FXR Activation Research Articles

FXR activation remodels hepatic and intestinal transcriptional landscapes in metabolic dysfunction-associated steatohepatitis.

The escalating obesity epidemic and aging population have propelled metabolic dysfunction-associated steatohepatitis (MASH) to the forefront of public health concerns. The activation of FXR shows promise to combat MASH and its detrimental consequences. However, the specific alterations within the MASH-related transcriptional network remain elusive, hindering the development of more precise and effective therapeutic strategies. Through a comprehensive analysis of liver RNA-seq data from human and mouse MASH samples, we identified central perturbations within the MASH-associated transcriptional network, including disrupted cellular metabolism and mitochondrial function, decreased tissue repair capability, and increased inflammation and fibrosis. By employing integrated transcriptome profiling of diverse FXR agonists-treated mice, FXR liver-specific knockout mice, and open-source human datasets, we determined that hepatic FXR activation effectively ameliorated MASH by reversing the dysregulated metabolic and inflammatory networks implicated in MASH pathogenesis. This mitigation encompassed resolving fibrosis and reducing immune infiltration. By understanding the core regulatory network of FXR, which is directly correlated with disease severity and treatment response, we identified approximately one-third of the patients who could potentially benefit from FXR agonist therapy. A similar analysis involving intestinal RNA-seq data from FXR agonists-treated mice and FXR intestine-specific knockout mice revealed that intestinal FXR activation attenuates intestinal inflammation, and has promise in attenuating hepatic inflammation and fibrosis. Collectively, our study uncovers the intricate pathophysiological features of MASH at a transcriptional level and highlights the complex interplay between FXR activation and both MASH progression and regression. These findings contribute to precise drug development, utilization, and efficacy evaluation, ultimately aiming to improve patient outcomes.

Emodin alleviates cholestatic liver injury by modulating Sirt1/Fxr signaling pathways

Emodin (EMO) has the effect of anti-cholestasis induced by alpha-naphthylisothiocyanate (ANIT). But its mechanism is still unclear. The farnesoid X receptor (Fxr) is the master bile acid nuclear receptor. Recent studies have reported that Sirtuin 1 (Sirt1) can regulate the activities of Fxr. The purpose of the current study was to investigate the mechanism of EMO against ANIT-induced liver injury based on Sirt1/Fxr signaling pathway. The ANIT-induced cholestatic rats were used with or without EMO treatment. Serum biochemical indicators, as well as liver histopathological changes were examined. The genes expressions of Sirt1, Fxr, Shp, Bsep and Mrp2 were detected. The expressions of Sirt1, Fxr and their downstream related genes were investigated in vitro. The results showed that EMO significantly alleviated ANIT-induced liver injury in rats, and increased Sirt1, Fxr, Shp, Bsep and Mrp2 gene expression in liver, while decreased the expression of Cyp7a1. EMO significantly activated Fxr, while Sirt1 inhibitor and Sirt1 gene silencing significantly reduced Fxr activity in vitro. Collectively, EMO in the right dose has a protective effect on liver injury induced by ANIT, and the mechanism may be through activation of Fxr by Sirt1, thus regulating bile acid metabolism, and reducing bile acid load in hepatocytes.

HSDL2 links nutritional cues to bile acid and cholesterol homeostasis.

In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase-like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

Multiomics integration identifies regulatory factors underlying reproductive disorders in geese1

Geese, descendants of migratory birds, have preserved the distinct reproductive and lipid metabolism traits of their wild ancestors. Therefore, compared to other poultry, geese have lower egg production ability and a more sensitive susceptibility to fatty liver. Recent research underscores the impact of lipid metabolism disorders on female reproductive health. In this context, we observed reproductive disorders (RD) and lipid metabolism anomalies in certain geese populations. This study systematically elucidated the differences between RD and normal geese at various levels, including genomics, transcriptomics, bile acid metabolomics, and microbiomics, revealing the crucial role of microorganisms. Our study provides a thorough examination of the ovarian anatomical, histological, and transcriptomic profiles between normal and RD geese. Genomic analyses pinpoint mutations in genes associated with bile acid metabolism, highlighting their potential role in RD pathogenesis. The genomic discoveries are substantiated by precise bile acid assays and ileum transcriptome analyses, which expose a significant disruption in bile acid absorption, activation of FXR, and an increase in serum chenodeoxycholic acid (CDCA) concentrations within RD geese. Notably, 16S rRNA sequencing uncovers significantly greater beta diversity in the ileum microbiota of RD geese as compared to the normal group. Both Wilcoxon rank sum test and LEfSe analyses highlighted a marked increase in Romboutsia abundance in RD geese. Experimental cultivation of microbiota with CDCA supplementation confirms the impact of CDCA on Romboutsia lituseburensis (R. lituseburensis) proliferation. Gavage experiments with R. lituseburensis elucidates its involvement in primary follicle reduction via immune-mediated pathways. Collectively, our multi-faceted analysis unravels the intricate involvement of Romboutsia in goose RD, offering insights from genetic, physiological, and microbial dimensions. Our findings not only deepen our understanding of the etiology of RD in geese but also suggest potential avenues for therapeutic interventions targeting bile acid metabolism and the modulation of specific microbiota components.

The Secondary Bile acid, Lithocholic Acid, Inhibits Cl− Secretion Across Colonic Epithelial Cells in Vitro

Lithocholic acid (LCA) is a secondary bile acid formed in the colon by bacterial metabolism of chenodeoxycholic acid. Our previous studies have shown the bile acid to be protective under conditions of colonic inflammation. Here, we sought to determine if LCA might also play a role in regulating fluid and electrolyte transport. T84 cell monolayers were mounted in Ussing chambers for measurements of Cl− secretion, the primary driving force for fluid secretion into the colon. qRT-PCR and Western blotting were used to analyze mRNA and protein expression. Results were expressed as mean ± SEM and data were analyzed by one way ANOVA and Tukey’s post hoc test or by mixed-effects analysis and Dunnett's post hoc test. To assess the effects of LCA on CFTR promoter activity, we used a luciferase promoter/reporter system, where HEK293 cells were transfected with a plasmid containing the firefly luciferase gene under control of the CFTR promoter. Co-transfections were carried out with 2 additional plasmids expressing either the nuclear bile acid receptor, FXR, or its dimerization partner, RXR. Pretreatment of T84 cell monolayers with LCA inhibited subsequent Cl− secretory responses to the cAMP-dependent agonist, forskolin (FSK; 10 μM), in a concentration (1 – 10 μM) and time-dependent (3 - 24 hrs) manner. Maximal effects of LCA were observed at a concentration of 10 μM after treatment for 24 hrs, when responses to FSK were reduced to 50.9 ± 8.5% of those in controls (n = 6; p < 0.01). LCA (10 μM; 24 hrs) also inhibited responses to the Ca2+-dependent secretagogues, thapsigargin (2 μM) and histamine (100 μM), by 59.4 ± 2.4% (n = 4; p < 0.001) and 52.2 ± 1.9% (n = 5; p < 0.001), respectively. In further experiments, using nystatin-permeabilized T84 monolayers to isolate apical Cl− conductances, LCA (10 μM; 24 hrs) reduced FSK-stimulated responses to 72.7 ± 6.6% (n = 17; p < 0.001) of those in control cells. Analysis of CFTR expression, the primary exit pathway for Cl− in colonic epithelial cells, revealed that LCA treatment reduced mRNA and protein expression of the channel to 0.65 ± 0.05 fold (n = 7; p < 0.01) and 0.43 ± 0.06 fold (n = 6; p < 0.001), respectively. In CFTR promoter assays, LCA (10 μM) reduced CFTR promoter activity to 0.7 ± 0.02 fold of that in control cells (n = 5; p < 0.01), with co-expression of FXR being required for this effect to be observed. Finally, while LCA activated both FXR and the vitamin D receptor (VDR) in T84 cells, its effects in downregulating CFTR expression and Cl− conductances were mimicked by the FXR agonist, GW4064, but not by the VDR agonist, calcitriol. In conclusion, LCA, at physiologically relevant concentrations, inhibits Cl− secretion across colonic epithelial cells, likely through a mechanism involving FXR activation and inhibition of CFTR expression. These data add to the growing pool of knowledge regarding important regulatory actions of LCA in the colon and highlight its potential role as a target for the treatment of intestinal disorders. This work was supported by a grant from Science Foundation Ireland. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hammerhead-type FXR agonists induce an enhancer RNA Fincor that ameliorates nonalcoholic steatohepatitis in mice

The nuclear receptor, farnesoid X receptor (FXR/NR1H4), is increasingly recognized as a promising drug target for metabolic diseases, including nonalcoholic steatohepatitis (NASH). Protein-coding genes regulated by FXR are well known, but whether FXR also acts through regulation of long non-coding RNAs (lncRNAs), which vastly outnumber protein-coding genes, remains unknown. Utilizing RNA-seq and global run-on sequencing (GRO-seq) analyses in mouse liver, we found that FXR activation affects the expression of many RNA transcripts from chromatin regions bearing enhancer features. Among these we discovered a previously unannotated liver-enriched enhancer-derived lncRNA (eRNA), termed FXR-induced non-coding RNA (Fincor). We show that Fincor is specifically induced by the hammerhead-type FXR agonists, including GW4064 and tropifexor. CRISPR/Cas9-mediated liver-specific knockdown of Fincor in dietary NASH mice reduced the beneficial effects of tropifexor, an FXR agonist currently in clinical trials for NASH and primary biliary cholangitis (PBC), indicating that amelioration of liver fibrosis and inflammation in NASH treatment by tropifexor is mediated in part by Fincor. Overall, our findings highlight that pharmacological activation of FXR by hammerhead-type agonists induces a novel eRNA, Fincor, contributing to the amelioration of NASH in mice. Fincor may represent a new drug target for addressing metabolic disorders, including NASH.

Hammerhead-type FXR agonists induce an enhancer RNA Fincor that ameliorates nonalcoholic steatohepatitis in mice.

The nuclear receptor, farnesoid X receptor (FXR/NR1H4), is increasingly recognized as a promising drug target for metabolic diseases, including nonalcoholic steatohepatitis (NASH). Protein-coding genes regulated by FXR are well known, but whether FXR also acts through regulation of long non-coding RNAs (lncRNAs), which vastly outnumber protein-coding genes, remains unknown. Utilizing RNA-seq and global run-on sequencing (GRO-seq) analyses in mouse liver, we found that FXR activation affects the expression of many RNA transcripts from chromatin regions bearing enhancer features. Among these we discovered a previously unannotated liver-enriched enhancer-derived lncRNA (eRNA), termed FXR-induced non-coding RNA (Fincor). We show that Fincor is specifically induced by the hammerhead-type FXR agonists, including GW4064 and tropifexor. CRISPR/Cas9-mediated liver-specific knockdown of Fincor in dietary NASH mice reduced the beneficial effects of tropifexor, an FXR agonist currently in clinical trials for NASH and primary biliary cholangitis (PBC), indicating that amelioration of liver fibrosis and inflammation in NASH treatment by tropifexor is mediated in part by Fincor. Overall, our findings highlight that pharmacological activation of FXR by hammerhead-type agonists induces a novel eRNA, Fincor, contributing to the amelioration of NASH in mice. Fincor may represent a new drug target for addressing metabolic disorders, including NASH.

Inhibition of ACOX1 enhances the therapeutic efficacy of obeticholic acid in treating non-alcoholic fatty liver disease and mitigates its lipotoxicity.

High-dose Obeticholic acid exhibits promise for non-alcoholic fatty liver disease (NAFLD) treatment but can induce lipotoxicity. Our study sought to understand this mechanism and propose a solution. In a non-alcoholic fatty liver disease (NAFLD) model induced by a high-fat diet in FXR-/- mice, we pinpointed that FXR regulated the expression of ACOX1 through RNA-Seq analysis. In the livers of FXR-/- mice, both ACOX1 mRNA and protein expression notably decreased. In both HL-7702 and HEP-G2 cells, the silencing of FXR through shRNA plasmids decreased ACOX1 expression, while FXR activation with GW4064 increased it. These effects were reversible with the ACOX1-specific inhibitor, 10,12-Tricosadiynoic acid. In the NAFLD model of FXR-/- mice, The activation of ACOX1 is correlated with elevated serum LDL, triglycerides, and aggravated hepatic steatosis. However, the combination of 10,12-Tricosadiynoic acid with low-dose obeticholic acid effectively treated hepatic steatosis, reducing LDL levels in the NAFLD model of wild-type mice. This combination therapy demonstrated efficacy comparable to high-dose obeticholic acid alone. Notably, the combined drug regimen treats hepatic steatosis by inhibiting the IL-1β and α-SMA pathways in NAFLD. Combining ACOX1-specific inhibitors with low-dose obeticholic acid effectively treats high-fat diet-induced hepatic steatosis and reduces serum LDL. This approach enhances the therapeutic effects of obeticholic acid and mitigates its lipotoxicity by inhibiting the IL-1β and α-SMA pathways.

WESTERN DIET DEPLETES SMALL INTESTINAL INTRAEPITHELIAL LYMPHOCYTES VIA FXR-INTERFERON PATHWAY

Abstract BACKGROUND The prevalence of obesity has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar “Western” diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed adaptive immunity. METHODS Small intestine samples from non-inflammatory bowel disease (IBD) subjects who underwent resection were used for CD3 immunohistochemistry (IHC), and the CD3+ intraepithelial lymphocytes (IEL) densities were correlated with the patients’ body mass index (BMI). Mice were fed with either standard diet (SD) or WD for various durations, and IELs were isolated for flow cytometry analysis. RESULTS In non-IBD subjects, high BMI correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to deplete IEL (including major subsets CD4+, CD8+, CD8α+8β-CD4+, and γδCD8+ T cells) but not splenic lymphocytes, and the effect was reversible after another 4 weeks of SD washout. ob/ob mice that spontaneously developed obesity while fed with SD did not show IEL depletion, suggesting that the quality, not the quantity of the diet is key to this phenotype. Importantly, WD-associated IEL depletion was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL depletion. We previously showed that a key molecular pathway activated by WD in the ileum is the FXR pathway. Phagocyte-specific Fxr conditional knockout (cKO) mice were protected from WD-mediated IEL depletion, suggesting that FXR activation in the phagocytes is critical in mediating this phenotype. Activated FXR signaling stimulated phagocytes to secrete type I interferon (IFN), and inhibition of type I IFN signaling within the phagocytes (type I IFN monoclonal antibody treatment and Stat1 cKO mice) prevented WD-mediated IEL loss. CONCLUSION WD consumption depletes IEL by a FXR-IFN pathway mediated by gut phagocytes. These findings have significant clinical implications in understanding the pathogenesis of gut inflammatory disorders such as IBD.

Barley consumption under a high-fat diet suppresses lipogenic genes through altered intestinal bile acid composition

We evaluated whether barley flour consumption in a high-fat environment affects lipid metabolism through signals mediated by bile acids. Four-week-old mice were fed a high-fat diet supplemented with cellulose (HC) or β-glucan-rich barley flour (HB) for 12 weeks. Bile acid composition in the intestinal tract and feces was measured by GC/MS. Gene expression levels involved in bile acid metabolism in the liver and intestinal tract were determined by RT-PCR. Similar parameters were measured in mice treated with antibiotics (antibiotics-cellulose [AC] and antibiotics-barley [AB]) to reduce the activity of intestinal bacteria. The Results showed that the HB group had lower liver blood cholesterol and triglyceride levels than the HC group. The HB group showed a significant decrease in primary bile acids in the gastrointestinal tract compared to the HC group. On the other hand, the concentration of secondary bile acids relatively increased in the cecum and feces. In the liver, Fxr activation suppressed gene expression levels in synthesizing bile acids and lipids. Furthermore, in the gastrointestinal tract, Tgr5 was activated by increased secondary bile acids. Correspondingly, AMP levels were increased in the HB group compared to the HC group, AMPK was phosphorylated in the liver, and gene expression involved in lipid synthesis was downregulated. A comparison of the AC and AB groups treated with antibiotics did not confirm these effects of barley intake. In summary, our results suggest that the prevention of lipid accumulation by barley consumption involves signaling through changes in bile acid composition in the intestinal tract.

OR33-06 Pioneer Factor Foxa2 Mediates Chromatin Conformation Changes In Ligand-dependent Activation Of FXR

Abstract Disclosure: Y. Hao: None. L. Han: None. C. Wu: None. I. Bochkis: None. Activation of nuclear receptors, a family of ligand-dependent transcription factors, is used extensively in pharmacology to develop drug targets for diverse medical conditions, including metabolic disease and cancer. We have previously shown that pioneer factor Foxa2 opens chromatin for binding of nuclear receptors FXR and LXRα during acute ligand activation (Kain et al., Molecular Metabolism, 2021). FXR is activated by bile acids and we have demonstrated that Foxa2 plays an important role in bile acid metabolism, as deletion of Foxa2 in the liver results in intrahepatic cholestasis (Bochkis et al., Nature Medicine, 2008). We hypothesized that in addition to increasing chromatin accessibility, Foxa2 also enables chromatin conformational changes during ligand activation. We performed Foxa2 HiChIP (chromatin conformation capture, Hi-C, combined with chromatin immunoprecipitation, ChIP) assay to assess Foxa2-dependent long-range interactions in mouse livers treated with vehicle and FXR agonist GW4064. HiChIP contact analysis shows that global chromatin interactions are dramatically increased during FXR activation. In addition, ligand-treated livers have more Foxa2-anchored loops, suggesting Foxa2 is involved in dynamic chromatin conformational changes in ligand-dependent FXR activation. We show that chromatin conformation, including genome-wide interactions, intra-chromosomal and inter-chromosomal loops, drastically changes upon addition of FXR agonist. Hence, Foxa2 enables these conformational changes and plays an extended role in bile acid metabolism. Presentation: Sunday, June 18, 2023

Kaempferol ameliorated alcoholic liver disease through inhibiting hepatic bile acid synthesis by targeting intestinal FXR-FGF15 signaling

BackgroundAlcoholic liver disease (ALD) is characterized by the disturbance of bile acids homeostasis, which further deteriorates ALD. Bile acid metabolism and its related signal molecules have become new therapeutic targets for alcoholic liver disease. This study aimed to investigate the impact of kaempferol (KAE) on ALD and elucidate its underlying mechanisms. MethodsC57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. KAE was administered as an interventional drug to chronic alcohol-fed mice for four weeks to assess its effects on liver damage and bile acid metabolism. And Z-Guggulsterone (Z-Gu), a global FXR inhibitor, was used to investigate the impact of intestinal FXR-FGF15 signal in ALD mice. Additionally, intestinal epithelial cells were exposed to alcohol or specific bile acid to induce the damage of FXR activity in vitro. The dual luciferase activity assay was employed to ascertain the interplay between KAE and FXR activity. ResultsThe results indicated that KAE treatment exhibited a significant hepatoprotective effect against chronic alcohol-fed mice. Accompanied by the intestinal FXR activation, the administration of KAE suppressed hepatic bile acid synthesis and promoted intestinal bile acid excretion in chronic ALD mice. And the notable alterations in total bile acid levels and composition were observed in mice after chronic alcohol feeding, which were reversed by KAE supplementation. And more, the protective effects of KAE on ALD mice were deprived by the inhibition of intestinal FXR activation. In vitro experiments demonstrated that KAE effectively activated FXR-FGF15 signaling, mitigated the damage to FXR activity in intestinal epithelial cells caused by alcohol or specific bile acids. Additionally, luciferase activity assays revealed that KAE directly promoted FXR expression, thereby enhancing FXR activity. ConclusionKAE treatment inhibited hepatic bile acids synthesis, maintained bile acids homeostasis in ALD mice by directly activating intestinal FXR-FGF15 signaling, which effectively alleviated liver injury induced by chronic alcohol consumption.

FXR Maintains the Intestinal Barrier and Stemness by Regulating CYP11A1-Mediated Corticosterone Synthesis in Biliary Obstruction Diseases.

Biliary obstruction diseases are often complicated by an impaired intestinal barrier, which aggravates liver injury. Treatment of the intestinal barrier is often neglected. To investigate the mechanism by which intestinal bile acid deficiency mediates intestinal barrier dysfunction after biliary obstruction and identify a potential therapeutic modality, we mainly used a bile duct ligation (BDL) mouse model to simulate biliary obstruction and determine the important role of the bile acid receptor FXR in maintaining intestinal barrier function and stemness. Through RNA-seq analysis of BDL and sham mouse crypts and qRT-PCR performed on intestinal epithelial-specific Fxr knockout (FxrΔIEC) and wild-type mouse crypts, we found that FXR might maintain intestinal stemness by regulating CYP11A1 expression. Given the key role of CYP11A1 during glucocorticoid production, we also found that FXR activation could promote intestinal corticosterone (CORT) synthesis by ELISA. Intestinal organoid culture showed that an FXR agonist or corticosterone increased crypt formation and organoid growth. Further animal experiments showed that corticosterone gavage treatment could maintain intestinal barrier function and stemness, decrease LPS translocation, and attenuate liver injury in BDL mice. Our study hopefully provides a new theoretical basis for the prevention of intestinal complications and alleviation of liver injury after biliary obstruction.

Hyodeoxycholic acid attenuates cholesterol gallstone formation via modulation of bile acid metabolism and gut microbiota

Background & aimsHyodeoxycholic acid (HDCA), a hydrophilic bile acid (BA), may prevent and suppress the formation of cholesterol gallstones (CGs). However, the mechanism by which HDCA prevents CGs formation remains unclear. This study aimed to investigate the underlying mechanism of HDCA in preventing CG formation. MethodsC57BL/6J mice were fed either a lithogenic diet (LD), a chow diet, or LD combined with HDCA. The concentration of BAs in the liver and ileum were determined using liquid chromatography-mass spectrometry (LC-MS/MS). Genes involved in cholesterol and BAs metabolism were detected using polymerase chain reaction (PCR). The gut microbiota in the faeces was determined using 16S rRNA. ResultsHDCA supplementation effectively prevented LD-induced CG formation. HDCA increased the gene expression of BA synthesis enzymes, including Cyp7a1, Cyp7b1, and Cyp8b1, and decreased the expression of the cholesterol transporter Abcg5/g8 gene in the liver. HDCA inhibited LD-induced Nuclear farnesoid X receptor (Fxr) activation and reduced the gene expression of Fgf15 and Shp in the ileum. These data indicate that HDCA could prevent CGs formation partly by promoting BA synthesis in the liver and reduced the cholesterol efflux. In addition, HDCA administration reversed the LD-induced decrease in the abundance of norank_f_Muribaculaceae, which was inversely proportional to cholesterol levels. ConclusionsHDCA attenuated CG formation by modulating BA synthesis and gut microbiota. This study provides new insights into the mechanism by which HDCA prevents CG formation. Lay summaryIn this study, we found that HDCA supplementation suppressed LD-induced CGs in mice by inhibiting Fxr in the ileum, enhancing BA synthesis, and increasing the abundance of norank_f_Muribaculaceae in the gut microbiota. HDCA can also downregulate the level of total cholesterol in the serum, liver, and bile.

FXR activation enhances ACE2 expression and inhibits viral-induced IL-6 release from colonic epithelial cells in vitro: implications for gastrointestinal symptoms of SARS-CoV-2

Introduction: While better known for its pulmonary symptoms, SARS-CoV-2 also adversely affects the gastrointestinal tract, causing diarrhoea with evidence of inflammation. The nuclear bile acid receptor, farnesoid x receptor (FXR), is expressed in colonic epithelial cells and has been previously shown to inhibit cytokine production and promote barrier function, thereby preventing inflammatory responses in the gut. In the current studies, we set out to investigate a potential role for FXR in modulating epithelial responses to SARS-CoV-2. Methods: Undifferentiated human colonic or ileal enteroids, colonic epithelial T84, or Caco-2 cells were all grown as monolayers on permeable supports. Cells were treated with the FXR agonists, obeticholic acid (OCA, 10 μM) or GW4064 (5 μM), or infected with live SARS-CoV-2 (2019-nCoV/USA_WA1/2020). Changes in cellular mRNA, protein or secreted cytokines were measured by qPCR, western blotting, or ELISA. SARS-CoV-2 levels were quantified by qPCR using primers for envelope (E) and nucleocapsid (N) protein. Data are expressed as mean ± SEM and statistical analysis was performed using paired t-tests or one-way ANOVA with Dunnett’s or Tukey’s multiple comparisons tests. Results: Treatment of colonic or ileal enteroids with the FXR agonist, OCA, increased expression of mRNA for the SARS-CoV-2 receptor, ACE2, by 2.1 ± 0.4 (n = 3; p = 0.08) and 2.3 ± 0.2 (n = 3; p < 0.05) fold, respectively. Similarly, treatment of T84 cells with another FXR agonist, GW4064, increased expression of ACE2 mRNA with a maximal response of 1.8 ± 0.2 fold (n = 8; p < 0.01) occurring after 72 hrs. Increased mRNA was accompanied by a 2.3 ± 0.7 fold (n = 6; p < 0.01) increase in cellular protein expression and a 1.4 ± 0.1 fold (n = 9; p < 0.001) increase in ACE2 protein secretion into the apical medium. Effects of FXR on ACE2 expression were confirmed in monolayers of Caco-2 cells where GW4064 induced a 1.5 ± 0.1 fold (n = 3; p < 0.001) increase in mRNA for the receptor. GW4064 treatment did not affect mRNA levels for SARS-CoV-2 E or N protein in the infected Caco-2 cells. However, further experiments revealed that treatment with GW4064 inhibited release of the proinflammatory cytokine, IL-6, from either Caco-2 cells infected with SARS-CoV-2 or from T84 cells treated with the viral mimic, polyinosinic-polycytidylic acid (poly (I:C)) (25 μg/ml) by 46 ± 12 % (n = 3, p < 0.05) and 35 ± 6 % (n = 8; p < 0.01), respectively. Conclusion: Our data suggest that FXR activation upregulates expression of ACE2 in intestinal and colonic epithelial cells but did not directly affect SARS-CoV-2 cellular levels. Rather, FXR activation inhibits viral-induced proinflammatory cytokine secretion and may therefore represent a good target for the development of new approaches to alleviate inflammatory diarrhoea associated with SARS-CoV-2 infection. This work was funded by grants from Science Foundation Ireland and the National Institutes of Health (DK047987). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A174 A NOVEL MECHANISM OF CROHN’S DISEASE SEVERITY IN WOMEN: EVALUATING THE IMPACT OF AN ESTROGEN-FARNESOID X RECEPTOR INTERACTION ON INTESTINAL BARRIER FUNCTION

Abstract Background Crohn’s disease (CD) is associated with deficits in intestinal barrier function. Activation of bile acid-sensing nuclear receptor Farnesoid X receptor (NR1H4, FXR) is associated with protective effects against reduced intestinal barrier function; namely through promoting tight junction complex genes (TJCGs) and reducing expression of inflammatory cytokines. The FXR -1G>T variant is associated with decreased FXR activation and increased risk of, and early progression to, CD-related surgery in females only. It is hypothesized an estrogen-FXR interaction is mediating this effect. Purpose We aimed to assess the combined effect of estrogen and FXR genetic variation on intestinal barrier function using a cell-based model and its impact in a clinical cohort. Method Caco-2 cells were characterized for expression of TJCGs (zonula occludens-1, occludin, junctional adhesion molecule A, and claudin-1 and claudin-2), FXR, and nuclear estrogen receptors (ERα, ERβ) by qPCR. The influence of FXR activation on TJCGs was characterized by incubation of Caco-2 cells with chenodeoxycholic acid (CDCA). FXR-knockout stable Caco-2 line was developed using CRISPR-Cas9 methods and verified by qPCR and genotyping. The effect of estradiol on expression of TJCGs in FXR-knockout and wildtype Caco-2 cell monolayers was compared by qPCR. Future experiments include comparison of FXR-knockout and wildtype monolayer permeability with estrogen exposure by transwell permeability assay. The effect of FXR genotype and exogenous estrogen CD severity (surgery, hospitalization, fistulizing disease) was evaluated in our female cohort by multivariate analysis. Result(s) Increased expression of TJCGs was seen in native Caco-2 monolayers incubated with CDCA. FXR-knockout cell line was then successfully created and confirmed. FXR-knockout cells showed decreased expression of TJCGs with the exception of zonula occludens-1. Estradiol exposure resulted in a dose-dependent decline in TJCGs expression in the wildtype Caco-2 cell line, however this effect was lost in the FXR-knockout cell line. Preliminary analysis of patient cohort data (n=359) showed exogenous estrogen was associated with lower surgery risk (OR = 0.603, 95% CI= 0.373–0.964, p < 0.05; Fischer’s exact test) and trended towards decreasing fistulizing disease risk in a multiple logistic regression model which included FXR genotype. The association of FXR genotype with increased surgery risk was also confirmed in this logistic regression model. Conclusion(s) Herein, we show that FXR activity affects expression of TJCGs, and this effect is attenuated by estrogen interactions. Our patient cohort preliminary analysis confirmed an increased CD severity risk associated with FXR genotype and demonstrated a trend of decreasing CD severity with exogenous estrogen exposure. Further studies will assess the mechanisms by which FXR and estrogen interact to influence intestinal permeability. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; Department of Medicine, Western University Disclosure of Interest D. Taylor: None Declared, L. Donovan: None Declared, J. Choi: None Declared, R. Kim: None Declared, U. Schwarz: None Declared, A. Wilson Consultant of: Consulting fees from Fresenius Kabi, Speakers bureau of: Speaking fees from Takeda and Pfizer

Bile duct ligation differently regulates protein expressions of organic cation transporters in intestine, liver and kidney of rats through activation of farnesoid X receptor by cholate and bilirubin.

Body is equipped with organic cation transporters (OCTs). These OCTs mediate drug transport and are also involved in some disease process. We aimed to investigate whether liver failure alters intestinal, hepatic and renal Oct expressions using bile duct ligation (BDL) rats. Pharmacokinetic analysis demonstrates that BDL decreases plasma metformin exposure, associated with decreased intestinal absorption and increased urinary excretion. Western blot shows that BDL significantly downregulates intestinal Oct2 and hepatic Oct1 but upregulates renal and hepatic Oct2. Invitro cell experiments show that chenodeoxycholic acid (CDCA), bilirubin and farnesoid X receptor (FXR) agonist GW4064 increase OCT2/Oct2 but decrease OCT1/Oct1, which are remarkably attenuated by glycine-β-muricholic acid and silencing FXR. Significantly lowered intestinal CDCA and increased plasma bilirubin levels contribute to different Octs regulation by BDL, which are confirmed using CDCA-treated and bilirubin-treated rats. A disease-based physiologically based pharmacokinetic model characterizing intestinal, hepatic and renal Octs was successfully developed to predict metformin pharmacokinetics in rats. In conclusion, BDL remarkably downregulates expressions of intestinal Oct2 and hepatic Oct1 protein while upregulates expressions of renal and hepatic Oct2 protein in rats, finally, decreasing plasma exposure and impairing hypoglycemic effects of metformin. BDL differently regulates Oct expressions via Fxr activation by CDCA and bilirubin.

Gut-Bile Acid-Immune Axis Regulates T Cell Homeostasis in the Pathogenesis of Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by isolated thrombocytopenia. In a previous study, we observed dysbiosis in the phylogenetic composition and function of the gut microbiome of patients with ITP [Sci China Life Sci, 2020]. It is suggested that impaired intestinal barrier as well as the gut microbiota-related metabolites may provide signaling molecules and facilitate communication between the intestinal tract and the immune system. For example, bile acids (BAs), which are synthesized in the liver and transformed into secondary BAs in the gut, have recently been considered to be important signaling metabolites for the immune system. The bile acid receptor FXR can also inhibit the TLR4/MAPK pathway and participate in the regulation of the immune response. Here, we provide evidence that the gut-bile acid-immune axis plays a role in the pathogenesis of ITP and that obeticholic acid (OCA) treatment can repair the gut barrier and restore the gut-bile acid balance to reduce the destruction of platelets. Methods After establishing an active ITP mouse model, pathological analyses of biopsies, 16S amplicon sequencing, targeted bile acid metabolomics and real-time fluorescent quantitative PCR were used to explore the changes in intestinal barrier function, gut microbiota composition, and bile acid metabolites in feces and plasma. OCA treatment of the ITP mouse model and bone marrow-derived dendritic cells was used to investigate the effect of OCA on gut bile acid metabolism and T cell differentiation. In a clinical sample study, 65 treatment-naïve ITP patients and 62 healthy controls were enrolled. Metagenomic sequencing, bile acid-targeted metabolomics and mass cytometry were used to explore the characteristics of the "gut-bile acid-immune” axis in the ITP patients. Results In the mouse model of ITP, the gut barrier was damaged, as shown by exfoliation of IECs according to H&E staining and greater FITC-dextran leakage from the intestinal mucosa into the peripheral circulation. RNA sequencing of the IEC transcriptome revealed that the expression of claudin-4 and claudin-8 was significantly diminished in ITP mice. As a result of the impaired gut barrier, plasma levels of LPS were elevated in ITP mice. Moreover, the Firmicutes:Bacteroidetes ratio was elevated in the gut microbiota according to 16S amplicon sequencing, and increased fecal levels of BAs were observed, which gave priority to primary BAs like β-MCA. The accumulation of β-MCA, which is an FXR antagonist, inhibited the activity of FXR in IECs, hepatocytes and immune cells such as DCs. After stimulation with LPS in vitro, murine bone marrow-derived DCs were observed to induce greater Th1 differentiation, which could be reversed by an FXR agonist (GW4064 or OCA). The secretion of IL-12 by DCs induced Th1 differentiation and was regulated by the TLR4/MAPK/p38 pathway, and this effect was inhibited by OCA through the activation of FXR. In vivo, OCA increased the expression of claudin-4 and claudin-8, and restored the balance of bile acid metabolism in the mice with ITP. After OCA treatment, the Th1/Th2 imbalance in the bone marrow of the mice with ITP was corrected, and reduced platelet destruction in the spleen and liver was observed with an in vivo imaging system. Clinical studies have shown that the gut microbiota in ITP patients is dysregulated, which showed an increased abundance of Firmicutes. Scanning electron microscopy showed that the gaps between IECs were increased in the ITP patients. The bile acid pool of the ITP patients was shown to have an increased proportion of primary BAs in the bone marrow. After in vitro treatment with OCA for 72 h, the proportion of human Th1 cells among human bone marrow mononuclear cells was significantly reduced, which suggested the therapeutic effect of OCA in ITP patients. Conclusions This study preliminarily revealed the characteristics of the "gut-bile acid-immune” axis in ITP patients and a mouse model. In vitro and in vivo studies confirmed that OCA could repair the intestinal barrier, correct gut microbiota dysbiosis and bile acid metabolism abnormalities, and decrease the proportion of peripheral Th1 cells via MAPK signaling in DCs, resulting in a decrease in the excessive destruction of platelets. This study provides a theoretical basis for targeting the gut microbiota in the clinical treatment of ITP.

Fucoidan Ameliorated Dextran Sulfate Sodium-Induced Ulcerative Colitis by Modulating Gut Microbiota and Bile Acid Metabolism.

Gut dysbiosis and bile acid (BA) metabolism disturbance are involved in the pathogenesis of ulcerative colitis. This study aimed to investigate the effect of fucoidan on BA metabolism and gut microbiota in dextran sulfate sodium-induced colitis mice. Our results showed that fucoidan effectively suppressed colonic inflammation and repaired the gut barrier. In addition, fucoidan increased the relative abundance of the Lachnospiraceae family, such as Turicibacter, Muribaculum, Parasutterella, and Colidextribacter, followed by an increase in short-chain fatty acids, especially in butyrate. Moreover, fucoidan modulated bile acid metabolism by elevating cholic acid, ursodeoxycholic acid, deoxycholic acid, and lithocholic acid and decreasing β-muricholic acid, which led to activation of FXR and TGR5 and further enhanced the gut barrier and suppressed colonic inflammation. Our results revealed that the effect of fucoidan alleviating colitis was largely mediated by gut microbiota, which was confirmed by the fecal transplantation experiment. Collectively, these findings provided the basis for fucoidan as a potential functional food for colitis.