Abstract
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.
Highlights
Alcohol-associated liver disease (ALD) includes a spectrum of liver pathologies including steatosis, fibrosis, cirrhosis, and the most severe manifestation known as alcohol-associated 79 hepatitis (AH)
It is well appreciated that chronic alcohol use can elicit structural alterations in the gut barrier, allowing either live bacteria themselves or microbe-associated molecule patterns (MAMPs), such as lipopolysaccharide (LPS), to enter the portal circulation where they can directly engage pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) or NOD-like 87 receptors (NLRP3, NLRP6, etc.) to promote hepatic inflammation and tissue injury[15,16,17,18,19,20]
TMA infusion was associated with altering the phosphorylation of several guanine nucleotide exchange factors (GEF), including Rac/Cdc[42] guanine nucleotide exchange factor 6 (Arhgef6) and Rho GTPase activating protein 17 (ARHGAP17)[63,64], and proteins involved in RNA processing/splicing including signal recognition particle 14 (SRP14)[65] and serine and arginine rich splicing factor 1 (SRSF1)[66] (Figure 6B and 6C). These data have identified acute TMA-driven signaling events in the liver in vivo, and potentially link TMA to acute alterations in protein kinase A (PKA), insulin, and GEF-driven signaling cascades that deserve further exploration. 286 DISCUSSION drug discovery has historically targeted pathways in the human host, there is untapped potential in therapeutically targeting the gut microbial endocrine organ to treat advanced liver disease. This paradigm shift is needed in light of the clear and reproducible associations between the gut microbiome in viral, alcohol-associated, and non-alcohol associated liver diseases[3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]
Summary
Alcohol-associated liver disease (ALD) includes a spectrum of liver pathologies including steatosis, fibrosis, cirrhosis, and the most severe manifestation known as alcohol-associated 79 hepatitis (AH). In addition to MAMP-PRR interactions, gut microbes can act as a collective endocrine organ, producing a vast array of small molecules, proteins, and lipid metabolites that can engage 90 dedicated host receptor systems to impact liver disease progression[21]. There is clear evidence that microbe-host interactions play a key role in liver disease progression[3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22], ALD drug discovery to this point has focused primarily on targets encoded by the human genome. Whereas pathways encoded by the host genome have long been pursued as drug targets, this work provides proof of concept that rationally designed drugs that target bacterial metabolism likely have untapped therapeutic potential in ALD and beyond
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