Isolithocholic Acid Research Articles

Ileal microbial microbiome and its secondary bile acids modulate susceptibility to nonalcoholic steatohepatitis in dairy goats

BackgroundLiver damage from nonalcoholic steatohepatitis (NASH) presents a significant challenge to the health and productivity of ruminants. However, the regulatory mechanisms behind variations in NASH susceptibility remain unclear. The gut‒liver axis, particularly the enterohepatic circulation of bile acids (BAs), plays a crucial role in regulating the liver diseases. Since the ileum is the primary site for BAs reabsorption and return to the liver, we analysed the ileal metagenome and metabolome, liver and serum metabolome, and liver single-nuclei transcriptome of NASH-resistant and susceptible goats together with a mice validation model to explore how ileal microbial BAs metabolism affects liver metabolism and immunity, uncovering the key mechanisms behind varied NASH pathogenesis in dairy goats.ResultsIn NASH goats, increased total cholesterol (TC), triglyceride (TG), and primary BAs and decreased secondary BAs in the liver and serum promoted hepatic fat accumulation. Increased ileal Escherichia coli, Erysipelotrichaceae bacterium and Streptococcus pneumoniae as well as proinflammatory compounds damaged ileal histological morphology, and increased ileal permeability contributes to liver inflammation. In NASH-tolerance (NASH-T) goats, increased ursodeoxycholic acid (UDCA), isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) in the liver, serum and ileal contents were attributed to ileal secondary BAs-producing bacteria (Clostridium, Bifidobacterium and Lactobacillus) and key microbial genes encoding enzymes. Meanwhile, decreased T-helper 17 (TH17) cells and increased regulatory T (Treg) cells proportion were identified in both liver and ileum of NASH-T goats. To further validate whether these key BAs affected the progression of NASH by regulating the proliferation of TH17 and Treg cells, the oral administration of bacterial UDCA, isoDCA and isoLCA to a high-fat diet-induced NASH mouse model confirmed the amelioration of NASH through the TH17 cell differentiation/IL-17 signalling/PPAR signalling pathway by these bacterial secondary BAs.ConclusionThis study revealed the roles of ileal microbiome and its secondary BAs in resilience and susceptibility to NASH by affecting the hepatic Treg and TH17 cells proportion in dairy goats. Bacterial UDCA, isoDCA and isoLCA were demonstrated to alleviate NASH and could be novel postbiotics to modulate and improve the liver health in ruminants.A8SM95maBgMZc1-WqV_tnDVideo

Apple Polyphenol Extracts Attenuated Depressive-Like Behaviors of High-Sucrose Diet Feeding Mice by Farnesoid X Receptor-Mediated Modulation of Bile Acid Circulation within the Liver-Gut-Brain Axis.

Although the association between high-sugar diets and depression has been verified, few studies have explored the antidepressant mechanisms of apple polyphenol extracts (APE). Therefore, fifty-four C57BL/6 male mice aged 5 weeks were randomly assigned into five groups: the control group with the standard diet (CON), the constant high-sucrose diet group (HSD), the "2 + 5" alternate diet group (A-HSD), and the 500 mg/(kg·bw) APE treatment for the HSD group (APE) and the A-HSD group (A-APE), respectively. The data of hypothalamic-pituitary-adrenal (HPA) axis function and behavioral experiments confirmed the success in the establishment of depression-like mouse models in both HSD and A-HSD groups, which were significantly alleviated after APE treatment. Meanwhile, APE reduced serum levels of corticosterone and adrenocorticotrophic hormone, alleviated histopathological damage of the liver, colon, and brain, respectively, elevated the protein expressions of Occludin, ZO-1, and MUC-2, and decreased Firmicutes/Bacteroidota ratio and Dubosiella abundance with the increased microbiota of Tannerellaceae_unclassified, Muribaculum, and Lachnospiraceae_unclassified. Moreover, APE treatment reduced Farnesoid X receptor (FXR) protein levels along with the increased expressions of CYP7A1 and TGR5, lowered the contents of serum and fecal total bile acids, and modulated fecal BA compositions, particularly glycocholic acid (GCA) and isolithocholic acid (ILCA). Thus, both the constant and alternate high-sucrose diets successfully induced depression-like behaviors in mice, and APE might be a potential nutraceutical to attenuate high-sucrose diet-induced depression by regulating BAs circulation within the liver-gut-brain axis mediated by FXR.

Diagnostic Potential of Alternations of Bile Acid Profiles in the Plasma of Patients with Huntington's Disease.

Huntington's disease (HD) is characterized by progressive involuntary chorea movements and cognitive decline. Recent research indicates that metabolic disturbance may play a role in its pathogenesis. Bile acids, produced during cholesterol metabolism in the liver, have been linked to neurodegenerative conditions. This study investigated variations in plasma bile acid profiles among individuals with HD. Plasma levels of 16 primary and secondary bile acids and their conjugates were analyzed in 20 healthy controls and 33 HD patients, including 24 with symptoms (symHD) and 9 carriers in the presymptomatic stage (preHD). HD patients exhibited significantly higher levels of glycochenodeoxycholic acid (GCDCA) and glycoursodeoxycholic acid (GUDCA) compared to healthy controls. Conversely, isolithocholic acid levels were notably lower in the HD group. Neurotoxic bile acids (glycocholic acid (GCA) + glycodeoxycholic acid (GDCA) + GCDCA) were elevated in symHD patients, while levels of neuroprotective bile acids (ursodeoxycholic acid (UDCA) + GUDCA + tauroursodeoxycholic acid (TUDCA)) were higher in preHD carriers, indicating a compensatory response to early neuronal damage. These results underscore the importance of changes in plasma bile acid profiles in HD and their potential involvement in disease mechanisms. The identified bile acids (GCDCA, GUDCA, and isolithocholic acid) could potentially serve as markers to distinguish between HD stages and healthy individuals. Nonetheless, further research is warranted to fully understand the clinical implications of these findings and their potential as diagnostic or therapeutic tools for HD.

Changes of bacterial communities and bile acid metabolism reveal the potential “intestine-hepatopancreas axis” in shrimp

The interaction between the gut and the liver plays a significant role in individual health and diseases. Mounting evidence supports that bile acids are important metabolites in the bidirectional communication between the gut and the liver. Most of the current studies on the “gut-liver axis” have focused on higher vertebrates, however, few was reported on lower invertebrates such as shrimp with an open circulatory system. Here, microbiomic and metabolomic analyses were conducted to investigate the bacterial composition and bile acid metabolism in intestine, hemolymph and hepatopancreas of Penaeus vannamei fed diets supplemented with octanoic acid and oleic acid. After six days of feeding, the bacterial composition in intestine, hemolymph and hepatopancreas changed at different stages, with significant increases in the relative abundance of several genera such as Pseudomonas and Rheinheimera in intestine and hepatopancreas. Notably, there was a more similar bacterial composition in intestine and hepatopancreas at the genus level, which indicated the close communication between shrimp intestine and hepatopancreas. Meanwhile, higher content of some bile acids such as lithocholic acid (LCA) and α-muricholic acid (α-MCA) in intestine and lower content of some bile acids such as taurohyocholic acids (THCA) and isolithocholic acid (IsoLCA) in hepatopancreas were detected. Furthermore, Spearman correlation analysis revealed a significant correlation between bacterial composition and bile acid metabolism in intestine and hepatopancreas. The microbial source tracking analysis showed that there was a high proportion of intestine and hepatopancreas bacterial community as the source of each other. Collectively, these results showed a strong crosstalk between shrimp intestine and hepatopancreas, which suggests a unique potential “intestine-hepatopancreas axis” in lower invertebrate shrimp with an open circulatory system. Our finding contributed to the understanding of the interplay between shrimp intestine and hepatopancreas in the view of microecology and provided new ideas for shrimp farming and disease control.

Comprehensive Clinical and Genetic Analyses of Circulating Bile Acids and Their Associations With Diabetes and Its Indices.

Bile acids (BAs) are cholesterol-derived compounds that regulate glucose, lipid, and energy metabolism. Despite their significance in glucose homeostasis, the association between specific BA molecular species and their synthetic pathways with diabetes mellitus (DM) is unclear. Here, we used a recently validated stable-isotope dilution highperformance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify a panel of BAs in fasting plasma from subjects (n=2,145) and explored structural and genetic determinants of BAs linked to DM, insulin resistance and obesity. Multiple 12α-hydroxylated BAs were associated with DM [adjusted odds ratios (aORs):1.3-1.9 (all P<0.05)] and insulin resistance [aORs:1.3-2.2 (all P<0.05)]. Conversely, multiple 6a-hydroxylated BAs and isolithocholic acid (Iso-LCA) were inversely associated with DM and obesity [aORs:0.3-0.9 (all P<0.05)]. Genome-wide association studies (GWAS) revealed multiple genome-wide significant loci linked with nine of the 14 DM-associated BAs, including a locus for Iso-LCA (rs11866815). Mendelian randomization analyses showed genetically elevated DCA levels were causally associated with higher BMI, and Iso-LCA levels were causally associated with reduced BMI and DM risk. In conclusion, comprehensive large-scale quantitative mass spectrometry and genetics analyses show circulating levels of multiple structurally specific BAs, especially DCA and Iso-LCA, are clinically associated with and genetically linked to obesity and DM.

Bile acid metabolites enhance expression of cathelicidin antimicrobial peptide in airway epithelium through activation of the TGR5-ERK1/2 pathway

Signals for the maintenance of epithelial homeostasis are provided in part by commensal bacteria metabolites, that promote tissue homeostasis in the gut and remote organs as microbiota metabolites enter the bloodstream. In our study, we investigated the effects of bile acid metabolites, 3-oxolithocholic acid (3-oxoLCA), alloisolithocholic acid (AILCA) and isolithocholic acid (ILCA) produced from lithocholic acid (LCA) by microbiota, on the regulation of innate immune responses connected to the expression of host defense peptide cathelicidin in lung epithelial cells. The bile acid metabolites enhanced expression of cathelicidin at low concentrations in human bronchial epithelial cell line BCi-NS1.1 and primary bronchial/tracheal cells (HBEpC), indicating physiological relevance for modulation of innate immunity in airway epithelium by bile acid metabolites. Our study concentrated on deciphering signaling pathways regulating expression of human cathelicidin, revealing that LCA and 3-oxoLCA activate the surface G protein-coupled bile acid receptor 1 (TGR5, Takeda-G-protein-receptor-5)—extracellular signal-regulated kinase (ERK1/2) cascade, rather than the nuclear receptors, aryl hydrocarbon receptor, farnesoid X receptor and vitamin D3 receptor in bronchial epithelium. Overall, our study provides new insights into the modulation of innate immune responses by microbiota bile acid metabolites in the gut-lung axis, highlighting the differences in epithelial responses between different tissues.

Bacteroides uniformis-induced perturbations in colonic microbiota and bile acid levels inhibit TH17 differentiation and ameliorate colitis developments

Inflammatory bowel disease (IBD) is associated with gut dysbiosis and can lead to colitis-associated malignancies. Bacteroides uniformis (Bu) regulates animal intestinal homeostasis; however, the mechanism by which it alleviates colitis in mice remains unknown. We investigated the effects of B. uniformis JCM5828 and its metabolites on female C57BL/6J mice with dextran sulfate sodium salt (DSS) induced colitis. Treatment with Bu considerably alleviated colitis progression and restored the mechanical and immune barrier protein expression. Additionally, Bu increased the abundance of the symbiotic bacteria Bifidobacterium and Lactobacillus vaginalis while decreasing that of pathogenic Escherichia-Shigella, and modulated intestinal bile acid metabolism. Bu largely regulated the expression of key regulatory proteins of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways in colonic tissues and the differentiation of TH17 cells. However, Bu could not directly inhibit TH17 cell differentiation in vitro; it modulated the process in the lamina propria by participating in bile acid metabolism and regulating key metabolites (alpha-muricholic, hyodeoxycholic, and isolithocholic acid), thereby modulating the intestinal immune response. Our findings suggest that Bu or bile acid supplements are potential therapies for colitis and other diseases associated with intestinal barrier dysfunction.

A Novel Serum Metabolomic Panel for the Diagnosis of Crohn's Disease.

A distinctive metabolic phenotype provides the opportunity to discover noninvasive biomarkers for the diagnosis of Crohn's disease (CD) and for differentiating it from other intestinal inflammatory diseases. The study sought to identify new biomarkers for CD diagnosis. Serum metabolites from 68 newly diagnosed and treatment-naïve patients with CD and 56 healthy control (HC) subjects were profiled using targeted liquid chromatography-mass spectrometry. Five metabolic biomarkers were identified to distinguish patients with CD from the HC subjects and validated in a separate cohort consisting of 110 patients with CD and 90 HC subjects using a combination of univariate analysis, orthogonal partial least-squares discriminant analysis, and receiver-operating characteristic curve analysis. Differences in the 5 metabolites were evaluated among patients with CD and patients with ulcerative colitis (n = 62), intestinal tuberculosis (n = 48), and Behçet's disease (n = 31). Among the 185 quantified metabolites, a panel of 5 (pyruvate, phenylacetylglutamine, isolithocholic acid, taurodeoxycholic acid, and glycolithocholic acid) were found to distinguish patients with CD with high accuracy from HC subjects, with an area under the curve of 0.861 (P < .001). The performance of the model in assessing clinical disease activity was comparable to that of the present biomarkers: C-reactive protein and erythrocyte sedimentation rate. The 5 metabolites were significantly different among the patients and were valuable in the differentiation between CD and other chronic intestinal inflammatory diseases. The combination of 5 serum metabolite biomarkers for the diagnosis of CD has the potential to provide an accurate, noninvasive, and inexpensive alternative to conventional tests and might be valuable for the differentiation from other diagnostically challenging intestinal inflammatory diseases.

Bile acid alterations associated with indolent course of inflammatory bowel disease

Background The indolent course of treatment-naive patients with inflammatory bowel disease (IBD) is confirmed predictable based on clinical characteristics. Current evidences supported that bile acids (BAs) alteration might be promising biomarkers in the field of IBD. We aimed to analyze the alterations of BAs as the disease progresses and explore their predictive value for indolent course of IBD. Methods The indolent course of IBD was defined as a disease course without need for strict interventions throughout the entire follow-up. A targeted metabolomics method was used to detect the concentration of 27 BAs from serum sample in treatment-naive patients with IBD (Crohn’s disease [CD], n = 27; ulcerative colitis [UC], n = 50). Patients with CD and UC were individually divided into two groups for further study according to the median time of indolent course. The overall BAs profile and the clinical value of BAs in predicting indolent course of IBD were identified between different groups. Results For CD, the levels of deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycolithocholic acid-3-sulfate disodium salt and iso-lithocholic acid were significantly increased in patients with indolent course > 18 M (p < 0.05). These five BAs owned 83.5% accuracy for predicting indolent course over 18 months in CD. For UC, the concentration of deoxycholic acid and glycodeoxycholic acid were significantly higher, while dehydrocholic acid were lower in patients with indolent course > 48 M (p < 0.05). These three BAs predicted indolent course over 48 months of 69.8% accuracy in UC. Conclusion The specific BAs alterations might be potential biomarkers in predicting disease course of IBD patients.

Gut commensal Parabacteroides distasonis alleviates inflammatory arthritis

ObjectiveGut microbiota dysbiosis is closely linked to the pathogenesis of rheumatoid arthritis (RA). We aimed to identify potential probiotic gut microbes that can ameliorate the development of RA.DesignMicrobiota profiling in...

Microbial Bile Acid Metabolism Shapes Effector T Cell Responses during Inflammation in Mouse and Human

Microbial transformation of bile acids (BAs) regulates intestinal immune homeostasis but their effect on T cell-driven pathologies is unknown. We previously reported that gut dysbiosis in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) is associated with increased graft-versus-host disease (GVHD), in which graft-derived T cells recognize the host as foreign. We hypothesized that the dramatic changes in intestinal microbiome composition during GVHD would lead to changes in the BA pool, which would alter immunoregulatory mechanisms governing T cell responses. Using a preclinical MHC-disparate GVHD model (B6 into BALB/c) and quantifying BAs with liquid chromatography-mass spectrometry, we found that GVHD was associated with a significant decrease in total BA concentration with a preferential loss of microbiota-dependent unconjugated primary BA (PBAs) and secondary BA (SBAs) (p=0.0002 and p<0.001, n=10/group). Metagenomic sequencing revealed changes in microbiota composition, including reduced abundance of Lactobacillus johnsonii and expansion of Enterococcus faecalis in GVHD mice (FDR=0.03, n=5/group) as previously reported. These changes were accompanied by a reduction in the abundance of bile salt hydrolase (BSH) genes (p=0.008) encoded by the gut microbiome (mainly Lactobacillus johnsonii). BSH catalyzes the deconjugation of PBAs, which is the gateway reaction leading to SBAs formation, suggesting that GVHD affects the BA pool by compromising early steps in microbial BA metabolism. To investigate the effects of GVHD on host metabolism, we profiled the liver transcriptome by bulk RNA-sequencing analysis. Pathways involved in BA metabolism were reduced in the livers of GVHD mice, which was associated with reduced levels of transcripts for genes involved in BA synthesis (Abcd3, Hsd17b11, Cyp7b1, Hsd17b6) and secretion (Abcg8, Slc22a18). As BAs regulate their synthesis via farnesoid X receptor (FXR) in a negative feedback loop, these results suggested that FXR activation may be increased because of loss of SBAs. Indeed, SBAs counteracted the activation of FXR by the natural agonist chenodeoxycholic acid, in FXR reporter assay and FXR TR-FRET coactivator assay, suggesting that bacterial transformation of BAs generally decreases FXR activity in vivo. Pharmacological activation of FXR (B6 into BALB/c, treatment with selective FXR agonist GW4064 day -1 to day 7, p<0.0001, n=20/group) showed increased mortality, whereas genetic ablation of FXR in donor T cells decreased GVHD and improved OS in two preclinical models (B6 into BALB/c, p<0.0001, n=30/group, Fig. 1a and B6 into 129, p<0.0001, n=20/group). Mice transplanted with TΔFXRcells had reduced GVHD-related pathology compared to recipients of TWT on day 28 post allo-HCT (p=0.006, n=10/group). Furthermore, we found reduced IFNγ production by CD4+ and CD8+ TΔFXR cells in the lamina propria of the large intestine on day 14 post-transplant (p=0.01 and p=0.02, n=10/group). Finally, we set out to corroborate our preclinical findings in allo-HCT patients. We compared 63 fecal samples from allo-HCT patients with lower gastrointestinal GVHD obtained at peri-GVHD onset (+/- 10 days) with 61 samples from control allo-HCT patients without GVHD, who were matched for age, graft type and HLA compatibility. Samples from controls were selected +/-10 days from pseudo-GVHD day that was assigned based on their paired GVHD patient. Most of the samples were exposed to the SBA ursodeoxycholic acid (UDCA, 93%). Similar to our findings in mice, we found that SBAs (non-UDCA, Fig. 1b) and unconjugated PBAs, as well as BSH abundances were decreased in GVHD patients compared to controls (p=0.007, p=0.008, and p=0.02 respectively). Notably, we found that important immunomodulatory SBAs were reduced in GVHD patients compared to controls, including 3-oxolithocholic acid, isolithocholic acid and ω-muricholic acid (p=0.01, p=0.004, and p<0.001). Lastly, in 747 allo-HCT recipients, we found that UDCA exposure was associated with a lower incidence of Enterococcus sp. domination (p=0.001), lower aGVHD cumulative incidence (p=0.005) and improved OS (p=0.017) as previously reported. Our results in mice and humans suggest that loss of SBA and increased FXR activation may amplify T-cell driven disease such as GVHD and provide a rationale to target the host-microbiota BA network to improve clinical outcomes in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Gene Alignment in Dorea sp. AM58-8 Produces 7-Dehydroxy-3β Bile Acids from Primary Bile Acids.

Bile acids are essential metabolites and signaling molecules in mammals. Primary bile acids are synthesized from cholesterol in the liver. At the same time, the microbiota in the mammalian gut has many interactions with bile acid, including various biotransformation processes such as 7-dehydroxylation and 3-epimerization. 7-Dehydroxylation is mediated by a bile acid-inducible (bai) operon, while 7-dehydroxylation and 3-epimerization are independently observed in only a few strains. Herein, we describe a novel microbe, Dorea sp. AM58-8, that can accomplish a two-step transformation and turn primary bile acids into both 3α secondary bile acids like deoxycholic acid and lithocholic acid, and 3β secondary bile acids like isodeoxycholic acid and isolithocholic acid. We subsequently characterized BaiA, BaiB, BaiE, and their substrate profiles biochemically. The potential bai gene clusters in the metagenomes were further mined. Their evolution, potential functions, and possible regulatory pathways were predicted using bioinformatics based on our understanding of the 7-dehydroxylation pathway in Dorea sp. AM58-8. This study of Dorea sp. AM58-8 also helps us distinguish the inactive bacteria that seem to have the 7-dehydroxylation pathway proteins and discover the 7-dehydroxylation pathway in other mammalian gut microbes.

Characterization of long-chain fatty acid-linked bile acids: a major conjugation form of 3β-hydroxy bile acids in feces

Although most bile acids (BAs) in feces are present in noncovalent forms that can be extracted with ethanol, non-negligible amounts of saponifiable BAs are also present. It is a major concern that such saponifiable BAs are routinely omitted from fecal BA measurements. We compared the BA profiles of healthy stools that were obtained with/without alkaline hydrolysis and found that as much as 29.7% (2.1–67.7%) of total BAs were saponifiable. Specifically, alkaline treatment led to significant elevations of isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) concentrations, suggesting that considerable proportions of isoDCA and isoLCA were esterified. Precursor ion scan data from LC/MS suggested the presence of long-chain FA-linked BAs. We chemically synthesized a series of fatty acid 3β-acyl conjugates of isoDCA and isoLCA as analytical standards and analyzed their fecal profiles from newborns to adults (n = 64) by LC/MS. FA-conjugated isobile acids (FA-isoBAs) were constantly present from 2 years of age to adulthood. C16- and C18-chain FA-isoBA esters were predominantly found regardless of age, but small amounts of acetic acid esters were also found. FA-isoBA concentrations were not correlated to fecal FA concentrations. Interestingly, there were some adults who did not have FA-isoBAs. Gut bacteria involved in the production of FA-isoBAs have not been identified yet. The present study provides insight into the establishment of early gut microbiota and the interactive development of esterified BAs.The contribution of FA-isoBAs to gut physiology and their role in pathophysiologic conditions such as inflammatory bowel disease are currently under investigation.

A dynamic multiple reaction monitoring strategy to develop and optimize targeted metabolomics methods: Analyzing bile acids in capecitabine-induced diarrhea

ObjectiveWe sought to develop and optimize a targeted bile acids (BAs) metabolomics method based on a dynamic multiple reaction monitoring (dMRM) strategy and explored the dynamic alterations of BAs in diarrhea induced by capecitabine in a mouse model. MethodThe targeted metabolomics method was developed using an Agilent 6460A triple quadrupole mass spectrometer, and 41 types of BAs were monitored in negative ionization mode. The mass spectrometer detection was optimized using dMRM to enhance the responses, separation, and peak shape and to shorten the analysis time. A mouse model of diarrhea was established by multiple administration of capecitabine, and plasma samples were collected at baseline and the end of drug administration for subsequent BAs analysis. ResultsThe targeted BA metabolomics method achieved shorter chromatographic separation time (10 min) for 41 BAs, with good peak shapes and response increases of 3- to 10-fold after application of dMRM. The mouse model of capecitabine-induced diarrhea was established, and the three BAs 23-norcholic acid, isolithocholic acid, and isodeoxycholic acid in the baseline samples contributed the most to differentiating mice with diarrhea from those without diarrhea. For mice that ultimately developed diarrhea, apocholic acid, isodeoxycholic acid, and 7-ketodeoxycholic acid exhibited the largest change in concentrations compared with their baseline concentrations. ConclusionThe dMRM strategy has obvious advantages compared with common MRM. The results in model mice showed that a differentiated profile of BAs in the baseline may indicate biomarkers of diarrhea induced by capecitabine, and disturbed homeostasis may explain the metabolomic mechanism of diarrhea occurrence.

The gut microbiota-bile acid axis links the positive association between chronic insomnia and cardiometabolic diseases

Evidence from human cohorts indicates that chronic insomnia is associated with higher risk of cardiometabolic diseases (CMD), yet whether gut microbiota plays a role is unclear. Here, in a longitudinal cohort (n = 1809), we find that the gut microbiota-bile acid axis may link the positive association between chronic insomnia and CMD. Ruminococcaceae UCG-002 and Ruminococcaceae UCG-003 are the main genera mediating the positive association between chronic insomnia and CMD. These results are also observed in an independent cross-sectional cohort (n = 6122). The inverse associations between those gut microbial biomarkers and CMD are mediated by certain bile acids (isolithocholic acid, muro cholic acid and nor cholic acid). Habitual tea consumption is prospectively associated with the identified gut microbiota and bile acids in an opposite direction compared with chronic insomnia. Our work suggests that microbiota-bile acid axis may be a potential intervention target for reducing the impact of chronic insomnia on cardiometabolic health.

Human gut bacteria produce ΤΗ17-modulating bileacid metabolites.

The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (TH17 cells). We previously reported that the bileacid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed TH17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key TH17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of TH17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit TH17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease.

Alterations in the gut microbiome and metabolome profiles of septic rats treated with aminophylline

The treatment of sepsis remains a major challenge worldwide. Aminophylline has been shown to have anti-inflammatory effects; however, the role of aminophylline in sepsis, a disease characterized by immune dysregulation, is unknown. In this study, we combined microbiome sequencing and metabolomic assays to investigate the effect of aminophylline administration on the intestinal flora and metabolites in septic rats. Sixty SD rats were randomly divided into three groups: a sham-operated (SC) group, a sepsis model (CLP) group and a CLP + aminophylline treatment (Amino) group. The intestinal flora and metabolic profile of rats in the CLP group were significantly different than those of the SC group, while aminophylline administration resulted in a return to a state similar to healthy rats. Differential abundance analysis showed that aminophylline significantly back-regulated the abundance of Firmicutes, unidentified_Bacteria, Proteobacteria, Lactobacillus, Escherichia-Shigella and other dominant bacteria (P < 0.05) and altered chenodeoxycholic acid, isolithocholic acid and a total of 26 metabolites (variable importance in the projection (VIP) > 1, P < 0.05). In addition, we found that there were significant correlations between differential metabolites and bacterial genera of the Amino and CLP groups. For example, Escherichia-Shigella was associated with 12 metabolites, and Lactobacillus was associated with two metabolites (P < 0.05), suggesting that differences in the metabolic profiles caused by aminophylline were partly dependent on its influence on the gutmicrobiome. In conclusion, this study identified a novel protective mechanism whereby aminophylline could regulate disordered intestinal flora and metabolites in septic rats.

Steroids aid in human decomposition fluid identification in soils of temporary mass graves from World War II

Steroids are widely used for the detection of faecal matter and also – in recent years – for characterising human decomposition in the terrestrial environment. Until now it was not clear whether all commonly used faecal (i.e. 5β-stanols, 5β-stanones and bile acids) and tissue derived steroids (i.e. cholesterol, 5α-cholestanol and 5α-cholestanone) could reveal the presence of human decomposition products in temporary graves. In this study, soil was sampled from three putative mass graves where concentration camp prisoners were temporarily buried for 10 months at the end of World War II (1944 – 45). We hypothesised that soil from the putative temporary mass graves exhibits elevated concentrations of faecal and tissue steroids compared to reference samples, thereby reflecting the former input of human decomposition products. Steroids (Δ5-sterols, 5β-stanols, 5α-stanols, stanones and bile acids) from soil samples from three soil pits suspected to be putative mass graves, as well as reference soil samples, were extracted and analysed with gas chromatography-mass spectrometry (GC–MS). Cholesterol, 5α-cholestanol and 5α-cholestanone were ubiquitously present in all soil pits and reference samples and therefore not indicative for the former input of human decomposition products. Compared to the reference, increased concentrations of selected faecal steroids (coprostanol, 5β-stigmastanol, 5β-cholestanone, lithocholic acid, isolithocholic acid) provided a strong indication of the former input of human decomposition products for one soil pit (pit 2). Additionally, epicoprostanol and isolithocholic acid were unique components for soil pit 2 as they were found neither in the other pits nor in the reference soil. In combination with earlier findings, steroid and bile acid patterns indicate an input of faecal and tissue constituents from former buried bodies in at least one of the three soil pits.

185 Evaluating Dynamics of Bile Acid Metabolism to Predict Recurrence of Clostridioides difficile Infection

INTRODUCTION: Recurrent Clostridioides difficile infection (CDI) is a major public health problem. The ability of commensal gut microbiota to metabolize primary into secondary bile acids plays a role in protection against this infection. Current clinical prediction tools for CDI recurrence do not incorporate biomarkers predictive of protective microbiota functionalities. We investigated metabolomic predictors of C. difficile recurrence. METHODS: We conducted a prospective longitudinal study of patients experiencing a first CDI episode. Patients testing positive with either enzyme immunoassay (EIA) toxin or polymerase chain reaction (PCR), and being treated for CDI, were eligible for inclusion. Serial stool samples were collected at diagnosis through week-8 post-completion of anti-CDI therapy if no recurrence, or until the point of recurrence (defined as diarrhea with positive C. difficile EIA toxin stool test). Liquid chromatography-mass spectrometry was performed to profile fecal bile acids. The week 1 post-antibiotic time point was chosen to assess for potential predictors. We derived a univariate logistic regression model predicting recurrence and computed the AUC (c-statistic) on discriminatory ability. The Youden index was calculated as the value that maximizes sensitivity and specificity. RESULTS: 29 first episode CDI patients were enrolled. 10 patients recurred (34.5%) during 8-weeks of follow-up. Average time to recurrence was 1.9 weeks. Two clinical factors were assessed a-priori for recurrence: use of metronidazole vs vancomycin (OR: 3.56, 95% CI 0.61–20.81, P = 0.15) and diagnosis with EIA vs PCR (OR 8.75, 95% CI 1.53–50.1, P = 0.01). At week 1, significantly more of the secondary bile acids lithocholic acid (LCA) and isolithocholic acid were found in non-recurrers vs recurrers (relative abundance per mg of stool: 20395.0 vs 4542.5, P = 0.037 and 4318.0 vs 1122.7, P = 0.031 respectively) (Table 1). As these are co-linear, we proceeded with LCA alone. With Youden index 17025, sensitivity was 80% and specificity was 57.9%, and the overall AUC resulted in a c-statistic of 0.753 (Figure 1a). However, when clinical risk factors were combined with LCA, sensitivity became 90% and specificity was 79%, with c-statistic of 0.91 (Figure 1b). CONCLUSION: The secondary bile acid, LCA, a known inhibitor of C. difficile's vegetative growth, predicted eventual recurrence of CDI with moderate accuracy; however, the model improved significantly with the addition of simple clinical variables.

Wash-Free Multiplexed Mix-and-Read Suspension Array Fluorescence Immunoassay for Anthropogenic Markers in Wastewater.

Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers. Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer. For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.