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.
This paper aims to understand the mechanisms by which gut microbes synergize with excessive alcohol intake to cause liver injury, and whether drugs that selectively target gut microbial metabolism can improve alcohol-associated liver disease (ALD). The authors used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in controls and patients with alcohol-associated hepatitis (AH). They also treated mice with bacterial choline trimethylamine (TMA) lyase inhibitors to reduce gut microbe-dependent TMA production, followed by measurement of Indices of liver injury. They showed that gut microbial choline metabolite TMA is increased in AH patients, which correlates with reduced liver expression of the TMA oxygenase Flavin-containing monooxygenase 3 (FMO3). They also show that inhibition of gut microbial CutC/D activity protects from ethanol-induced liver injury in mouse models, which was associated with reorganization of the gut microbiome and host liver transcriptome. The authors conclude that microbial TMA is elevated in patients with AH, and inhibition of TMA production by gut microbes protects against ethanol-induced liver injury.https://doi.org/10.7554/eLife.76554.sa0
Alcohol-associated liver disease (ALD) includes a spectrum of liver pathologies including steatosis, fibrosis, cirrhosis, and the most severe manifestation known as alcohol-associated hepatitis (AH). Shortly after diagnosis AH patients die at a staggering rate of 40–50% (Masarone et al., 2016; Kochanek et al., 2017). Despite many attempts, an effective therapy for this deadly disease has been elusive. Similar to other components of the spectrum of ALD, AH has consistently been linked to reorganization of the gut microbiome and dysregulation of microbe-host interactions (Chen et al., 2011; Yan et al., 2011; Mutlu et al., 2009; Mutlu et al., 2012; Tripathi et al., 2018; Ciocan et al., 2018; Llopis et al., 2016; Duan et al., 2019; Smirnova et al., 2020; Gao et al., 2019; Puri et al., 2018; Lang and Schnabl, 2020). 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 receptors (NLRP3, NLRP6, etc.) to promote hepatic inflammation and tissue injury (Wilkinson et al., 1974; Tarao et al., 1979; Uesugi et al., 2001; Paik et al., 2003; DeSantis et al., 2013; Knorr et al., 2020). 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 dedicated host receptor systems to also impact liver disease progression (Brown and Hazen, 2015). Collectively, these MAMP-PRR and microbial metabolite-host receptor interactions converge to promote ALD and many other diseases of uncontrolled inflammation (Brown and Hazen, 2015; Gilbert et al., 2018).
Although there is now clear evidence that microbe-host interactions play a key role in liver disease progression (Chen et al., 2011; Yan et al., 2011; Mutlu et al., 2009; Mutlu et al., 2012; Tripathi et al., 2018; Ciocan et al., 2018; Llopis et al., 2016; Duan et al., 2019; Smirnova et al., 2020; Gao et al., 2019; Puri et al., 2018; Lang and Schnabl, 2020; Wilkinson et al., 1974; Tarao et al., 1979; Uesugi et al., 2001; Paik et al., 2003; DeSantis et al., 2013; Knorr et al., 2020; Brown and Hazen, 2015; Gilbert et al., 2018), ALD drug discovery to this point has focused primarily on targets encoded by the human genome. Our knowledge is rapidly expanding as to how microbes intersect with ALD progression, including cataloging microbial genomes. We also now understand the repertoire of MAMPs gut microbes harbor as well as the vast array of metabolites that they produce in both patients with ALD and animal models of ethanol-induced liver injury (Chen et al., 2011; Yan et al., 2011; Mutlu et al., 2009; Mutlu et al., 2012; Tripathi et al., 2018; Ciocan et al., 2018; Llopis et al., 2016; Duan et al., 2019; Smirnova et al., 2020; Gao et al., 2019; Puri et al., 2018; Lang and Schnabl, 2020; Wilkinson et al., 1974; Tarao et al., 1979; Uesugi et al., 2001; Paik et al., 2003; DeSantis et al., 2013; Knorr et al., 2020; Brown and Hazen, 2015; Gilbert et al., 2018). However, there are very few examples of where this information has been leveraged into safe and effective therapeutic strategies. In general, the microbiome-targeted therapeutic field has primarily focused on either anti-, pre-, or pro-biotic approaches, yet these microbial community-restructuring approaches have resulted in very modest or non-significant effects in clinical studies of liver disease (Kwak et al., 2014; Asgharian et al., 2020; Reijnders et al., 2016; Madjd et al., 2016). As an alternative microbiome-targeted approach, we and others have begun developing non-lethal selective small molecule inhibitors of bacterial enzymes with the intention of reducing levels of disease-associated microbial metabolites with mechanistic rationale for contribution to disease pathogenesis (Roberts et al., 2018; Wang et al., 2015; Gupta et al., 2020; Organ et al., 2020; Orman et al., 2019). In fact, we have recently shown that small molecule inhibition of the gut microbial transformation of choline into trimethylamine (TMA), the initial and rate-limiting step in the generation of the cardiovascular disease (CVD)-associated metabolite trimethylamine N-oxide (TMAO), can significantly reduce disease burden in animal models of atherosclerosis, thrombosis, heart failure, and chronic kidney disease (Roberts et al., 2018; Wang et al., 2015; Gupta et al., 2020; Organ et al., 2020). Although the gut microbial TMAO pathway has been studied mostly in the context of CVD (Wang et al., 2011; Koeth et al., 2013; Zhu et al., 2016; Zhu et al., 2017; Tang et al., 2013; Wang et al., 2014b; Trøseid et al., 2015; Tang and Hazen, 2014), recent studies found that breath levels of the primary metabolite TMA and other related co-metabolites are elevated in patients with ALD (Hanouneh et al., 2014; Ascha et al., 2016). These data showed promise, but whether the gut microbial TMAO pathway is causally related to ALD has never been explored. Hence, here we set out to understand how the gut microbial TMA/TMAO pathway may play a contributory role in ALD susceptibility and progression, and to test whether selective drugs that lower gut microbial production of TMA can be an effective therapeutic strategy. In an era when host genetics/genomics approaches dominate, this work reminds us that genes and metabolic products produced by gut bacteria play equally important roles in modulating disease susceptibility. 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.
Circulating levels of the gut microbial metabolite TMA are elevated in AH
In a previous collaborative study, we reported that the highly volatile microbial metabolite TMA is elevated in exhaled breath of patients with AH (Hanouneh et al., 2014), and related co-metabolites, such as trimethyllysine and carnitine, can serve as prognostic indicators of mortality in AH (Ascha et al., 2016). Given the extremely volatile nature of TMA, it is readily detectable in breath, but is challenging to accurately quantitate levels in the circulation because TMA rapidly dissipates during collection and storage. To reduce the volatility of TMA and enable its analysis in the circulation, we coordinated patient blood collection utilizing rapid acidification of separated plasma (protonated TMA has a lower vapor pressure) across a large multi-center AH consortium (Defeat Alcoholic Steatohepatitis [DASH] consortium) (Crabb et al., 2016; Vatsalya et al., 2020; Saha et al., 2019). This provided us the unique opportunity to accurately quantify circulating TMA levels in human subjects, including those with moderate or severe AH for the first time. Patient demographics and clinical characteristics for the cohort examined are summarized in Figure 1—source data 1; Figure 1—source data 2. Importantly, MELD score, Maddrey’s discriminant function score, Child-Pugh score, aspartate aminotransferase (AST), total bilirubin, creatinine, and international normalized ratio were higher in patients with severe AH compared to moderate AH patients, while serum albumin was lower in severe AH compared to moderate AH patients. In agreement with previous breath metabolomics studies (Hanouneh et al., 2014; Ascha et al., 2016), plasma TMA levels were significantly elevated in moderate and severe AH patients compared to healthy controls (Figure 1A). However, the CVD-related co-metabolite TMAO was reciprocally decreased in AH patients (Figure 1B). Given the reciprocal alterations in plasma TMA and TMAO levels, we next examined the expression of the host liver enzyme flavin-containing monooxygenase 3 (FMO3) which is the predominant TMA to TMAO converting enzyme in the adult liver (Cashman, 2002). Interestingly, mRNA levels for FMO3 are uniquely repressed in patients with more severe AH (AH with liver failure [MELD 22–28] and AH with emergency liver transplant [MELD 18–21]), but not in other liver disease etiologies such as non-alcoholic fatty liver disease (NAFLD) or viral hepatitis (Figure 1C). In agreement with reduced mRNA levels (Figure 1C), patients with severe AH undergoing emergency liver transplant have marked reduction in FMO3 protein (Figure 1D), which likely contributes to elevations in plasma TMA (Figure 1A). Although ethanol feeding in mice does not consistently result in reduced hepatic Fmo3 expression (data not shown), a single injection of lipopolysaccharide (LPS) to induce acute hepatic inflammation is associated with both a reduction in the expression of Fmo3 and a significant increase in the TMA receptor trace amine-associated receptor 5 (Taar5) (Figure 1E). It is important to note that circulating choline levels was not significantly altered in patients with AH compared to healthy controls (Figure 1—figure supplement 1). However, plasma levels of one of the gut microbial substrates for TMA production (carnitine) and other TMA pathway co-metabolites (e.g. betaine and γ-butyrobetaine) were elevated in patients with AH compared to healthy controls (Figure 1—figure supplement 1). These findings, in addition to previous breath metabolomic studies (Hanouneh et al., 2014; Ascha et al., 2016), provide evidence that TMA and related co-metabolites may allow for discrimination of AH from other liver diseases.
Figure 1—source data 1
Figure 1—source data 2
Figure 1—source data 3
Figure 1—source data 4
Figure 1—source data 5
Figure 1—source data 6
Microbial choline TMA lyase inhibition protects mice from ethanol-induced liver injury
We next sought to establish whether a causal relationship between gut microbial TMA production and ALD progression exists, and to test the hypothesis that selectively drugging microbial choline transformation can serve as a mechanism for improving host liver disease and attenuating ethanol-induced liver injury in mice. Mice were individually treated with two recently reported non-lethal bacterial choline TMA lyase inhibitors, iodomethylcholine (IMC) and fluoromethylcholine (FMC) (Roberts et al., 2018). These small molecule inhibitors exhibit potent in vivo inhibition of the gut microbial choline TMA lyase enzyme CutC (Craciun and Balskus, 2012), and have been shown to effectively block bacterial choline to TMA conversion in vivo (Roberts et al., 2018). Designed as suicide substrate mechanism-based inhibitors, past studies reveal that the vast majority of IMC and FMC is retained in the gut within luminal bacteria and excreted in the feces with limited systemic exposure of the polar drug in the host (Roberts et al., 2018; Gupta et al., 2020; Organ et al., 2020).
IMC treatment effectively blunted ethanol-induced increases in plasma TMA and TMAO (Figure 2A and B). IMC also produced modest increases in plasma choline and betaine, while reducing plasma carnitine, particularly in pair-fed mice (Figure 2C–E). IMC also prevented ethanol-induced increases in alanine aminotransferase (ALT) and hepatic steatosis (Figure 2F, G, and K). Interestingly, IMC treatment prevented ethanol-induced increases in hepatic triglycerides (Figure 2G), and reduced hepatic total and cholesterol esters, but not free cholesterol, in both pair- and ethanol-fed conditions (Figure 2H–I). IMC treatment also reduced the expression levels of the pro-inflammatory cytokine tumor necrosis factor α (Tnfα) (Figure 2L). Although IMC was well tolerated in several previous mouse studies in the setting of standard rodent chow-feeding (Roberts et al., 2018; Gupta et al., 2020; Organ et al., 2020), here we found an unexpected reduction in food intake and body weights in mice receiving both IMC and ethanol (Figure 2—figure supplement 1A, B). Although IMC was clearly protective against ethanol-induced liver injury, this potential drug-ethanol interaction prompted us to test another structurally distinct gut microbe-targeted choline TMA lyase inhibitor FMC (Roberts et al., 2018; Figure 3 and Figure 3—figure supplement 1).
Importantly, FMC was well tolerated and did not significantly alter liquid diet intake or body weights throughout the 25-day chronic ethanol feeding study (Figure 2—figure supplement 1C, D). FMC treatment trended toward reducing plasma TMA (Figure 3A), and more dramatically suppressed plasma TMAO levels (Figure 3B). Unlike IMC, which also altered other co-metabolites such as choline, betaine, and carnitine (Figure 2B-E), FMC did not significantly alter these TMA co-metabolites (Figure 3C-E). More importantly, as with IMC (Figure 2F–K), FMC treatment significantly protected against ethanol-induced ALT elevations (Figure 3F), hepatic steatosis (Figure 3G and K), and reduced total and esterified cholesterol levels without altering free cholesterol (Figure 3H–J). However, FMC trended to reduce but did not significantly alter Tnfα expression (Figure 3L). To determine whether these effects were generalizable in other models of ethanol-induced liver injury, we exposed control and FMC-treated mice to a 10-day chronic model in which mice were allowed free access to a 5% vol/vol (27% kcal) for 10 days (Figure 3—figure supplement 1). In this 5%–10-day ethanol feeding model FMC treatment did not significantly alter food intake, body weight, or blood ethanol levels, but was able to selectively suppress TMA and TMAO levels (Figure 3—figure supplement 1). FMC treatment in the 5%–10-day model significantly reduced plasma AST and ALT levels, and trended toward lowering liver triglycerides (Figure 3—figure supplement 1). However, in this short-term model there were no apparent differences in hepatic cytokine/chemokine gene expression with either ethanol exposure or FMC treatment (Figure 3—figure supplement 1 and data not shown). Collectively, these data demonstrate that gut microbe-targeted choline TMA lyase inhibition with two structurally distinct inhibitors (IMC or FMC) can generally protect mice against ethanol-induced liver injury.
Microbial choline TMA lyase inhibitors promote remodeling of the gut microbiome and host liver transcriptome in an ethanol-dependent manner
One theoretical advantage of the selective microbe-targeted choline TMA lyase inhibitors, compared to antibiotic or MAMP-PRR-targeted therapies, is that they are anticipated to exert less selective pressure for development of drug resistance given their non-lethal nature. However, microbes that preferentially utilize choline as a carbon or nitrogen source might be anticipated to have reduced competitive advantage in the presence of the inhibitor. We therefore next examined whether IMC or FMC treatment was associated with alterations in choline utilizers and other members of the murine gut microbiome community that are known to be correlated with ethanol-induced liver injury (Chen et al., 2011; Yan et al., 2011; Mutlu et al., 2009; Mutlu et al., 2012; Tripathi et al., 2018; Ciocan et al., 2018; Llopis et al., 2016; Duan et al., 2019; Smirnova et al., 2020; Gao et al., 2019; Puri et al., 2018; Lang and Schnabl, 2020). It is important to note that both IMC (Figure 4A–E) and FMC (Figure 4F–J) altered the gut microbiome, with some consistent, yet several distinct differences. Non-metric multidimensional scaling (NMDS) of microbial taxa revealed distinct clusters, indicating that both IMC and FMC promoted clear restructuring of the cecal microbiome in an ethanol-dependent manner (Figure 4A and F). Under pair-feeding conditions, both IMC and FMC caused a reciprocal decrease in the relative abundance of Bacteroidetes and increase in Firmicutes (Figure 4B and G). However, under ethanol-fed conditions IMC resulted in increased Bacteroidetes and reduced Firmicutes, and FMC treatment resulted in more modest reductions in Bacteroidetes and increased Firmicutes (Figure 4B and G). When examining drug-specific alterations at the genus level, we found that under both pair- and ethanol-fed conditions, IMC treatment promoted significant increases in Faecalibaculum and Escherichica/Shigella, and reductions in Bacteroidales_S24-7 (Figure 4C–E , and H–I). FMC, however, most significantly altered Turicibacter, Oscillibacter, and Lachnospiraceae, and it is important to note that these FMC-induced alterations were different between pair- and ethanol-fed groups (Figure 4C–E , and H–I). Collectively, these data demonstrate that inhibition of gut microbial choline to TMA transformation with a selective non-lethal small molecule inhibitor promotes restructuring of the gut microbiome in an ethanol-dependent manner.
To more globally understand the effects of choline TMA lyase inhibitors on the host liver, we performed unbiased RNA sequencing in mice undergoing pair or ethanol feeding treated either with or without IMC (Figure 5). NMDS and hierarchical clustering analysis showed clear separation between all four groups (Figure 5A and B). In pair-fed mice, IMC treatment caused significant decreases in several genes encoding major urinary proteins (Mup2, Mup10, Mup11, and Mup18) and cytochrome p450 enzymes (Cyp3a16, Cyp3a44), while increasing other genes involved in xenobiotic metabolism (Ephx1, Cyp4a31) and hormone/cytokine signaling (Lepr, Fgf21, Il22ra1) (Figure 5C). Under ethanol-feeding conditions, IMC treatment most significantly altered genes involved in hepatocyte metabolism (Cyp8b1, Ugt1a5, Pnpla5, Sult2a8, Ces3a, and Cmah), RNA processing (Ddx21, Ftsj3, Dus1l, and Cmah), and again major urinary proteins (Mup2, Mup10, Mup11, and Mup20) (Figure 5D and E). These unbiased RNASeq data demonstrate that gut microbe-targeted choline TMA lyase inhibitors can alter the host liver transcriptome in an ethanol feeding-dependent manner.
The microbe-derived metabolite TMA elicits rapid hormone-like signaling effects in mouse liver
The gut microbe-derived co-metabolites TMA and TMAO are generated postprandially in both rodents and humans after a substrate-rich meal is ingested (Schugar et al., 2018; Boutagy et al., 2015). Given the acute meal-related production and recent identification of candidate host receptors for TMA (Li et al., 2013; Wallrabenstein et al., 2013) and TMAO (Chen et al., 2019), we hypothesized that TMA may be acting as a gut microbe-derived hormone to promote liver injury. However, currently nothing is known regarding the acute hormone-like signaling effects stimulated by TMA in the liver. To address this gap, we infused TMA directly into the portal circulation draining the gut (i.e. portal vein) of fasted mice and examined global phosphorylation events stimulated in the liver 10 min later using a phosphoproteomics approach (Figure 6A). It is important to note that this experiment provided high levels of exogenous TMA via direct injection, and future studies should focus on more physiologically relevant modes of TMA production like provision of gut bacteria that can naturally or be genetically engineered to produce high levels. A total of 36 liver proteins exhibited site-specific hypo- or hyper-phosphorylation 10 min after administration of TMA relative to vehicle-injected mice (Figure 6B). Several of the TMA-driven phosphorylation events represented proteins that are enriched in key hormonal signaling pathways known to impact hepatic metabolism. For example, portal vein infusion of TMA resulted in altered phosphorylation of proteins implicated in protein kinase A (PKA) signaling, including A kinase anchor protein 1 (AKAP1) (Huang et al., 1999) and FK506-binding protein 15 (FKBP15) (Nooh and Bahouth, 2017), and insulin signaling including insulin receptor substrate 2 (IRS2) (Araki et al., 1994; Figure 6B–D). TMA infusion was also associated with altering the phosphorylation of several guanine nucleotide exchange factors (GEF), including Rac/Cdc42 guanine nucleotide exchange factor 6 (Arhgef6) and Rho GTPase activating protein 17 (ARHGAP17) (Zhou et al., 2016; Aslan, 2019), and proteins involved in RNA processing/splicing including signal recognition particle 14 (SRP14) (Strub and Walter, 1990) and serine- and arginine-rich splicing factor 1 (SRSF1) (Cho et al., 2011; Figure 6B and C). These data have identified acute TMA-driven signaling events in the liver in vivo, and potentially link TMA to acute alterations in PKA-, insulin-, and GEF-driven signaling cascades that deserve further exploration.
Although 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 (Chen et al., 2011; Yan et al., 2011; Mutlu et al., 2009; Mutlu et al., 2012; Tripathi et al., 2018; Ciocan et al., 2018; Llopis et al., 2016; Duan et al., 2019; Smirnova et al., 2020; Gao et al., 2019; Puri et al., 2018; Lang and Schnabl, 2020; Wilkinson et al., 1974; Tarao et al., 1979; Uesugi et al., 2001; Paik et al., 2003; DeSantis et al., 2013; Knorr et al., 2020; Brown and Hazen, 2015). Now we are faced with both the challenge and opportunity to test whether microbe-targeted therapeutic strategies can improve health in the human metaorganism without negatively impacting the symbiotic relationships between microbes and host. Although traditional microbiome manipulating approaches such as antibiotics, prebiotics, probiotics, and fecal microbial transplantation have shown their own unique strengths and weaknesses, each of these presents unique challenges particularly for use in chronic diseases such as end stage liver disease. As we move toward selective non-lethal small molecule therapeutics, the goal is to have exquisite target selectivity and limited systemic drug exposure given that the targets are microbial in nature. This natural progression parallels the paradigm shifts in oncology which have transitioned from broadly cytotoxic chemotherapies to target-selective small molecule and biologics-based therapeutics. Here, we provide the first evidence that the gut microbial choline metabolite TMA is elevated in the plasma of patients with AH, which corroborates previous reports showing that TMA is also prominent in the breath of patients with AH (Hanouneh et al., 2014). Hence, further studies are warranted to determine whether combined measures of breath and blood TMA can serve as a prognostic biomarker to accurately predict AH-related mortality. Here, we also show for the first time that a selective non-lethal small molecule drug that reduces bacterial production of TMA can prevent ethanol-induced liver injury in mice. We also demonstrate that direct administration of TMA can elicit rapid signaling effects in the liver, supporting the notion that gut microbial metabolites produced postprandially can act in an endocrine-like manner to alter host signal transduction and associated disease pathogenesis. Collectively, these studies suggest that selective drugs targeting the gut microbial TMA pathway may hold promise for treating AH.
As drug discovery advances in the area of small molecule non-lethal bacterial enzyme inhibitors, it is key to understand how these drugs impact microbial ecology in the gut and other microenvironments. As we have previously reported (Roberts et al., 2018; Gupta et al., 2020; Organ et al., 2020), gut microbe-targeted choline TMA lyase inhibitors (IMC and FMC) induced a significant remodeling of the cecal microbiome in mice. In the current studies there were some consistent, but many different cecal microbiome alterations when comparing IMC and FMC (Figure 4), yet both drugs similarly improved ethanol-induced liver injury. As small molecule bacterial enzymes inhibitors are developed, it will be extremely important to understand their effects on microbial ecology, and it is expected that some of the beneficial effects of these drugs will indeed originate from the restructuring of gut microbiome communities. In fact, this is not an uncommon mechanism by which host targeted drugs impact human health. A recent study showed that nearly a quarter of commonly used host-targeted drugs have microbiome-altering properties (Maier et al., 2018), and in the context of diabetes therapeutics it is important to note metformin’s anti-diabetic effects are partially mediated by the drug’s microbiome altering properties (Wu et al., 2017). Given the strong association between gut microbiome and liver disease (Chen et al., 2011; Yan et al., 2011; Mutlu et al., 2009; Mutlu et al., 2012; Tripathi et al., 2018; Ciocan et al., 2018; Llopis et al., 2016; Duan et al., 2019; Smirnova et al., 2020; Gao et al., 2019; Puri et al., 2018; Lang and Schnabl, 2020; Wilkinson et al., 1974; Tarao et al., 1979; Uesugi et al., 2001; Paik et al., 2003; DeSantis et al., 2013; Knorr et al., 2020; Brown and Hazen, 2015; Gilbert et al., 2018), it may prove advantageous to find therapeutics that beneficially remodel the gut microbiome as well as engage either their microbe or host target of interest.
The metaorganismal TMA/TMAO pathway represents only one of many microbial metabolic circuits that have been associated with human disease (Figure 7). In fact, many microbe-associated metabolites such as short chain fatty acids, secondary bile acids, phenolic acids, polyamines, and others have more recently been associated with many human diseases (Brown and Hazen, 2015; Gilbert et al., 2018). In an ethanol- and meal-related manner, gut microbes produce a diverse array of metabolites that reach micromolar to millimolar concentrations in the blood, making the collective gut microbiome an active endocrine organ (Brown and Hazen, 2015). Small molecule metabolites are well known to be mediators of signaling interactions in the host, and this work provides evidence that diet/ethanol-microbe-host metabolic interplay can be causally linked to ethanol-induced liver injury. Our work, and that of many others, demonstrates that there is clear evidence of bi-directional crosstalk between the gut microbial endocrine organ and host liver metabolism. As drug discovery advances, it will be important to move beyond targets based solely in the human host. This work highlights that non-lethal gut microbe-targeted enzyme inhibitors may serve as effective therapeutics in AH and provides proof of concept that this may be a generalizable approach to target metaorganismal crosstalk in other disease contexts. In fact, selective inhibition of bacterial enzymes has the advantage over host targeting given that small molecules can be designed to avoid systemic absorption and exposure, thereby minimizing potential host off target effects. As shown here with the gut microbial TMA/TMAO pathway, it is easy to envision that other microbe-host interactions are mechanistically linked to host disease pathogenesis, serving as the basis for the rational design of microbe-targeted therapeutics that improve human health.
Overview of human study populations
We made use of three different human study populations, detailed below, that included patients with severities of AH/ALD. It must be noted that one limitation of our study is that each of these cohorts used slightly different diagnostic criteria for defining the severity/stage of AH/ALD.
Human study populations and sample collection for TMA measurement
A total of 285 subjects were included in this study. De-identified plasma samples, along with clinical and demographic data, were obtained from (1) the Northern Ohio Alcohol Center (NOAC) at the Cleveland Clinic biorepository including 21 healthy individuals and 15 patients diagnosed and (2) the Defeat Alcoholic Steatohepatitis (DASH) consortium (Cleveland Clinic, University of Louisville School of Medicine, University of Massachusetts Medical School, and University of Texas Southwestern Medical Center) including 249 patients with AH. Diagnosis with AH was performed using clinical and laboratory criteria, with MELD score utilized for distinguishing moderate (MELD < 20) and severe (MELD > 20) AH, as recommended by the NIAAA Alcoholic Hepatitis consortia (Crabb et al., 2016). A detailed description of patient recruitment, inclusion and exclusion criteria for the DASH consortium has been reported in previous studies (Vatsalya et al., 2020). Patients with AH were classified as moderate (MELD < 20, n = 112) and severe (MELD ≥ 20, n = 152) according to the MELD score at admission as part of either of two independent clinical trials (ClincalTrials.gov identifier # NCT01809132 and NCT03224949) or the NOAC biorepository. These studies were approved by the Institutional Review Boards of all four participating institutions and all study participants consented prior to collection of data and blood samples. Clinical and demographic data for the entire cohort is presented in Figure 1—source data 1 and for the sub-set of subjects used for TMA analysis is presented in Figure 1—source data 2.
In order to be able to measure volatile compounds such as TMA, blood was collected in EDTA-coated tubes and immediately placed on ice. Plasma was separated by centrifugation at 1200× g for 15 min at 4°C. Plasma was rapidly acidified by adding 25 mL of 1 M hydrochloric acid (HCL) to 500 mL of aliquoted plasma, followed by vigorous vortexing. Acidified plasma samples were stored at –80°C in air-tight O-ring cryovials (Fisher Scientific, product # 02-681-373) until being processed for quantification of TMA and other volatile compounds. A non-acidified sample was also collected for standard plasma biochemistries.
Analysis of hepatic FMO3 expression across different liver disease etiologies
For data shown in main Figure 1 panel C, we leveraged access to publicly available bulk liver RNA sequencing data from patients with different liver disease etiologies (Argemi et al., 2019). For this cohort, early AH (EAH) was defined as MELD 7–8, severe AH with liver failure (AHL) with MELD 22–28, and AHL with emergency liver transplant (ExAH) with MELD 18–21. All raw fastq files were downloaded from SRA (PRJNA531223) and dbGAP (phs001807.v1.p1) (Argemi et al., 2019). Fastq files were aligned to the human genome (GRCh38, indices downloaded from https://github.com/pachterlab/kallisto-transcriptome-indices/releases/download/ensembl-96/homo_sapiens.tar.gz; Pachter, 2018) using Kallisto version 0.44.0 with 100 bootstraps calculated (Bray et al., 2016). Data were then merged with clinical data and analyzed with Sleuth in gene_mode with aggregation_column set to Ensemble Gene ID; in addition, extra_bootstrap_summary and read_bootstrap_tpm were set to true (Pimentel et al., 2017). Differential expression was measured with Sleuth using a cutoff of q < 0.05.
Human study populations and sample collection for liver Western blotting
De-identified samples from five livers explanted from severe AH patients during liver transplantation or five wedge biopsies from healthy donor livers were snap-frozen in liquid nitrogen and stored at –80°C. Samples were provided by the Clinical Resource for Alcoholic Hepatitis Investigations at Johns Hopkins University (R24 AA0025107, Z. Sun PI). Written informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institutional Review Boards at Johns Hopkins Medical Institutions. This cohort utilized Maddrey’s Discriminant Function as the primary indicator of disease severity, with an average score of 102.5 ± 27.7. MELD scores are not available for this cohort. Descriptive biochemical and clinical data for this cohort have been reported previously (Tripathi et al., 2018).
Whole tissue homogenates were made from tissues in a modified RIPA buffer as previously described (Warrier et al., 2015; Helsley et al., 2019; Schugar et al., 2017; Lord et al., 2016), and protein was quantified using the bicinchoninic assay (Pierce). Proteins were separated by 4–12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and then proteins were detected after incubation with specific antibodies as previously described (Warrier et al., 2015; Helsley et al., 2019; Schugar et al., 2017; Lord et al., 2016) and listed in the Key resources table.
Real-time PCR analysis of gene expression
Tissue RNA extraction and qPCR analysis was performed as previously described (Helsley et al., 2019). The mRNA expression levels were calculated based on the ΔΔ-CT method using cyclophilin A as the housekeeping gene. qPCR was conducted using the Applied Biosystems 7500 Real-Time PCR system. All primer sequences are listed in the Key resources table.
Chemical synthesis of gut microbe-targeted choline TMA lyase inhibitors
The small molecule choline TMA lyase inhibitors IMC and FMC have been previously described as potent and selective mechanism-based inhibitors targeted microbial CutC (Roberts et al., 2018). Here, IMC and FMC were synthesized and structurally characterized as outlined below using both multinuclear NMR analysis and high-resolution mass spectrometry. 1H- and 13C-NMR spectra for IMC and FMC were recorded on a Bruker Ascend spectrometer operating at 400 MHz. Chemical shifts are reported as parts per million (ppm). IMC iodide was prepared using a previously reported method using 2-dimethylethanolamine and diiodomethane as reactants in acetonitrile followed by recrystallization from dry ethanol. 1H- and 13C-NMRs of IMC were both consistent with that in the reported literature (Mistry et al., 2002), as well as consistent based on proton and carbon chemical shift assignments indicated below. High-resolution MS corroborated the expected cation mass and provided further evidence of structural identity. 1H-NMR (400 MHz, D2O): 5.24 (s, 2H, -N-CH2-I), 4.06–3.99 (m, 2H, -CH2-CH2-OH), 3.68–3.62 (m, 2H, -N-CH2-CH2-), 3.29 (s, 6H, -N(CH3)2); 13C-NMR (100 MHz, D2O): 66.1 (-CH2-CH2-OH), 55.8 (-N-CH2-CH2-), 52.9 (-N(CH3)2), 33.0 (-N-CH2-I); HRMS (ESI/TOF): m/z (M+) calculated for C5H13INO, 230.0036; found, 230.0033. The synthesis of fluoromethylcholine chloride was performed using the procedure below. 1H- and 13C-NMRs of FMC were consistent with that in the reported literature (Gao et al., 2019). High-resolution MS was also consistent with the expected cation mass. Chloro(fluoro)methane (2.05 kg, 29.9 mol, 6 eq) was bubbled into a solution of 2-dimethylaminoethanol (444.0 g, 4.98 mol, 500 mL, 1 eq) in THF (1000 mL) at –70°C for 4 hr. The mixture was then transferred to an autoclave and heated to 80°C and stirred for 18 hr (pressure: ~15–50 psi). During this period, a white precipitate formed. The solid was isolated by filtration, washed with cold THF (600 mL), and dried under vacuum to give fluoromethylcholine chloride as a white solid (1.14 kg, 70.7% yield, 98.0% purity). 1H-NMR (400 MHz, D2O): 5.44 (s, 1H, -N-CH2-F), 5.32 (s, 1H, -N-CH2-F), 4.04–3.98 (m, 2H, -CH2-CH2-OH), 3.60–3.54 (m, 2H, -N-CH2-CH2-), 3.19 (s, 6H, -N(CH3)2); 13C-NMR (100 MHz, D2O): 97.8 and 95.6 (-N-CH2-F), 62.9 (-CH2-CH2-OH), 55.1 (-N-CH2-CH2-), 48.0 (-N(CH3)2); HRMS (ESI/TOF): m/z (M+) calculated for C5H13FNO (M+) 122.0976, found 122.0975.
Ethanol feeding trials in mice
All mice were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care, International-approved animal facility. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the Cleveland Clinic. Age- and weight-matched female C57BL6/J mice were randomized into pair- and ethanol-fed groups and adapted to control liquid diet for 2 days. Two models of chronic ethanol feeding were used. (1) A 25-day chronic model in which mice were allowed free access to increasing concentrations of ethanol for 25 days (i.e. chronic feeding model) as previously described (McCullough et al., 2018). In this model, the ethanol-fed mice were acclimated to ethanol as follows: 1% vol/vol for 2 days, 2% vol/vol for 2 days, 4% vol/vol (22% kcal) for 1 week, 5% vol/vol (27% kcal) for 1 week, and last 6% vol/vol (32% kcal) for 1 week and is denoted as 32%, day 25. (2) A 10-day chronic model in which mice were allowed free access to a 5% vol/vol (27% kcal) for 10 days (Bertola et al., 2013). Ethanol-fed mice were allowed ad libitum access to liquid diet. Control mice were pair-fed a diet that received isocalorically substituted maltose dextrin for ethanol. Some cohorts received choline TMA lyase inhibitors IMC (0.06% wt/wt) or FMC (0.006% wt/wt) in these liquid diets throughout the entire 10- to 25-day feeding period. Lieber-DeCarli high-fat ethanol and control diets were purchased from Dyets (catalog number 710260; Bethlehem, PA).
Female C57BL6/J mice at 10.5 weeks of age were injected intraperitoneally with either 15 mg/kg LPS (500 µg/mL, Sigma L4391) or a matched volume (30 mL/kg) of sterile saline. After 6 hr, mice were euthanized with ketamine/xylazine and the liver was immediately collected and homogenized in TRIzol. RNA was extracted using chloroform phase separation and purified using Qiagen RNeasy kit.
Liver histology and immunohistochemistry
For histological analysis, formalin-fixed tissues were paraffin embedded, sectioned, and stained with hematoxylin and eosin. Formalin-fixed samples are coded at the time of collection for blinded analysis.
Measurement of plasma aminotransferase levels
To determine the level of hepatic injury in mice, plasma was used to quantify ALT and AST levels using a commercially available enzymatic assay (Sekisui Diagnostics, Lexington, MA) according to manufacturer’s instruction.
Measurement of hepatic lipid levels
Extraction of liver lipids and quantification of total plasma and hepatic triglycerides, cholesterol, and cholesterol esters was conducted using enzymatic assays as described previously (Warrier et al., 2015; Helsley et al., 2019; Schugar et al., 2017; Lord et al., 2016).
Quantification of TMA-related metabolites in acidified plasma
Stable isotope dilution high-performance liquid chromatography with on-line tandem mass spectrometry (LC-MS/MS) was used for quantification of levels of TMAO, TMA, choline, carnitine, betaine, and γ-butyrobetaine in plasma, as previously described (Wang et al., 2014a). Their d9(methyl) isotopologues were used as internal standards. LC-MS/MS analyses were performed on a Shimadzu 8050 triple quadrupole mass spectrometer. IMC and d2-IMC, along with other metabolites, were monitored using multiple reaction monitoring of precursor and characteristic product ions as follows: m/z 230.0 → 58.0 for IMC; m/z 232.0 → 60.1 for d2-IMC; m/z 76.0 → 58.1 for TMAO; m/z 85.0 → 66.2 for d9-TMAO; m/z 60.2 → 44.2 for TMA; m/z 69.0 → 49.1 for d9-TMA; m/z 104.0 → 60.1 for choline; m/z 113.1 → 69.2 for d9-choline; m/z 118.0 → 58.1 for betaine; m/z 127.0 → 66.2 for d9-betaine.
Cecal microbiome analyses
16S rRNA amplicon sequencing were done for V4 region using via miSEQ from mouse cecal contents. Raw 16S amplicon sequence and metadata were demultiplexed using split_libraries_fastq.py script implemented in QIIME1.9.1 (Caporaso et al., 2010). Demultiplexed fastq file was split into sample specific fastq files using split_sequence_file_on_sample_ids.py script from Qiime1.9.1 (Caporaso et al., 2010). Individual fastq files without non-biological nucleotides were processed using Divisive Amplicon Denoising Algorithm (DADA) pipeline (Callahan et al., 2016). The output of the dada2 pipeline (feature table of amplicon sequence variants [an ASV table]) was processed for alpha and beta diversity analysis using phyloseq (McMurdie and Holmes, 2013), and microbiomeSeq (http://www.github.com/umerijaz/microbiomeSeq) packages in R. Alpha diversity estimates were measured within group categories using estimate_richness function of the phyloseq package (McMurdie and Holmes, 2013). Multidimensional scaling (also known as principal coordinate analysis [PCoA]) was performed using Bray-Curtis dissimilarity matrix (Knorr et al., 2020) between groups and visualized by using ggplot2 package (Wickham, 2009). We assessed the statistical significance (p < 0.05) throughout and whenever necessary, we adjusted p-values for multiple comparisons according to the Benjamini and Hochberg method to control false discovery rate (Benjamini, 2010) while performing multiple testing on taxa abundance according to sample categories. We performed an analysis of variance (ANOVA) among sample categories while measuring the of alpha diversity measures using plot_anova_diversity function in microbiomeSeq package (http://www.github.com/umerijaz/microbiomeSeq). Permutational multivariate analysis of variance (PERMANOVA) with 999 permutations was performed on all principal coordinates obtained during PCoA with the ordination function of the microbiomeSeq package. Wilcoxon (non-parametric) test was performed on ASV’s abundances against metadata variables levels using their base functions in R (Tilt, 1999).
RNA sequencing in mouse tissues
RNA sequencing libraries were generated from mouse liver using the Illumina mRNA TruSeq Directional library kit and sequenced using an Illumina HiSeq4000 (both according to the manufacturer’s instructions). RNA sequencing was performed by the University of Chicago Genomics Facility, and data analysis and data availability are described in detail in the online supplement. Briefly, RNA samples were checked for quality and quantity using the Bio-analyzer (Agilent). RNA sequencing libraries were generated using the Illumina mRNA TruSEQ Directional library kit and sequenced using an Illumina HiSEQ4000 (both according to the manufacturer’s instructions). RNA sequencing was performed by the University of Chicago Genomics Facility. Raw sequence files will be deposited in the Sequence Read Archive before publication (SRA). Single-end 100 bp reads were trimmed with Trim Galore (v.0.3.3, https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/. ) and controlled for quality with FastQC (v0.11.3, http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc) before alignment to the Mus musculus genome (Mm10 using UCSC transcript annotations downloaded July 2016). Reads were aligned using the STAR alignerSTAR in single pass mode (v.2.5.2a_modified, RRID:SCR_004463, https://github.com/alexdobin/STAR) with standard parameters. Raw counts were loaded into R (http://www.R-project.org/) and edgeR was used to perform upper quantile, between-lane normalization, and DE analysis. Values generated with the cpm function of edgeR, including library size normalization and log2 conversion, were used in figures. Heat maps were generated of top 50 differentially expressed transcripts using pheatmap. Reactome-based pathway analysis was performed using an open-sourced R package: ReactomePA. RNA sequencing data have been deposited into the National Institutes of Health (NIH)-sponsored GEO repository (accession number GSE157681).
Phosphoproteomics analyses to examine TMA-induced signaling events in mouse liver
The goal of this experiment was to unbiasedly identify TMA-responsive signaling events in mouse liver after an acute exposure (10 min) of TMA. To closely mimic physiological route of delivery, we delivered saline or TMA directly into the portal vein in fasted mice. Briefly, C57BL/6 mice were fasted overnight (12 hr fast), and between the hours of 9:00–10:00 am (2–3 hr into light cycle), mice were anesthetized using isoflurane (4% for induction and 2% for maintenance). Once fully anesthetized, a midline laparotomy was performed, and the portal vein was visualized under a Leica M650 surgical microscope. Briefly, a fresh 10 mM stock of trimethylamine hydrochloride (TMA-HCL) made in sterile saline, and the pH of stock solution was adjusted to 7.4. Mice then received 20 μL of either saline vehicle or TMA-HCL via direct syringe infusion (Becton-Dickson product #309306); 9.75 min later a small aliquot (50 μL) of portal blood was collected by pulling back on injection syringe left in place following injection. In saline vehicle injected mice, portal blood levels of TMA ranged from 0.49 to 2.22 μM and TMAO levels ranged from 2.53 to 7.14 μM. In mice injected with TMA-HCL, portal blood levels of TMA ranged from 125.36 to 319.55 μM and TMAO levels ranged from 9.68 to 17.48 μM. Exactly 10 min after initial injection, the liver was rapidly snap-frozen by immersion in liquid nitrogen. Liver samples were homogenized, the protein was precipitated with acetone, and the protein concentration was measured. A total of 1 mg of protein from each sample was digested with trypsin and the resulting tryptic peptides were subjected to phosphoserine and phosphothreonine enrichment using the Thermo Scientific Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit (Fisher # PI88301). The enrichment was performed based on the manufacturer’s instructions. The enriched peptide samples were subjected to C18 clean-up prior to LC-MS analysis. The LC-MS system was a Finnigan LTQ-Obitrap Elite hybrid mass spectrometer system. The HPLC column was a Dionex 15 cm × 75 µm id Acclaim Pepmap C18, 2 μm, 100 Å reversed-phase capillary chromatography column. Five μL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.25 μL/min were introduced into the source of the mass spectrometer on-line. The microelectrospray ion source is operated at 1.9 kV. The digest was analyzed using the data-dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. The LC-MS/MS data files were searched against the mouse UnitProtKB database (downloaded in December 2019 contains 17,017 sequences) using Sequest bundled into Proteome Discoverer 2.4. Cysteine carbamidomethylation was set as a fixed modification and oxidized methionine, protein N-terminal acetylation, and phosphorylation of serine, threonine, and tyrosine were considered as dynamic modification. A maximum of two missed cleavages were permitted. The peptide and protein false discovery rates were set to 0.01 using a target-decoy strategy. Phosphorylation sites were identified using ptmRS node in PD2.4. The relative abundance of the positively identified phosphopeptides was determined using the extracted ion intensities (Minora Feature Detection node) with Retention time alignment. All peptides were included in the quantitation, the peptide intensities were normalized to total peptide amount. Missing values were imputed in Perseus using a normal distribution. A total of 789 phosphopeptides were identified with 36 phosphopeptides determined to be two-fold different in the TMA and saline samples with a p-value < 0.05 (t-test).
All statistical analyses were performed using GraphPad Prism and p < 0.05 was considered statistically significant. All data are presented as mean ± SEM, unless otherwise noted in the figure legends. All data were tested for equal variance and normality. For two-group comparison of parametric data, a two-tailed Student’s t-test was performed, while non-parametric data were analyzed with Mann-Whitney U test (also called the Wilcoxon rank-sum test). For studies comparing vehicle and TMA lyase inhibitors in pair- and ethanol-fed mice, a two-way ANOVA was performed, followed by Tukey’s tests for post hoc analysis. For human studies in AH patients, statistical significance was determined by ANOVA and a Tukey’s honest significant difference post hoc test (p < 0.05).
Sequencing data have been deposited in GEO under accession code GSE157681.
NCBI Gene Expression OmnibusID GSE157681. The Gut Microbe-Derived Metabolite Trimethylamine is a Biomarker of and Therapeutic Target in Alcohol-Associated Liver Disease.
Metabolomics studies identify novel diagnostic and prognostic indicators in patients with alcoholic hepatitisWorld Journal of Hepatology 8:499–508.https://doi.org/10.4254/wjh.v8.i10.499
Discovering the false discovery rateJournal of the Royal Statistical Society 72:405–416.https://doi.org/10.1111/j.1467-9868.2010.00746.x
Mouse model of chronic and binge ethanol feeding (the NIAAA model)Nature Protocols 8:627–637.https://doi.org/10.1038/nprot.2013.032
Short-term high-fat diet increases postprandial trimethylamine-N-oxide in humansNutrition Research (New York, N.Y.) 35:858–864.https://doi.org/10.1016/j.nutres.2015.07.002
Near-optimal probabilistic RNA-seq quantificationNature Biotechnology 34:525–527.https://doi.org/10.1038/nbt.3519
The gut microbial endocrine organ: bacterially derived signals driving cardiometabolic diseasesAnnual Review of Medicine 66:343–359.https://doi.org/10.1146/annurev-med-060513-093205
Characterization of fecal microbial communities in patients with liver cirrhosisHepatology (Baltimore, Md.) 54:562–572.https://doi.org/10.1002/hep.24423
Alcohol-induced liver injury is modulated by Nlrp3 and Nlrc4 inflammasomes in miceMediators of Inflammation 2013:751374.https://doi.org/10.1155/2013/751374
Serum and Fecal Oxylipins in Patients with Alcohol-Related Liver DiseaseDigestive Diseases and Sciences 64:1878–1892.https://doi.org/10.1007/s10620-019-05638-y
Current understanding of the human microbiomeNature Medicine 24:392–400.https://doi.org/10.1038/nm.4517
Targeted Inhibition of Gut Microbial Trimethylamine N-Oxide Production Reduces Renal Tubulointerstitial Fibrosis and Functional Impairment in a Murine Model of Chronic Kidney DiseaseArteriosclerosis, Thrombosis, and Vascular Biology 40:1239–1255.https://doi.org/10.1161/ATVBAHA.120.314139
The breathprints in patients with liver disease identify novel breath biomarkers in alcoholic hepatitisClinical Gastroenterology and Hepatology 12:516–523.https://doi.org/10.1016/j.cgh.2013.08.048
The NLRP3 Inflammasome in Alcoholic and Nonalcoholic SteatohepatitisSeminars in Liver Disease 40:298–306.https://doi.org/10.1055/s-0040-1708540
BookDeaths: Final Data for 2017. National Vital Statistics ReportsNational Center for Health Statistics.
Short-term probiotic therapy alleviates small intestinal bacterial overgrowth, but does not improve intestinal permeability in chronic liver diseaseEuropean Journal of Gastroenterology & Hepatology 26:1353–1359.https://doi.org/10.1097/MEG.0000000000000214
Microbiota and Fatty Liver Disease-the Known, the Unknown, and the FutureCell Host & Microbe 28:233–244.https://doi.org/10.1016/j.chom.2020.07.007
Comparison of the effect of daily consumption of probiotic compared with low-fat conventional yogurt on weight loss in healthy obese women following an energy-restricted diet: a randomized controlled trialThe American Journal of Clinical Nutrition 103:323–329.https://doi.org/10.3945/ajcn.115.120170
Epidemiology and Natural History of Alcoholic Liver DiseaseReviews on Recent Clinical Trials 11:167–174.https://doi.org/10.2174/1574887111666160810101202
Complement Factor D protects mice from ethanol-induced inflammation and liver injuryAmerican Journal of Physiology. Gastrointestinal and Liver Physiology 315:G66–G79.https://doi.org/10.1152/ajpgi.00334.2017
Intestinal dysbiosis: a possible mechanism of alcohol-induced endotoxemia and alcoholic steatohepatitis in ratsAlcoholism, Clinical and Experimental Research 33:1836–1846.https://doi.org/10.1111/j.1530-0277.2009.01022.x
Colonic microbiome is altered in alcoholismAmerican Journal of Physiology. Gastrointestinal and Liver Physiology 302:G966–G978.https://doi.org/10.1152/ajpgi.00380.2011
Nonlethal Inhibition of Gut Microbial Trimethylamine N-oxide Production Improves Cardiac Function and Remodeling in a Murine Model of Heart FailureJournal of the American Heart Association 9:e016223.https://doi.org/10.1161/JAHA.119.016223
Structure-Guided Identification of a Small Molecule That Inhibits Anaerobic Choline Metabolism by Human Gut BacteriaJournal of the American Chemical Society 141:33–37.https://doi.org/10.1021/jacs.8b04883
Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cellsHepatology (Baltimore, Md.) 37:1043–1055.https://doi.org/10.1053/jhep.2003.50182
The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitisHepatology (Baltimore, Md.) 67:1284–1302.https://doi.org/10.1002/hep.29623
Biomarkers of Macrophage Activation and Immune Danger Signals Predict Clinical Outcomes in Alcoholic HepatitisHepatology (Baltimore, Md.) 70:1134–1149.https://doi.org/10.1002/hep.30617
Fecal Microbiome Distinguishes Alcohol Consumption From Alcoholic Hepatitis But Does Not Discriminate Disease SeverityHepatology (Baltimore, Md.) 72:271–286.https://doi.org/10.1002/hep.31178
Intestinal microbial metabolism of phosphatidylcholine and cardiovascular riskThe New England Journal of Medicine 368:1575–1584.https://doi.org/10.1056/NEJMoa1109400
The contributory role of gut microbiota in cardiovascular diseaseThe Journal of Clinical Investigation 124:4204–4211.https://doi.org/10.1172/JCI72331
The gut-liver axis and the intersection with the microbiomeNature Reviews. Gastroenterology & Hepatology 15:397–411.https://doi.org/10.1038/s41575-018-0011-z
Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in miceHepatology (Baltimore, Md.) 34:101–108.https://doi.org/10.1053/jhep.2001.25350
Keratin 18 Is a Diagnostic and Prognostic Factor for Acute Alcoholic HepatitisClinical Gastroenterology and Hepatology 18:2046–2054.https://doi.org/10.1016/j.cgh.2019.11.050
BookGgplot2: Elegant Graphics for Data AnalysisNew York, NY: Springer Publishing Company, Incorporated.https://doi.org/10.1007/978-0-387-98141-3
Enteric dysbiosis associated with a mouse model of alcoholic liver diseaseHepatology (Baltimore, Md.) 53:96–105.https://doi.org/10.1002/hep.24018
[Editors' note: we include below the reviews that the authors received from another journal, along with the authors’ responses.]
We have spent the last 9 months performing additional experiments to address reviewer concerns, including several additional mouse studies as well as increasing the number of patient samples under investigation. Here we provide a point-by-point response to each reviewer comment.
Board of Editors:
Interesting study, which is unfortunately too preliminary for some aspects of the work. Some additional analyses are needed based on the following major comments:
1. Many mouse experiments have limited sample size (n=4 to 5) without replication. Increase in the sample size and/or independent replications are needed.
To address this valid point, we have conducted additional mouse experiments to increase the reproducibility of the results. As reviewer #2 astutely pointed out, there is an interaction with IMC + ethanol leading to reduced food intake (Supplemental Figure 2). Given this, we chose to focus the additional mouse experiments on the more potent TMA lyase inhibitor, FMC. Data shown in the original submission indicate that FMC treatment reduces ethanol-induced liver injury in a chronic model of ethanol feeding (Figure 3). In the revised manuscript, we have treated mice with the TMA lyase inhibitor FMC in a separate model of ethanol-induced liver injury. This has allowed us to both replicate findings in another model and also increase sample size. These data are included in Supplementary Figure 3.
2. Alcohol increases TMA in Figure 2 but not in Figure 3. Please justify this discrepancy. It might resolve, if the sample size is expanded to sufficient numbers.
We agree that there is a discrepancy between TMA and TMAO level across the different studies done. We suspect that this is in large part due to the timing of when the blood samples were collected, which is a critical determinant of circulating TMA and TMAO. We and others have previously reported (PMIDs: 29172946, 26265295, 26465927, 34405585, 23614584 ) that TMA and TMAO levels are highly dependent on the time of the last meal (i.e. when dietary substrates are presented to gut microbes). Furthermore, in another project we have under late stage revision at a preprint server (https://www.biorxiv.org/content/10.1101/2020.12.04.411546v1) we have found that the TMAO pathway is under complex circadian regulation and the highest levels are actually at night when mice are actively eating. Collectively, we have come to appreciate that plasma TMA and TMAO levels can be dramatically different based on the timing of the last meal as well under circadian regulation. However, it is important to note that the studies here were not designed to evaluate the peak levels of TMA and TMAO, and instead aimed to test the ability of TMA lyase inhibitors (IMC and FMC) to blunt the chronic effects of ethanol feeding. It is most common to perform necropsies for such ethanol feeding trials during the early morning to avoid circadian changes, so we felt it was important to stay consistent in our goal to focused on ethanol-induced liver injury phenotypes. Although the levels of TMA are not consistently altered across all studies in this manuscript, we do not feel this negatively impacts the primary outcome of the study that gut microbe-targeted TMA lyase inhibitors improve ethanol-induced liver injury. Regardless of whether ethanol either acutely or chronically elevates TMA levels, our data show that gut microbe-targeted TMA lyase inhibitors can provide some benefit in different mouse models of ethanol-induced liver injury which is an important advance.
3. Another discrepancy is the lack of difference in circulating TMA between moderate and severe alcoholic hepatitis while hepatic FMO3 is reduced in severe AH but not in "early AH". This suggests that a decreased FMO3 does not explain why TMA is high during AH.
The liver FMO3 mRNA levels in the early AH start to decrease compared to healthy controls, albeit not statistically significant. We suspect that the level of hepatic inflammation likely dictates the level of FMO3 expression. To directly test this hypothesis, we performed a new experiment in mice where we injected a high dose of the toll like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) to induce strong hepatic inflammation, and see that Fmo3 mRNA expression is reduced by ~ 70% in mouse liver (Figure 1E). These new data show that activation of TLR4-dependent signaling, which is a hallmark of ethanol consumption, promotes reduction in Fmo3 expression. Serendipitously, in this same experiment we also found that the only known host TMA receptor (PMID: 23393561, 23177478) called trace-amine associated receptor 5 (TAAR5) was upregulated by LPS treatment. We agree with the possibility that decreased FMO3 may not be the only reason for increased TMA in alcohol-associated hepatitis patient or in mouse models of ethanol consumption, and it is very likely that broad changes in gut microbiome community structure (i.e. overgrowth of TMA producers) is also likely involved.
4. In line with the reviewers' comments, more experiments are needed to provide mechanistic insight (see comments by reviewer #1, more severe ALD models, FMO3-ko mice).
We appreciate these suggestions and have spent the past 9 months performing additional models of ethanol-induced liver injury using our lead TMA lyase inhibitor (FMC), and also performed ethanol feeding studies in mice genetically lacking Fmo3 (Fmo3-ko). Results from these studies are discussed below and/or included in the revised manuscript.
Several recent studies reported that breath levels of the primary metabolite TMA and other related cometabolites are elevated in ALD patients. In the current paper, the authors reported that serum levels of TMA not TMAO were elevated in patients with moderate or severe AH compared to healthy controls. By using chronic ethanol feeding model with the treatment of CutC/D inhibitors, the authors found that small molecule inhibition of gut microbial CutC/D activity protected mice from ethanol-induced liver injury. Finally, the authors performed cecal microbiome analyses (Fig, 4) and liver RNA seq analyses (Figure 5) from CutC/D inhibitor-treated ethanol-fed mice and phosphoproteomics analyses of TMA infused liver (Fig, 6).
Although the authors confirmed elevation of TMA in patients with AH and in chronic ethanol-fed mice, the role of TMA was tested in chronic ethanol-fed mice with two inhibitors, the molecular mechanisms by which TMA contributes to the pathogenesis of AH are not clear. The important role of TMA in AH should be tested in more severe ALD models and in FMO3 knockdown or knockout mice.
1. The liver enzyme flavin-containing monooxygenase 3 (FMO3) is the predominant TMA to TMAO converting enzyme in the adult liver. In Figure 1C, the authors show that mRNA levels for FMO3 are uniquely repressed in patients with AH, but not in other liver disease etiologies such as NAFLD or viral hepatitis. However, are the expression levels of FMO3 related to the disease severity (such as the loss of hepatocytes)? In Figure 1C, the authors used AH with liver failure (AHL, n = 18), explant tissue from patients with severe AH; however, NAFLD, HCV, and HCV cirrhosis seem less severe compared to AH samples. It will be interested to test whether serum TMA levels are also elevated in patients with end-stages of NAFLD or HCV, and whether hepatic expression of FMO3 mRNA is decreased in these samples.
The reviewer raises a valid point about the loss of hepatocytes as the culprit behind the FMO3 expression levels across varying liver etiologies. However, this is a difficult thing to model in rodents as is the focus of this current study. We agree that it would be of interest to measure plasma TMA in the same patients described in Figure 1C (i.e. across different liver disease etiologies). However, the residual plasma samples from those patients were not properly acidified precluding us from being able to detect TMA. It is important to note that since our original submission several new papers have emerged showing that plasma TMAO levels are actually increased in subjects with non-alcoholic fatty liver disease (NAFLD) and are associated with non-alcoholic steatohepatitis (NASH) in obese subjects with type 2 diabetes (PMIDs: 32791310, 33993608). Interestingly, elevated levels of TMAO in NAFLD is associated with increase all-cause mortality (PMID: 33993608). Another recent study reported that circulating TMAO is reduced in subjects with advanced hepatitis B-related hepatocellular carcinoma (PMID: 27122669). Although beyond the scope of this body of work, these results indicate that additional validation cohorts are needed to determine whether the TMA-FMO3TMAO is specifically altered in different liver disease etiologies.
2. In Figure 1, serum TMA levels were much higher in patients with moderate or severe AH. How about serum TMA levels in alcoholics without liver diseases and alcoholic cirrhosis?
At this point we do not have access to acidified plasma or serum samples from alcoholics with liver disease and/or alcoholic cirrhosis. We are attempting to actively recruit such patients through our collaborative network, but feel this is beyond the scope of the current manuscript.
3. Hepatic expression of FMO3 is markedly downregulated in severe AH; How about hepatic expression of FMO3 in ethanol-fed mice in Figure 2, 3? It will be interesting to test whether knockdown or knockout of FMO3 elevates TMA and liver injury in mice after ethanol feeding.
We have spent substantial effort to address this extremely important issue, and provide here a summary of the findings. First, over 5 year ago we used an antisense oligonucleotide (ASO) knockdown approach that we have previously published (PMID: 25600868, 28636934) to reduce hepatic Fmo3 expression in ethanol-challenged mice. See Author response image 1, where there was a striking exacerbation of ethanol-induced inflammation and liver enzyme levels (ALT) in Fmo3 knockdown mice, yet an attenuation of ethanol-induced hepatic steatosis.
Although these data are striking, we have decided not to include these data in the revised manuscript here because we have discovered and published on the
potential off target effects of Fmo3 ASOs (PMID: 31070450) and want to ensure high rigor and reproducibility in our published results.
After receiving the original reviewer comments in January 2021, as a more rigorous genetic approach we immediately bred littermate Fmo3+/+ and Fmo3-/- for ethanol feeding trials. This has been no small task to complete during the COVID-19 pandemic, but we were able to get 3 separate cohorts of mice through the chronic Lieber DiCarli ethanol feeding model. However, the results are somewhat inconclusive because in these studies there was a very modest effect of ethanol even in wild type (Fmo3+/+) mice (see Author response image 2). Although our line of Fmo3-/- mice has been backcrossed to C57BL/6J background, we suspect that in this current state of backcrossing that this mouse line is simply resistant to ethanol-induced liver injury (which we have seen to be very strain specific), which preclude strong interpretations. Although none of these results with Fmo3 loss-of-function satisfactorily address the question at hand, we wanted to be completely transparent of our attempts to be responsive to the reviewers. If you do feel these Fmo3 loss-of-function experiments are important to show in the manuscript, we are happy to include these data with clear discussion of the caveats.
4. The authors show that serum levels of TMA and TMAO are elevated in ethanol-fed mice in Figure 2A, B, but such elevation was not observed in Figure 3A, B. These results are inconsistent.
As described above, we fully acknowledge that there is a discrepancy between TMA and TMAO level across the different studies done. We suspect that this is in large part due to the timing of when the blood samples were collected, which is a critical determinant of circulating TMA and TMAO. We and others have previously reported (PMIDs: 29172946, 26265295, 26465927, 34405585, 23614584 ) that TMA and TMAO levels are highly dependent on the time of the last meal (i.e. when dietary substrates are presented to gut microbes). Furthermore, in another project we have under late stage revision at a preprint server (https://www.biorxiv.org/content/10.1101/2020.12.04.411546v1) we have found that the TMAO pathway is under complex circadian regulation and the highest levels are actually at night when mice are actively eating. Collectively, we have come to appreciate that plasma TMA and TMAO levels can be dramatically different based on the timing of the last meal and circadian regulation. However, it is important to note that the studies here were not designed to look at the peak levels of TMA and TMAO, and instead designed to test the ability of TMA lyase inhibitors (IMC and FMC) to blunt the chronic effects of ethanol feeding. It is most common to perform necropsies for such ethanol feeding trials during the early morning, so we felt it was important to stay consistent in our goal to focused on ethanol-induced liver injury phenotypes. Although the levels of TMA are not consistently altered across all studies in this manuscript, we do not feel this negatively impacts the primary outcome of the study that gut microbe-targeted TMA lyase inhibitors improve ethanol-induced liver injury. Regardless of whether ethanol either acutely or chronically elevates TMA levels, our data show that gut microbe-targeted TMA lyase inhibitors can provide some benefit in different mouse models of ethanolinduced liver injury which is an important advance.
5. Although the authors performed cecal microbiome analyses (Fig, 4) and liver RNA seq analyses (Figure 5) from CutC/D inhibitor-treated ethanol-fed mice, and phosphoproteomics analyses of TMA infused liver (Fig, 6), these results did not really give clear mechanisms how TMA contributes to the pathogeneses of ALD.
We agree that our current data only provide potential clues into mechanism, and additional studies will be required to fully understand the mechanism by which TMA contributes to the pathogenesis of ALD. Despite this, we feel strongly that the major impact of this manuscript stems from our seminal observation that TMA is elevated in AH patients, and that selective inhibition of the gut microbial enzyme that produces TMA from choline (CutC/D) protects mice from ethanol-induced liver injury. This is the first ever demonstration that a bacterially-targeted enzyme inhibitor can provide benefit in preclinical animal models of ALD, and bolsters the emerging concept that drugging microbial metabolism can potentially improve human health (PMID: 28389555).
In abstract, line 27, "non-lethal non-lethal" is duplicate
This has been addressed in the revised abstract
Line 22 page 6, is "microbe-associated molecule patterns (MAMPs)" the same as " Pathogenassociated molecule patterns (PAMPs)"? if yes, it is better to use PAMPs.
We feel that MAMP is more appropriate here because PAMP also includes viral molecular patterns which is not the focus of this story, which is gut microbiome focused.
The aim of the paper was to understand how the gut microbioal TMA/TMAO pathway may contribute to alcoholic liver disease (ALD) susceptibility and progression.
The paper and the subject are really interesting. The authors demonstrate that this pathway is involved in alcohol-fed mice and correlated the results in human ALD.
1. The cohort of patients is not well described. Suppl Table 1 does not includes any clinical data (patients characteristics, alcohol consumption, liver function, liver enzymes…).
We have included a more detailed description of the study subjects in the revised Supplemental Tables 1 and 2.
2. Sometimes the authors use the words moderate/severe alcoholic hepatitis, and sometimes early/advanced/and liver failure. This is really unclear. What kind of patients are studied. Severe AH is not synonymous to advanced liver disease. Are several different cohorts of patients included? Are patients in Figure 1A and 1C similar of different. If yes, it must be explained. The number of patients is very low in Figure 1C.
We apologize for the lack of clarity and have removed any description of advanced liver disease. Instead, we have clearly indicated the subjects under study were patients with either moderate or severe alcoholic hepatitis.
3. Even if the “magic p< 0.05” is obtained in Figure 1A and 1B, the are many overlaps of values between healthy controls and alcoholic patients rendering unlikely the use of TMA and TMAO as marker of the disease. Moreover, what does it mean that TMA/TMAO is a marker? A marker of what? This overlap should be emphasized and discussed. The title, the section “what you need to know”, the discussion should be modified accordingly.
We agree that our study was not designed to establish TMA and TMAO as “biomarker” of alcoholic hepatitis, so the sections mentioned have been updated accordingly. We have removed any discussion of TMA or TMAO being a “marker” or “biomarker” throughout this manuscript.
4. Figure 2 and 3. Are control mice similar in these 2 figures? I don’t think so as the values of controls in the two figures are different. Therefore, how the authors explain twice the absence of Cyp2e1 induction in alcohol-fed mice in terms of mRNA (Figure 2N, 3N) and the clear induction of Cyp2e1 in terms of protein (Figure 2O, 3O). Accordingly, in FMC-treated mice, there is a slight increase of Cyp2e1 mRNA whereas the authors conclude to a blunted induction of CYP2E1 protein. I don’t understand these discrepancies. Figure 2M, what is the interpretation of TNFα decrease with treatment whereas alcohol intake does not increase its level ? Moreovere, there is no modification of TNFα in Figure 3M.
Ethanol feeding studies require mice to be pair-fed to control for caloric intake. Therefore, the control mice in Figures 2 and 3 are different. The pair-fed control mice in Figure 2 were used for the IMC experiment and the pair-fed control mice in Figure 3 were used for the FMC experiment. Upon further investigation into Cyp2e1 mRNA and CYP2E1 protein levels in additional mouse studies, we did not find a consistent difference in all studies so we have removed the Cyp2e1 data altogether. To further confirm that ethanol metabolism was not altered by our TMA lyase inhibitors, we also provide new data showing blood ethanol levels which were not altered by FMC treatment. Although less than satisfying, It is not uncommon to see variable ethanol-induced effects on cytokine levels (i.e. TNFa) in different ethanol-feeding trials in mice. We simply report the data for each study for full transparency.
5. There is a reduction of food intake in mice receiving IMC, therefore, all the results may be related to a decrease of alcohol consumption. Accordingly, food intake and weight gain/loss should be provided for FMC-treated mice. Is alcohol consumption and/or blood/urine concentration level similar between control and FMC-treated mice?
We do agree the protection with IMC is likely partly explained by the reduction in food intake. However, we feel it is important to include the IMC study data (in comparison to FMC effects) so that future research can focus on FMC or other downstream inhibitors that lack this potentially confounding effect. Food intake data (which directly reports on ethanol consumption) are included for all studies, and we have also now included blood alcohol levels for control versus FMC-treated mice which were similar.
6. Figure 6. Infusion of TMA into the portal circulation. No material/method are provided for this experiment. The authors write “we infused physiological levels of TMA directly into the portal circulation draining the gut (i.e. portal vein) of fasted mice”. What is physiological concentration of TMA in the portal vein in the model? In figures 2 and 3 plasma level are provided but not portal vein level. Which dose of TMA was infused? Is this dose similar to the physiological concentrations (in control and/or ethanol-fed) or higher? Does this injection modify lipid metabolism/genes as the major phenotype of alcohol-fed mice is liver steatosis ? I don’t understand the relationships between the reported modifications in this experiment and the phenotype of the mice.
We apologize for the limited details provided for this experiment, and apparent lack of clarity behind this study. The purpose of this experiment was to understand for the first time what signaling events occur acutely after TMA is sensed by the mouse liver. To address this we directly administered TMA via the portal vein (as it would be delivered from gut microbes) to unbiasedly characterize the acute (10 minute in vivo stimulation) signaling events in the liver. This study is important because there are essentially no data in the literature focused on defining TMA-induced signal transduction in the liver, other than the few papers describing that TMA can activate the G protein coupled receptor Trace amine-associated receptor 5 (TAAR5). Current published results only show that TMA can activate the Gs-α-coupled TAAR5 receptor using reporter systems (PMID: 23393561, 23177478). Our study is the first to characterize the acute effects of TMA on the global liver phosphoproteome, and provides the first clues into what major phosphorylation events occur acutely. These findings will allow downstream mechanistic investigation into TMA-induced signaling in future studies.
As described in the online supplement portal blood levels of TMA in normal fasted mice typically range from 0.49-2.2 mM (i.e. this is the baseline fasted level). Under our experimental conditions exactly 10 minutes after portal administration, we achieved a portal blood TMA range of 125.4-320.6 mM. Admittedly this is on the high end of physiological concentrations, we have seen TMA levels in the millimolar range in Fmo3-/- mice fed a high choline diet.
The detailed protocol description can be found in subsection “Phosphoproteomics Analyses to Examine TMA-Induced Signaling Events in Mouse Liver”
“The goal of this experiment was to unbiasedly identify TMA-responsive signaling events in mouse liver after an acute exposure (10 minutes) of TMA. […] Missing values were imputed in Perseus using a normal distribution. A total of 789 phosphopeptides were identified with 36 phosphopeptides determined to be two-fold different in the TMA and Saline samples with a p-value <0.05 (t-test).”
7. The Discussion section is too long and sometimes off topic.
We apologize for this; we have cut the discussion down to make it more clear and concise.
8. “What you need to know” section should be modified. The new findings cannot include TMA as a biomarker of advanced AH. 1- it has not been demonstrated; (2) “advanced” AH is not a disease; the name of the disease is “severe AH”. Impact/short summary section: too general; should focus on the results of the paper.
We apologize for this; all sections have been modified to reflect these suggestions. We have removed any discussion of “advanced AH” and have removed any indication of biomarker status.
Second decision letter and responses:
Response to Reviewer 2:
1. I have recommended to the author to clarify the definitions: alcoholic hepatitis / early / moderate / advanced. It has not been done in the whole manuscript: alcohol-associated hepatitis is not synonymous to severe alcoholic hepatitis: figure 1 C and its legend (early alcoholic hepatitis, alcoholic hepatitis with liver failure); lay summary… It really seems that these sections were not carefully reread.
We apologize for not completely responding to the reviewer’s suggestion in the revision. We have now carefully clarified the phenotypes of the patients with ALD used in our analysis. In part, some of the confusion may have from the fact that the available patient samples and datasets used slightly different enrollment criteria. We have clarified this and noted the different diagnostic criteria between data sets as a limitation to the study. We have included a revision of the supplemental Tables including more detailed description of the patients, and have updated the manuscript throughout to accurately reflect the patient diagnosis.
2. Supplemental Tables 1 and 2 are now written. The authors choose to define the severity alcoholic hepatitis according to the MELD score and not the Maddrey's discriminant function. Nevertheless, they obviously write (page 13 line 32) that Maddrey's discriminant function and Child-Pugh score are important. Therefore they should include these scores in supplemental tables 1 and 2. Moreover, in these 2 tables, the units of laboratory data must be given. The amount of alcohol consumption should be included.
These are now included as the reviewer requests. We have also included MDF and Child-Pugh, when available. Alcohol intake has been reported via an AUDIT questionnaire, which is an alcohol use disorders identification test; this has been provided in both supplemental tables. We had originally focused on MELD score in this Table, as that was the primary diagnostic indicator of disease severity used in the DASH consortium.
3. In this paper, the authors demonstrate in a mouse model that they can prevent alcoholinduced liver lesions. Therefore, in the "what you need to know – new findings", they should not write that production of TMA is a therapeutic target in advanced alcoholic hepatitis. It's a shortcut to the results: (i) again, in this section, I don't know what is "advanced" alcoholic hepatitis; (ii) the author describes a pathophysiological mechanisms and he should not write in this section of the manuscript that TMA production is a therapeutic target in patients with severe alcoholic hepatitis; (iii) The title of the manuscript should be changed accordingly.
We thank the reviewer for reminding us as to the purpose of the “new findings” section. We removed the “What you need to know” section and repurposed the “Highlights” section. Based off the reviewers’ suggestions, we have edited this to better reflect our actual results, rather than our interpretation of those results. For the additional comments:
i) The phrase Advanced hepatitis has been replaced with alcohol-associated hepatitis
ii) The “therapeutic target” hypothesis has been removed
iii) The title of the manuscript has been changed to “Gut Microbe-Derived Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury,” in order to better reflect the results of our studies.
4. TMA infusion in the portal vein. The "material and method" section is now written and included in the manuscript. Again, I am not sure of the relevance of such a way of administration. I don't understand the explanation of the authors who assert that they infused a physiological levels of TMA (page 18 line 32). I even think that the doses may be supraphysiological. The authors should be more clear and discuss the limitations of their results according to the dose of TMA and the way of administration. I think that giving a diet enriched in choline would have been more physiological than a direct infusion of TMA into the portal vein.
We apologize for the confusion in regards to this experiment. However, we do feel strongly that this experiment provides new important information for the field, and should be included in this body of work. The rationale for giving TMA directly into the portal vein is to mimic its delivery to the liver from the intestine. Given the fact that TMA smell like rotting fish it is nearly impossible to provide it in the diet (i.e. mice will not eat the diet). Furthermore, we also do not like the approach of peripheral administration (i.e. intraperitoneal or intravascular injection) because naturally TMA is made in the gut solely by bacteria and then enters the liver via the portal tract; making these peripheral approaches much less physiologic. We do agree with the reviewer however that this experiment is slightly above physiological levels, but by design we were trying to raise levels in vivo to very high levels to maximize responses. Given this key limitation, we have removed any mention of “physiological levels” and have included the following sentences in the results regarding the limitations of this experiment:
“It is important to note that in this experiment we provided very high levels of exogenous TMA via direct injection, and future studies should focus on more physiologically-relevant modes of TMA production like provision of gut bacteria that can naturally or be genetically engineered to produce high levels.”
The reviewer mentions that “a diet enriched in choline would have been more physiological than a direct infusion of TMA into the portal vein.” Although we agree that dietary choline supplementation can raise TMA and TMAO levels in animals, we respectfully disagree that this would provide relevant information to TMA-specific signal transduction like our experiment sets out to do. Provision of supplemental dietary choline impacts many downstream metabolites originating from bacteria (TMA and acetaldehyde) and the host (free choline, many species of phosphatidylcholine, acetylcholine) and also influences cholinergic signaling. If we simply provide choline, it is much more difficult to interrogate the specific signaling elicited by TMA. Although beyond the scope of this current study, we have designed a set of gut microbial communities that can or cannot make TMA, via genetic deletion of the gut microbial choline TMA lyase CutC. We have plans in the near future to follow up on the portal infusion studies to validate some the findings using this approach and others as well.
5. Graphical abstract / suppl Figure 5. the title "metaorgansim metabolism model" must be changed. A metaorganism model should include the other pathways involved in AH than the only TMA/TMAO pathway. Many other metabolites can be involved in ALD pathophysiology. The title should refer to the manuscript itself.
We have changed the headings/title of the graphical abstract to specifically refer to TMA/TMAO pathway rather than the broader term of “metaorganismal model”. The new graphical abstract now refers specifically the metaorganismal TMAO pathway which is the focus of this body of work.
6. The authors suggest that TMA and not TMAO is involved in the pathophysiology of ALD. Interestingly, it has been suggested in other papers that TMAO is involved in the pathophysiology of NAFLD/NASH. How can the authors reconcile these discrepancies?
We agree there are several reports that have shown an association between plasma TMAO and NAFLD/NASH (32791310, 26743949), but these are simply associations. To our knowledge there are no direct mechanistic links between TMAO and NAFLD/NASH. Given this manuscript is focused solely on alcohol-associated liver disease, we have focused our conclusion and discussion around ethanol-induced liver injury.
Third decision letter and responses:
The revised version of your manuscript entitled "Gut Microbial Trimethylamine is Elevated in Alcohol Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice " has been evaluated by the original reviewers and by the board of editors and we are sorry to say that we still see some significant issues that need to be addressed.
You will find the comments of Reviewer 2 below, who requests some changes in the graphical abstract and in Suppl Figure 5c.
In addition and more importantly, there is a global consensus between reviewers and the board of editors to consider that the interpretation of Figure 6 is challenging.
We feel that the final paper would be much improved by the addition of relatively simple experiments surrounding the conclusion of Figure 6:
–Adding an active control (i.e. another microbial metabolite) to confirm the specificity of the results obtained with TMA
–Adding additional doses of TMA to confirm the dose-dependent changes observed in the liver
1. We have indeed performed identical phosphoproteomic studies with other gut microbegenerated metabolites including a phenol acid called 4-hydroxyphenylacetic acid (4-HPAA). I have attached here this manuscript we have recently prepared for 4-HPAA in the context of NAFLD (currently under peer review). Included in this resubmission, we have included the manuscript (Osborn. et al. 2021) which is currently under revision at Cell Host Microbe. This manuscript uses a similar in vivo phosphoproteomic approach in Figure 5A-5C, and as you would expect uncovered many different pathways that are not overlapping with figure 6 here with TMA. Given this work is an integral part of another manuscript, it cannot be included here. However, the data does address the specificity question in your decision letter below.
2. Alternatively, if the editorial team thinks that the phosphoproteomic data provided in Figure 6 are hard to interpret, we would be happy to just remove that figure. However, we do think the data are useful for providing new leads to follow up on, but years of additional work will be needed to identify the TMA receptor systems and downstream signal transduction.
3. Unfortunately, we will not be able to perform the dose-response for in vivo phosphoproteomics as requested by the editors. Although we agree this would provide some additional information, it would be incremental in nature and would not change any interpretation of the current work. Given a backlog on our proteomics mass spectrometer and the holiday season we would not be able to finish those analyses until at least March of 2022, based on our current schedule. This timeline is simply not feasible for us with competitors manuscripts advancing to our stage, so these data would not be included in a revised submission. It is important for us to be first with this story so we will need to make a decision on publication route in the coming weeks.
The manuscript has been improved.
In his answer, the author writes that giving choline to induce an elevation of TMA would not be specific. This is correct. In this case, if the author thinks that giving choline is irrelevant, the graphical abstract and suppl Figure 5c which suggest that the intake of choline, the consumption of meat, eggs or cheese worsens alcoholic liver disease should be changed. This is not demonstrated at all and is a shortcut that may mislead the reader.
We have now changed the graphical abstract to remove any food items. This revised figure has now been uploaded.https://doi.org/10.7554/eLife.76554.sa2
Article and author information
National Institutes of Health (P50 AA024333)
- Arthur J McCullough
- Srinivasan Dasarathy
- Daniela S Allende
- Laura E Nagy
- Jonathan Mark Brown
National Institutes of Health (R01 DK120679)
- Jonathan Mark Brown
National Institutes of Health (P01 HL147823)
- Jonathan Mark Brown
- Stanley L Hazen
National Institutes of Health (U01 AA026938)
- Laura E Nagy
- Jonathan Mark Brown
National Institutes of Health (P50 CA150964)
- Jonathan Mark Brown
National Institutes of Health (U01 AA021890)
- Laura E Nagy
- Srinivasan Dasarathy
National Institutes of Health (U01 AA021893)
- Srinivasan Dasarathy
- Bruce Barton
- Craig J McClain
- Marko Mrdjen
- Gyongyi Szabo
- Arthur J McCullough
National Institutes of Health (R01 HL103866)
- Stanley L Hazen
National Institutes of Health (R01 HL144651)
- Zeneng Wang
National Institutes of Health (R01 HL130819)
- Zeneng Wang
National Institutes of Health (U01 AA026980)
- Craig J McClain
National Institutes of Health (P50 AA 024337)
- Craig J McClain
National Institutes of Health (R21 AR 071046)
- Srinivasan Dasarathy
National Institutes of Health (R01 GM119174)
- Srinivasan Dasarathy
National Institutes of Health (R01 DK113196)
- Srinivasan Dasarathy
National Institutes of Health (R56 HL141744)
- Srinivasan Dasarathy
National Institutes of Health (U01 DK061732)
- Srinivasan Dasarathy
National Institutes of Health (U01 AA026977)
- Gyongyi Szabo
National Institutes of Health (UH3 AA026970)
- Gyongyi Szabo
National Institutes of Health (K99 AA028048)
- Anagha Kadam
National Institutes of Health (1S10RR031537)
- Belinda Willard
National Institutes of Health (Leducq Transatlantic Networks of Excellence Award)
- Stanley L Hazen
JSPS Overseas Research Fellowship (201960331)
- Tatsunori Miyata
American Heart Association (17POST3285000)
- Robert N Helsley
American Heart Association (15POST2535000)
- Rebecca C Schugar
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
This work was supported in part by National Institutes of Health grants P50 AA024333 (AJM, SD, DSA, LEN, JMB), R01 DK120679 (JMB), P01 HL147823 (JMB, SLH), U01 AA026938 (LEN, JMB), P50 CA150964 (JMB), U01 AA021890 (LEN, SD), U01 AA021893 (SD, BB, CJM, MM, GS, and AJM), R01 HL103866 (SLH), R01 HL144651 (ZW), R01 HL130819 (ZW), U01 AA026980 (CJM), P50 AA 024337 (CJM), R21 AR 071046 (SD), R01 GM119174 (SD), R01 DK113196 (SD), R56 HL141744 (SD), U01 DK061732 (SD), U01 AA026977 (GS), UH3 AA026970 (GS), K99 AA028048 (AK), a Leducq Transatlantic Networks of Excellence Award (SLH), a JSPS Overseas Research Fellowship 201960331 (TM), and the American Heart Association (Postdoctoral Fellowships 17POST3285000 to RNH and 15POST2535000 to RCS). The Orbitrap Elite instrument used for proteomics was purchased via an NIH shared instrument grant 1S10RR031537 (BW).
Clinical trial registration NCT01809132; NCT03224949.
Human subjects: Patients with AH were classified as moderate (MELD < 20, n=112) and severe (MELD ≥20, n=152) according to the MELD score at admission as part of either of two independent clinical trials (ClincalTrials.gov identifier # NCT01809132 and NCT03224949) or the NOAC biorepository. These studies were approved by the Institutional Review Boards of all 4 participating institutions and all study participants consented prior to collection of data and blood samples. Written informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institutional Review Boards at Johns Hopkins Medical Institutions.
All mice were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care, International-approved animal facility. All experimental protocols were approved by the institutional animal care and use committee (IACUC) at the Cleveland Clinic.
- Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States
- Hossein Ardehali, Northwestern University, United States
- Received: December 21, 2021
- Accepted: December 31, 2021
- Preprint posted: January 1, 2022 (view preprint)
- Accepted Manuscript published: January 27, 2022 (version 1)
- Accepted Manuscript updated: February 4, 2022 (version 2)
- Version of Record published: February 17, 2022 (version 3)
© 2022, Helsley et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
- Page views
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
- Genetics and Genomics
Background: Causality between plasma triglyceride (TG) levels and atherosclerotic cardiovascular disease (ASCVD) risk remains controversial despite more than four decades of study and two recent landmark trials, STRENGTH and REDUCE-IT. Further unclear is the association between TG levels and non-atherosclerotic diseases across organ systems.
Methods: Here, we conducted a phenome-wide, two-sample Mendelian randomization (MR) analysis using inverse-variance weighted (IVW) regression to systematically infer the causal effects of plasma TG levels on 2,600 disease traits in the European ancestry population of UK Biobank. For replication, we externally tested 221 nominally significant associations (p < 0.05) in an independent cohort from FinnGen. To account for potential horizontal pleiotropy and the influence of invalid instrumental variables, we performed sensitivity analyses using MR-Egger regression, weighted median estimator, and MR-PRESSO. Finally, we used multivariable MR controlling for correlated lipid fractions to distinguish the independent effect of plasma TG levels.
Results: Our results identified 7 disease traits reaching Bonferroni-corrected significance in both the discovery (p < 1.92 × 10-5) and replication analyses (p < 2.26 × 10-4), suggesting a causal relationship between plasma TG levels and ASCVDs, including coronary artery disease (OR 1.33, 95% CI 1.24-1.43, p = 2.47 × 10-13). We also identified 12 disease traits that were Bonferroni-significant in the discovery or replication analysis and at least nominally significant in the other analysis (p < 0.05), identifying plasma TG levels as a novel potential risk factor for 9 non-ASCVD diseases, including uterine leiomyoma (OR 1.19, 95% CI 1.10-1.29, p = 1.17 × 10-5).
Conclusions: Taking a phenome-wide, two-sample MR approach, we identified causal associations between plasma TG levels and 19 disease traits across organ systems. Our findings suggest unrealized drug repurposing opportunities or adverse effects related to approved and emerging TG-lowering agents, as well as mechanistic insights for future studies.
Funding: RD is supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) (R35-GM124836) and the National Heart, Lung, and Blood Institute of the NIH (R01-HL139865 and R01-HL155915).
Background: Cancer patients show increased morbidity with COVID-19 and need effective immunization strategies. Many healthcare regulatory agencies recommend administering 'booster' doses of COVID-19 vaccines beyond the standard 2-dose series, for this group of patients. Therefore, studying the efficacy of these additional vaccine doses against SARS-CoV-2 and variants of concern is of utmost importance in this immunocompromised patient population.
Methods: We conducted a prospective single arm clinical trial enrolling patients with cancer that had received two doses of mRNA or one dose of AD26.CoV2.S vaccine and administered a 3rd dose of mRNA vaccine. We further enrolled patients that had no or low responses to three mRNA COVID vaccines and assessed the efficacy of a 4th dose of mRNA vaccine. Efficacy was assessed by changes in anti-spike antibody, T-cell activity and neutralization activity were again assessed at baseline and 4 weeks.
Results: We demonstrate that a 3rd dose of COVID-19 vaccine leads to seroconversion in 57% of patients that were seronegative after primary vaccination series. The immune response is durable as assessed by anti-S antibody titers, T-cell activity and neutralization activity against wild-type SARS-CoV2 and BA1.1.529 at 6 months of follow up. A subset of severely immunocompromised hematologic malignancy patients that were unable to mount an adequate immune response (titer <1000 AU/mL) after the 3rd dose and were treated with a 4th dose in a prospective clinical trial which led to adequate immune-boost in 67% of patients. Low baseline IgM levels and CD19 counts were associated with inadequate seroconversion. Booster doses induced limited neutralization activity against the Omicron variant.
Conclusions: These results indicate that 3rd dose of COVID vaccine induces durable immunity in cancer patients and an additional dose can further stimulate immunity in a subset of patients with inadequate response.
Funding: Leukemia lymphoma society, National Cancer Institute.
Clinical trial identifier: NCT05016622.