It has also been demonstrated that alcohol-induced dysbiosis is only partially reversible by alcohol withdrawal or probiotic treatment . Interestingly, patients dependent on alcohol also displayed reduced fungal diversity and Candida overgrowth, presenting the first evidence of the role of gut mycobiome in pathogenesis of liver diseases . Genetically modified murine models have advanced our mechanistic understanding of the contribution of various components of the gut-barrier in the etiology and progression of ALD. Using Reg3b-/- or Reg3g-/- mice, it was found that REG3 lectins protected against alcoholic steatohepatitis by reducing mucosa-associated microbiota, thereby preventing translocation of viable bacteria . Muc2-deficient mice were protected against alcohol-induced liver inflammation due to a compensatory increase in Reg3g and Reg3b lectins . Furthermore, IgA-knockout in mice led to increased levels of IgM and a net protective effect against ASH progression . In response to ethanol-induced gut-barrier dysfunction and translocation, TLRs and other PRRs activate hepatic Kupffer cells and macrophages, as was demonstrated in male Wistar rats . This step initiates inflammatory cascades releasing TNF, IL-1, IL-10, IL-12 and TGF-β . Using TLR4 chimeric mice, it was shown that endotoxin-induced release of TGF-β is mediated by a MyD88-NF-κB-dependent pathway, providing an explanatory mechanism for endotoxin-induced liver inflammation . Furthermore, increased translocation of fungal β- glucan also induced liver inflammation via CLEC7A receptor on hepatic Kupffer cells such that treatment of mice with antifungal agents reduced intestinal fungal overgrowth, decreased β-glucan translocation and ameliorated ethanol-induced liver disease .

Alongside immunological responses to barrier dysfunction, ALD is also marked by systemwide changes in many bio-active compounds. Alcohol consumption leads to an increase in hepatic bile acid synthesis in humans and mice .This increase could be explained by dysbiosis associated disruption in FXR activation in enterocytes as FXR-deficient mice were more likely to develop ethanol-induced steatohepatitis , and treatment with an FXR agonist had protective effects against liver damage . Alcohol-associated dysbiosis in mice was further linked to reduced LCFA biosynthesis such that LCFA supplementation restored eubiosis. In fact, a statistically significant correlation between Lactobacillus spp. and bacterial LCFA was found in patients with ALD but not in healthy individuals as controls . Butyrate production was also negatively altered following ethanol exposure and administration of butyrate in the form of tributyrin mitigated alcohol-induced liver injury in mice .With increasing evidence of mechanistic links between the gut microbiota and liver disease progression, fecal microbiota transplantation is being explored as a therapeutic option for ALD . However, larger, carefully designed trials across multiple ethnic groups are needed before FMT can be considered safe in routine clinical practice for managing ALD.Cirrhosis is an extreme manifestation of chronic liver injury characterized by loss of liver cells, thick fibrous scar and regenerating nodules; this topic has been extensively reviewed elsewhere so we only provide a brief discussion here . NAFLD, ALD, primary biliary cholangitis , primary sclerosing cholangitis or hepatitis can each progress to cirrhosis and constitute its subtypes. ASH and NASH have emerged as the second and third leading causes of cirrhosis in adults in the USA and based on the etiology there is a variable risk of developing HCC . Alterations in the gut microbiota including dysbiosis and SIBO have been associated with and its complications . Treatment for portal systemic encephalopathy and decompensated cirrhosis includes treatment with nonsystemic antibiotics such as rifaximin to reduce intestinal microbiota overgrowth . Gut microbiome alterations were observed in patients with alcohol-associated and hepatitis-associated cirrhosis in a Chinese cohort ,drainage gutter with an invasion of the lower intestinal tract by microbes associated with the oral cavity such as Veillonella and Steptococcus. Concordant with these findings, Chen and colleagues also found an over-representation of genera including Veillonella, Megasphaera, Dialister, Atopobium and Prevotella in the duodenum of patients with cirrhosis. The genera Neisseria and Gemella were discriminative between HBV-related and PBC-related cirrhosis . In 2017, Bajaj and colleagues observed statistically significant fungal dysbiosis in patients with cirrhosis and showed that Bacteroidetesto Ascomycota ratio could independently predict hospitalization in these patients . All experimental models of liver fibrosis result in gut microbial dysbiosis and increased intestinal permeability, and treatment of gastrointestinal tract with nonabsorbable antibiotics improved survival by immunomodulation, reducing translocation and incidences of infection . Mice with genetic ablations of the receptors for bacterial product ligands are protected from experimental liver fibrosis . The current treatment philosophy involves decreasing the bacterial product ligands or blocking their receptors, which results in decreased inflammatory and fibrogenic signaling in the liver, although no antifibrotic drug is currently available for routine clinical practice.The etiology of non-viral HCC follows a so-called multiplehit pathway, whereby liver steatosis, followed by oxidative stress, ER stress together with intestinal dysbiosis and inflammation contribute to the final manifestation of cancer.

The gut microbiota dramatically changes in composition in hosts with HCC. Clostridium species have been found to be enriched in obesity-induced mouse models of HCC , but clinical studies of patients with HCC detected an overgrowth of intestinal Escherichia coli . Murine models and human studies have reported a migration of Helicobacter species to HCC tumor tissues . Notably, members of this genus are known to promote tumor-development by activating NF-kB and WNT signaling and suppressing anti-tumor immunity, and might have a potential role in HCC development . To get insights into the molecular events explaining the progression of liver disease to HCC, various murine models have been explored. However, most of these models have proven suboptimal because they either do not develop all intermediate pathological & metabolic stages, or they manifest HCC incompletely . We have highlighted some frequently-used rodent models of liver disease, their usage and caveats in Table 1.2.2 to aide future research. Accumulating evidence suggests that HCC-associated dysbiosis is accompanied by gutbarrier dysfunction, bacterial translocation, systemic circulation of their tumor-promoting metabolites and activation of proinflammatory and oncogenic signaling pathways . The intestinal poly-immunoglobulin receptor regulates the transport of IgA into the intestinal lumen and maintains microbial homeostasis . PIgR-/- mice modelling NASH-induced HCC had increased levels of systemic and liver IgA, and a concomitant increase in hepatic tumorigenesis due to localized inhibition of liver cytotoxic T cells that prevent HCC development . Furthermore, the application of broad spectrum antibiotics has been shown to attenuate liver inflammation and HCC-development in mice , highlighting the role of the intestinal microbiome in liver tumorigenesis. In another mouse model in which HCC was induced by diethylnitrosamine , activation of TLR4 due to LPS translocation upregulated the hepatic mitogen EREG in hepatic stellate cells and activated NF-kB, resulting in enhanced tumor cell proliferation . Additionally, the secondary bile acid deoxycholic acid , a metabolic byproduct of gut bacteria, was shown to upregulate proinflammatory genes, such as IL6 and TNF, to provoke a senescence associated secretory phenotype in hepatic stellate cells suggesting that SASP could be playing a key role in at least obesity-linked HCC development . In addition to its role in HCC development, the gut microbiome also modulates protumorigenic adaptive immune response via type 17 T helper cells, which produce the proinflammatory cytokine IL-17A . The therapeutic efficacy of the anticancer drugcyclophosphamide depended on the interplay between Th17 signaling and gut microbiome such that germ-free tumor-bearing mice or mice given non-absorbable antibiotics had reduced Th17 response and a subsequent resistance to therapeutic effects of cyclophosphamide was seen . Increased understanding of the role of the gut microbiota has motivated successful microbiome-based therapeutic modalities for HCC, such as treatment with synthetic bile acids to reduce HCC risk in patients with NAFLD , non-selective beta-blockers in the intestinal mucosa which prevent bacterial translocation and liver inflammation and administration of probiotics in rodents models of HCC slowed tumor growth and reduced tumor size .