Gut-liver Axis: Recent concepts in pathophysiology in alcohol-associated liver disease.

The growing recognition of the role of the gut microbiome's impact on alcohol-related diseases, especially in alcohol-associated liver disease, emphasizes the need to understand molecular mechanisms involved in governing organ-organ communication to identify novel avenues to combat alcohol-related diseases. The gut-liver axis refers to the bidirectional communication and interaction between gut and liver. Intestinal microbiota play a pivotal role in maintaining homeostasis within the gut-liver axis and this axis plays a significant role in alcohol-associated liver disease. The intricate communication between intestine and liver involves communication between multiple cellular components in each organ that enable them to carry out their physiological functions. In this review, we focus on novel approaches to understanding how chronic alcohol exposure impacts the microbiome, and individual cells within the liver and intestine, as well as the impact of ethanol on the molecular machinery required for intra- and inter-organ communication.

Dysregulated meta-organismal metabolism of aromatic amino acids in alcohol-associated liver disease.

BACKGROUND: Chronic alcohol consumption impairs gut barrier function and perturbs the gut microbiome. Although shifts in bacterial communities in patients with alcohol-associated liver disease (ALD) have been characterized, less is known about the interactions between host metabolism and circulating microbe-derived metabolites during the progression of ALD. METHODS: A large panel of gut microbiome-derived metabolites of aromatic amino acids was quantified by stable isotope dilution liquid chromatography with online tandem mass spectrometry in plasma from healthy controls (n = 29), heavy drinkers (n = 10), patients with moderate (n = 16) or severe alcohol-associated hepatitis (n = 40), and alcohol-associated cirrhosis (n = 10). RESULTS: The tryptophan metabolites, serotonin and indole-3-propionic acid, and tyrosine metabolites, p-cresol sulfate, and p-cresol glucuronide, were decreased in patients with ALD. Patients with severe alcohol-associated hepatitis and alcohol-associated cirrhosis had the largest decrease in concentrations of tryptophan and tyrosine-derived metabolites compared to healthy control. Western blot analysis and interrogation of bulk RNA sequencing data from patients with various liver pathologies revealed perturbations in hepatic expression of phase II metabolism enzymes involved in sulfonation and glucuronidation in patients with severe forms of ALD. CONCLUSIONS: We identified several metabolites decreased in ALD and disruptions of hepatic phase II metabolism. These results indicate that patients with more advanced stages of ALD, including severe alcohol-associated hepatitis and alcohol-associated cirrhosis, had complex perturbations in metabolite concentrations that likely reflect both changes in the composition of the gut microbiome community and the ability of the host to enzymatically modify the gut-derived metabolites.

Metaorganismal choline metabolism shapes olfactory perception.

Microbes living in the intestine can regulate key signaling processes in the central nervous system that directly impact brain health. This gut-brain signaling axis is partially mediated by microbe-host-dependent immune regulation, gut-innervating neuronal communication, and endocrine-like small molecule metabolites that originate from bacteria to ultimately cross the blood-brain barrier. Given the mounting evidence of gut-brain crosstalk, a new therapeutic approach of "psychobiotics" has emerged, whereby strategies designed to primarily modify the gut microbiome have been shown to improve mental health or slow neurodegenerative diseases. Diet is one of the most powerful determinants of gut microbiome community structure, and dietary habits are associated with brain health and disease. Recently, the metaorganismal (i.e., diet-microbe-host) trimethylamine N-oxide (TMAO) pathway has been linked to the development of several brain diseases including Alzheimer's, Parkinson's, and ischemic stroke. However, it is poorly understood how metaorganismal TMAO production influences brain function under normal physiological conditions. To address this, here we have reduced TMAO levels by inhibiting gut microbe-driven choline conversion to trimethylamine (TMA), and then performed comprehensive behavioral phenotyping in mice. Unexpectedly, we find that TMAO is particularly enriched in the murine olfactory bulb, and when TMAO production is blunted at the level of bacterial choline TMA lyase (CutC/D), olfactory perception is altered. Taken together, our studies demonstrate a previously underappreciated role for the TMAO pathway in olfactory-related behaviors.

35kDa hyaluronan ameliorates ethanol driven loss of anti-microbial defense and intestinal barrier integrity in a TLR4-dependent manner.

Acute and chronic alcohol exposure compromise intestinal epithelial integrity, due to reduced expression of anti-microbial peptides (AMP) and loss of tight junction integrity. Ameliorating gut damage is beneficial in preventing associated distant organ pathologies. Orally administered purified hyaluronan (HA) polymers with an average size of 35 kDa have multiple protective effects in the gut and are well-tolerated in humans. Therefore, we tested the hypothesis that HA35 ameliorates ethanol-induced gut damage. Specifically, mechanisms that restore epithelial barrier integrity and normalize expression of the Reg3 class of C-type lectin AMPs (i.e. Reg3ß and Reg3γ) were investigated. Chronic ethanol feeding to mice reduced expression of C-type lectin AMPs in the proximal small intestine (jejunum), reduced expression of tight junction proteins and increased bacterial translocation to the mesenteric lymph node. Oral consumption of HA35 during the last 6 days of ethanol exposure ameliorated the effects of chronic ethanol. Similarly, in vitro challenge of isolated intestinal organoids from murine jejunum with ethanol reduced the expression of C-type lectin AMPs and impaired barrier integrity; these ethanol-induced responses were prevented by pre-treatment with HA35. Importantly, HA receptor null jejunum-derived organoids demonstrated that the HA receptor Tlr4, but not Cd44 nor Tlr2, was required for the protective effect of HA35. Consistent with the data from organoids, HA35 did not protect Tlr4-deficient mice from chronic ethanol-induced intestinal injury. Together, these data suggest therapeutic administration of HA35 is beneficial in restoring gut epithelial integrity and defense during the early stages of ethanol-driven intestinal damage.

Gut microbial trimethylamine is elevated in alcohol-associated hepatitis and contributes to ethanol-induced liver injury in mice.

There is mounting evidence that microbes residing in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite TMA is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

Identification and quantification of oligogenic loss-of-function disorders.

PURPOSE: Monogenic disorders can present clinically heterogeneous symptoms. We hypothesized that in patients with a monogenic disorder caused by a large deletion, frequently additional loss-of-function (LOF)-intolerant genes are affected, potentially contributing to the phenotype. METHODS: We investigated the LOF-intolerant gene distribution across the genome and its association with benign population and pathogenic classified deletions from individuals with presumably monogenic disorders. For people with presumably monogenic epilepsy, we compared Human Phenotype Ontology terms in people with large and small deletions. RESULTS: We identified LOF-intolerant gene dense regions that were enriched for ClinVar and depleted for population copy number variants. Analysis of data from >143,000 individuals with a suspected monogenic disorder showed that 2.5% of haploinsufficiency disorder-associated deletions can affect at least 1 other LOF-intolerant gene. Focusing on epilepsy, we observed that 13.1% of pathogenic and likely pathogenic ClinVar deletions <3 megabase pair, covering the diagnostically most relevant genes, affected at least 1 additional LOF-intolerant gene. Those patients have potentially more complex phenotypes with increasing deletion size. CONCLUSION: We could systematically show that large deletions frequently affected admditional LOF-intolerant genes in addition to the established disease gene. Further research is needed to understand how additional potential disease-relevant genes influence monogenic disorders to improve clinical care and the efficacy of targeted therapies.