Abnormalities in the gut microbiome and gut-brain axis have emerged as potentially important contributors to the pathophysiology of neuropsychiatric diseases. Several microbe-derived metabolites (e.g., neurotransmitters, short-chain fatty acids, indoles, bile acids [BAs], choline metabolites, lactate, and vitamins) play a significant role in the context of emotional and behavioral changes (Caspani et al., 2019). Both direct and indirect mechanisms have been proposed through which gut microbial metabolites can affect central nervous system (CNS) functions (Yarandi et al., 2016; Tognini, 2017; Tremlett et al., 2017; Caspani et al., 2019). These include activation of afferent vagal nerve fibers, stimulation of the mucosal immune system or circulatory immune cells after translocation from the gut into the circulation, and absorption into the bloodstream followed by uptake and biochemical interaction with a number of distal organs. In the brain, these metabolites may activate receptors on neurons or glia, modulate neuronal excitability, and change gene expression patterns via epigenetic mechanisms (Caspani et al., 2019).

A growing body of evidence indicates the various mechanisms related to bidirectional communication between the gut microbiome and the host's CNS with anxiety and depression (Dinan and Cryan, 2015, 2017; Rieder et al., 2017; Simpson et al., 2021). Certain gut bacteria regulate the production of neurotransmitters and their precursors, such as serotonin, gamma-aminobutyric acid and tryptophan, and they also regulate proteins such as brain-derived neurotrophic factor, a key molecule involved in neuroplastic changes in learning and memory (Bercik et al., 2010; O'Sullivan et al., 2011; Agus et al., 2018; Miranda et al., 2019). Metabolites such as short-chain fatty acids (Parada Venegas et al., 2019) are involved in neuropeptide and gut hormone release, and they modulate immune signaling along the gut-brain axis via cytokine production. Gut bacteria are thought to be involved in the development and functioning of the hypothalamic-pituitary-adrenal axis (Sudo et al., 2004; de Weerth, 2017; Foster et al., 2017a). Dysregulation of the hypothalamic-pituitary-adrenal axis has been implicated in anxiety and depressive disorders, being associated with higher cortisol levels, increased intestinal permeability, and a sustained proinflammatory state (Keller et al., 2017). Gastrointestinal conditions believed to involve gut-microbial dysbiosis and intestinal permeability, such as irritable bowel syndrome, co-occur at remarkably high rates with psychiatric disorders (Simpson et al., 2020). In addition, several animal studies have supported the possibility of gut dysbiosis having a causative role in depression-like behaviors. For example, mice exposed to antibiotics showed gut dysbiosis, depression-like behavior, and altered neuronal hippocampal firing, with reversal of this phenotype following probiotic treatment (Guida et al., 2018). Transplantation of gut microbiota from humans with major depressive disorder (MDD) to germ-free or microbiota-deficient rodents resulted in a depression-like phenotype, including anhedonia and anxiety-like behaviors (Kelly et al., 2016; Zheng et al., 2016). Despite the literature supporting the involvement of the microbiota-gut-brain axis in mental health disorders, the underlying mechanisms of bidirectional communication and the metabolite mediators by which the gut bacteria regulate the gut-brain connection are not fully understood. Therefore, characterizing the rich array of compounds produced by gut bacteria and defining their protective and cytotoxic effects on the CNS can effectively define targeted interventions.

A potential mechanism by which the gut microbiome may alter CNS function is its impact on BAs. BAs are the amphipathic end products of cholesterol metabolism and can contribute significantly to hepatic, intestinal, and metabolic disorders (Li and Chiang, 2014). Figure 1 shows how BAs are synthesized from cholesterol in the liver via two major pathways, the classical and the alternative; secondary BAs are metabolized by colonic bacteria through multiple and well-characterized enzymatic pathways (Lefebvre et al., 2009). Primary BAs are the direct products of cholesterol metabolites in hepatocytes, such as cholic acid (CA) and chenodeoxycholic acid (CDCA). In response to cholecystokinin after feeding, primary BAs are secreted by the liver into the small intestine to ensure absorption of dietary lipids. Accordingly, 95% of the BAs are actively absorbed in the terminal ileum and redirected into the portal circulation to reenter the liver. A small proportion pass into the colon where bacteria transform them into secondary BAs—lithocholic acid (LCA), deoxycholic acid (DCA), and ursodeoxycholic acid—via deconjugation and 7α-dehydroxylation (Hofmann and Hagey, 2008; Bajor et al., 2010). Although DCA and LCA are the most abundant secondary BAs, ~50 different secondary BAs have been detected in human feces (Devlin and Fischbach, 2015).

Although primary BAs like CDCA may be synthesized in the brain, no evidence so far supports the synthesis of secondary BAs in the brain (Baloni et al., 2020). This suggests that the major source of brain BAs is the systemic circulation, which functions as a direct communication bridge between the gut microbiome and the brain (Monteiro-Cardoso et al., 2021), thereby playing a vital role in brain health. Circulating BAs generated in the liver and intestine can reach the brain by crossing the blood-brain barrier, either by simple diffusion or through BA transporters (Monteiro-Cardoso et al., 2021). Higashi et al. (2017) recently found that levels of CA, CDCA, and DCA detected in the brain positively correlated with their serum levels. The liver-gut-brain axis is critical for the maintenance of metabolic homeostasis, yet much remains to be elucidated about how BAs that are synthesized in the liver and modified in the gut mediate the crosstalk between the peripheral and central nervous system and impact neuropsychiatric disorders like major depression and anxiety disorders.

Several lines of evidence implicate secondary BAs as contributors to CNS dysfunction. Hepatic encephalopathy is associated with elevated levels of ammonia and cytotoxic BAs, including several conjugated primary and secondary BAs (Xie et al., 2018). Post-mortem brain samples and serum concentrations of living Alzheimer's disease patients (compared to health controls) demonstrated lower levels of the primary bile acid, CA, and higher levels of its bacterially-derived secondary bile acid, DCA and its conjugated forms (MahmoudianDehkordi et al., 2019; Nho et al., 2019; Baloni et al., 2020). In contrast, ursodeoxycholic acid, the 7β isomer of CDCA, has antiapoptotic, anti-inflammatory, antioxidant, and neuroprotective effects in various models of neurodegenerative diseases (Ramalho et al., 2013; Daruich et al., 2019) and Huntington's disease (Rodrigues et al., 2000; Parry et al., 2010; Mortiboys et al., 2013; Ackerman and Gerhard, 2016). Taken together, these data indicate that BAs affect brain function under both normal and pathological conditions. However, the association of BAs on psychiatric diseases such as MDD has received little study to date.

In this study, we profiled baseline serum samples from 208 patients enrolled in a randomized controlled trial of treatment-naïve outpatients with MDD, measuring 36 primary and secondary BAs to address the following questions:

Mounting evidence indicates that gut dysbiosis and the bidirectional communication between brain and gut microflora play an important role in the development of neuropsychiatric diseases. Using targeted metabolomics in participants with MDD, we determined that increased levels of cytotoxic secondary BAs, bacterially-derived from the primary bile acid CDCA, correlated with anxiety symptom severity. The classical pathway that, predominantly, produces the primary bile acid CA seemed to be less impacted. Additionally, participants who did not benefit from treatment were found to have higher baseline levels of the cytotoxic secondary BAs derived from CDCA. Our findings suggest that alternate therapies might be needed that target the gut microbiome for patients who have gut dysbiosis.

We first addressed whether BA concentrations impacted depression and anxiety symptom severity. Overall, BA concentrations appeared to have a stronger impact on anxiety symptoms than on depressive symptoms. Several secondary BA concentrations, and the ratios of secondary to primary BAs, were significantly different between low vs. high-anxious MDD participants irrespective of depression severity. These secondary BAs included LCA and its derivatives, 7-keto-LCA, isoLCA, alloLCA, and 12-ketoLCA. The 7α-dehydroxylation reaction that results in the formation of the secondary BAs has been described as the most quantitatively important process performed by colonic bacteria belonging to the genus Clostridium, an enzymatic reaction that is impacted in many neurological diseases (Kiriyama and Nochi, 2019). LCA is produced by 7α-dehydroxylation of CDCA and is known to be cytotoxic in rodents as well as several human cell types.

Our second question addressed whether there were any associations of symptoms with the classical and alternate pathways of BA synthesis. In Alzheimer's disease, we had observed a significant shift in BA synthesis from classical to the alternative pathways in the Alzheimer's participants compared to healthy controls (MahmoudianDehkordi et al., 2019; Nho et al., 2019; Baloni et al., 2020). In these MDD participants, we observed that the alternate pathway that favors CDCA synthesis was significantly impacted in the highly-anxious participants. However, no shift from classical to alternate pathway could be observed in these participants since the ratio of CA/CDCA, indicative of such a shift, was not significantly associated with symptom severity. Lower CDCA levels and higher secondary metabolites derived from CDCA (and mostly higher ratios of these secondary BAs to CDCA) characterized the participants with higher symptom severity, which may indicate greater utilization of CDCA by the gut bacteria. We also found no significant impact of glycine and taurine conjugation of BA on symptom severity. Interestingly, dehydrolithocholic acid, a major metabolite of LCA, was strongly negatively correlated to anxiety levels in the MDD participants. It is an agonist of the nuclear receptors GPCR1, the farnesoid X receptor (FXR) and the pregnane X receptor, and has recently been shown to regulate adaptive immunity by inhibiting the differentiation of TH17 cells that are known to contribute autoimmunity and inflammation (Hang et al., 2019). Our final question examined whether any relationship exists between baseline metabolite levels and response to treatment. Remitters showed higher levels of CDCA and one of its gut microbial metabolites (isoLCA) compared to participants for whom treatment failed.

The enzymatic processes involved in altered BA metabolism in CNS diseases may be informed by the association of BAs with inborn errors of metabolism (IEM), in which reduced intestinal BA concentrations result in serious morbidity or mortality. To date, investigators have identified nine recognized IEMs of BAs that lead to enzyme deficiencies and impaired BA synthesis (Heubi et al., 2007; Sundaram et al., 2008). These diseases are characterized by a failure to produce primary BAs and an accumulation of unusual BAs and BA intermediaries. Administration of BAs for replacement therapy often improves the symptoms of IEM, such as cerebrotendinous xanthomatosis, with CDCA the predominant choice for treating both neurological and non-neurological symptoms (Nie et al., 2014). We have recently reported on a common link between IEM and depression through acylcarnitines and beta oxidation of fatty acids, in which medium-chain acyl-coenzyme A dehydrogenase, an enzyme involved in the production of medium chain acylcarnitines, was shown to be causally linked to depression and also to IEM. These emerging data linking metabolomic disturbances in CNS disorders and IEM provide novel insights into pathobiological processes that contribute to psychiatric disorders (Milaneschi et al., 2022).

BAs influence metabolic processes by acting as signaling molecules via the nuclear receptors FXR, the pregnane X receptor, the vitamin D receptor, Takeda G-protein-coupled bile acid receptor, and sphingosine-1-phosphate receptor 2, initiating a variety of signaling cascades relevant to metabolic and hepatic diseases such as obesity, steatosis and steatohepatitis, as well as liver and colon cancer (Lefebvre et al., 2009; Wan and Sheng, 2018). FXR plays many important roles in the regulation mechanisms of BA synthesis and transport. FXR activation represses the expression of the main enzymes in BA synthesis, CYP7A1 and CYP27A1 (Pauli-Magnus and Meier, 2005). In contrast, FXR activation upregulates UGT2B4, which is involved in the conversion of hydrophobic BAs to their less toxic glucuronide derivatives (Barbier et al., 2003). CDCA is the most potent activator of FXR. Studies in knockout mice suggest the involvement of FXR in modulating brain function. Deletion of FXR altered the levels of several neurotransmitters in the hippocampus and cerebellum, and impaired cognitive functioning and motor coordination (Huang et al., 2015), which suggests that FXR signaling is required for normal brain function. A recent study using a rat-model (Chen et al., 2018) found that over-expression of hippocampal FXR mediated chronic unpredictable stress-induced depression-like behaviors and decreased hippocampal brain-derived neurotrophic factor expression, and that knocking out of hippocampal FXR completely prevented depressive behaviors via brain-derived neurotrophic factor expression.

The decrease in CDCA with concomitant increase in LCA has particular pathognomonic significance in MDD patients. LCA is the most potent ligand for Takeda G-protein-coupled BA receptor (Kawamata et al., 2003), and BA-dependent Takeda G-protein-coupled BA receptor-mediated signaling has been shown to influence the brain by regulating the production of the gut peptide hormone GLP-1 (Monteiro-Cardoso et al., 2021), which potentiates glucose-stimulated insulin secretion. LCA is also a potent activator of pregnane X receptor and vitamin D receptor. Thus, largely through their binding and activation of these receptors, BAs regulate their own synthesis, conjugation, transport, and detoxification, as well as lipid, glucose, and energy homeostasis (Hylemon et al., 2009; Li and Chiang, 2015; Ridlon et al., 2016; Grant and DeMorrow, 2020).

LCA is formed in humans mainly from the intestinal bacterial 7α-dehydroxylation of CDCA and comprises <5% of the total BA pool in humans but is one of the most hydrophobic naturally occurring BAs (Ceryak et al., 1998).

LCA has been shown to induce double-strand breaks in DNA (Kulkarni et al., 1980). The mammalian host responds by metabolizing LCA, mainly through sulfation, enabling more efficient excretion and reduced hydrophobicity (Ridlon and Bajaj, 2015). BA sulfation is an important detoxification process that converts hydrophobic BAs into excretable metabolites in the liver. Sulfation is catalyzed by a group of enzymes called sulfotransferases (Ridlon and Bajaj, 2015). Although, only a small proportion of BAs in bile and serum are sulfated, more than 70% of BAs in urine are sulfated, indicating their efficient elimination in urine (Alnouti, 2009). It is estimated that 40–75% of the hydrophobic, hepatotoxic LCA in human bile is present in the sulfated form (Palmer and Bolt, 1971). The formation of BA-sulfates increases during cholestatic diseases. Therefore, sulfation may play an important role in maintaining BA homeostasis under pathologic conditions. In our study, we observed elevated levels of the sulfated form of the toxic LCA and GLCA in more severely anxious patients. We have also previously reported increased production of other bacterially-derived sulfates like p-cresol sulfate and indoxyl sulfates (Brydges et al., 2021) in the PReDICT study participants. Together, these findings suggest that alterations in sulfotransferase activities may occur in the liver of some patients.

The microbial conversion of CDCA to 7-keto-LCA, present at higher levels in highly-anxious MDD participants, is known to be reduced in the liver by human 11β-HSDH-1, an enzyme with the primary function of converting cortisone to the active glucocorticoid, cortisol (Odermatt et al., 2011). Microbial-derived 7-keto-LCA acts as a competitive inhibitor of 11β-HSDH-1, and thus may influence the ratio of cortisone/cortisol.

In contrast to toxic LCA and most of its derivatives, dehydrolithocholic acid was the only one that negatively correlated to anxiety levels and depression level indicating a protective metabolite. It is an agonist of the nuclear receptors, GPCR1, FXR, PXR, and has recently been shown to regulate adaptive immunity by inhibiting the differentiation of TH17 cells that are known to cause autoimmunity and inflammation (Hang et al., 2019).

There are a few limitations to our study. First, we lacked a healthy control group to compare with the participants who had MDD. Second, food and medication intake (e.g., use of pre/probiotics and antibiotics) can cause intestinal dysbiosis, thus influencing the metabolism of bile acids. In PReDICT, no food diary, record of physical activity, and medication were kept or used as inclusion/exclusion criteria of participants. Hence, we were not able to control for the effect of those potential confounders. Third, we did not apply multiple comparison adjustments due to the relatively small sample size and the exploratory nature of this study. Fourth, these findings will require replication in an independent cohort. Fifth, a number of novel BAs have recently been discovered and were not included in our metabolomic analyses; these compounds should be evaluated in future studies. Sixth, stool samples were not available in the PReDICT study. Considering the role of the gut microbiota in the synthesis and metabolism of bile acids, a microbiome and metabolomics analysis performed in stool samples, coupled with the analysis in the blood, would have provided a closer readout on the microbiota-related activity in bile acids production.

It has been suggested (Hibbing et al., 2010; Foster et al., 2017b) that in the highly evolutionary competitive environment of the human gut microbiome, the persistence of these microbial enzyme activities usually indicates that they increase the organism's ability to survive. However, dysbiosis in the gut is also possible. Our data suggest that low levels of CDCA might be a result of increased utilization for production of bacterial products in the intestine which, in turn, suggest gut-microbe composition changes or associated enzymatic changes. The underlying pathophysiological significance of BA pool changes remain to be determined, but a reasonable hypothesis emerging from this work is that increases in circulating BAs result from a more hydrophobic BA pool in the colon consequent to gut microbial dysbiosis. These BAs may then produce enhanced toxicity and pathophysiology to cells in the liver, gastrointestinal tract, and the brain.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Emory University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

SM and SB did analysis of data and helped write the manuscript. CB did PLS regression analysis. WJ and his team generated biochemical data. RK-D, BD, and AR helped with interpretation of findings and clinical relevance. RK-D is PI for project and helped with concept development, study design, data interpretation and connecting biochemical and clinical data, and with the writing of the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by grant support to RK-D (PI) through NIH grants R01MH108348, R01AG046171, and U01AG061359. BD has support from NIH grants P50-MH077083 (PI Mayberg), R01-MH080880 (PI Craighead), UL1-RR025008 (PI Stevens), M01-RR0039 (PI Stevens), and the Fuqua Family Foundations.

BD has received research support from Acadia, Compass, Aptinyx, NIMH, Sage, and Takeda, and has served as a consultant to Greenwich Biosciences, Myriad Neuroscience, Otsuka, Sage, and Sophren Therapeutics. AR has received consulting fees from Compass Inc., Curbstone Consultant LLC, Emmes Corp., Holmusk, Johnson and Johnson Janssen, Liva-Nova, Neurocrine Biosciences Inc., Otsuka-US, Sunovion; speaking fees from Liva-Nova, and Johnson and Johnson Janssen; and royalties from Guilford Press and the University of Texas Southwestern Medical Center, Dallas, TX for the Inventory of Depressive Symptoms and its derivatives. He is also named co-inventor on two patents: U.S. Patent No. 7,795,033: Methods to Predict the Outcome of Treatment with Antidepressant Medication, Inventors: McMahon FJ, Laje G, Manji H, AR, Paddock S, Wilson AS; and U.S. Patent No. 7,906,283: Methods to Identify Patients at Risk of Developing Adverse Events During Treatment with Antidepressant Medication, Inventors: McMahon FJ, Laje G, Manji H, AR, Paddock S. RK-D is an inventor on key patents in the field of Metabolomics and hold equity in Metabolon, a biotech company in North Carolina. In addition, she holds patents licensed to Chymia LLC and PsyProtix with royalties and ownership. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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