<jats:title>Abstract</jats:title> <jats:p>Inflammatory bowel diseases (IBDs) ar... more <jats:title>Abstract</jats:title> <jats:p>Inflammatory bowel diseases (IBDs) are chronic inflammatory multifactorial diseases caused by genetic, immune, and environmental factors. A decrease in intestinal serotonin transporter (SERT), which controls the extracellular availability of serotonin (5-HT) has been implicated in IBD. We previously showed that SERT deletion in mice altered gut bacterial community structure. The gut microbiota-derived metabolites are functional intermediaries between the microbiota and host. Here, we investigated the impact of SERT deficiency on gut metabolites under basal conditions and chronic colitis mimicking human IBD. Methods: Global metabolic profiles were analyzed by "Metabolon" (Durham, NC) on fecal samples from wild type littermates (WT) and SERT KO mice given water or chronic DSS (2.5% DSS for 5 weeks, n=7–9/group). Data were analyzed by ANOVA contrasts (difference between groups) and by two-way ANOVA (P&amp;lt;0.05, q&amp;lt;0.01). Results: SERT KO exhibited more severe colitis vs WT as assessed by histological score, myeloperoxidase activity and colon length. There was more pronounced decrease in the mRNA of tight junction proteins (TJs) occludin-1 and ZO-1 in SERT KO DSS vs WT DSS intestine. Metabolic profiling revealed that SERT deficiency alone resulted in extremely low levels of fecal ectoine, a bacterial derived solute that maintains TJ proteins expression. DSS treatment of WT (but not SERT KO) resulted in a significant increase in microbial derived metabolites including phenylalanine, N-acetylphenylalanine, tyrosine derivatives, glutamate, glutamine and benzoate derivatives. An increase in Trimethylamine N-oxide (TMAO), the short chain fatty acids butyrate/isobutyrate, TCA cycle metabolites; and a decrease in several metabolites including spermidine and various primary and secondary bile acids occurred in DSS treated WT and SERT KO to varying degrees; suggesting that these pathways may contribute to the colitis severity in SERT KO mice. Several secondary bile acids, ketone bodies, the metabolites of pterin, riboflavin pathway (FAD and FMN) and fatty acid metabolism pathways were increased in SERT KO (basal and DSS) suggesting genotype related differences in microbial community. We recently showed an impairment of Aryl hydrocarbon receptor (AhR), an IBD susceptible gene, in SERT KO mice, which could be partly due to altered availability of ligands. Indeed, the bacterial derived AhR ligand tryptamine was extremely low in SERT KO (basal and DSS). DSS increased the host derived AhR ligand, kynurenine in WT, but not in SERT KO.Conclusion:These data highlight the impact of serotonergic machinery and SERT inhibition on host physiology and pathophysiology of IBD. The results provide unique insights into gut bacteria derived metabolites and may aid in the development of novel treatment for disorders with altered SERT and 5-HT availability (Supported by CCFA and NIH).</jats:p>
American journal of physiology. Gastrointestinal and liver physiology, Jan 29, 2018
Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States.... more Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States. While C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile associated diarrhea remains poorly understood. Studies have shown that both NHE3 (Na/H exchanger) and DRA (Down Regulated in Adenoma, Cl/HCO exchanger) resulting in decreased electrolyte absorption are implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and down-regulated in patients with CDI, but the role of DRA in C. difficile infection remains unknown. In the current studies, we examined the effects of C. difficile toxins TcdA and TcdB on DRA protein and mRNA levels in intestinal epithelial cells (IECs). Our data demonstrates that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA,...
Journal of visualized experiments : JoVE, Mar 16, 2017
The intestinal epithelium has important transport and barrier functions that play key roles in no... more The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used i...
American journal of physiology. Gastrointestinal and liver physiology, Jun 14, 2016
SLC26A3 or Down-regulated in adenoma (DRA) is the major Cl(-)/HCO3 (-)exchanger involved in elect... more SLC26A3 or Down-regulated in adenoma (DRA) is the major Cl(-)/HCO3 (-)exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that up-regulate DRA activity may serve as potential anti-diarrheals. In this regard, Sphingosine-1-Phosphate (S1P), a member of bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, current studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-)exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-)exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promo...
American journal of physiology. Gastrointestinal and liver physiology, Jan 15, 2014
Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials... more Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus acidophilus (LA) and its culture supernatant (CS) stimulated Cl(-)/HCO3 (-) exchange activity, acutely via an increase in the surface levels of downregulated in adenoma (DRA, SLC26A3) and in long-term treatments via increasing its expression involving transcriptional mechanisms. However, the role of LA in modulating DRA activity under inflammatory conditions is not known. Current in vitro studies using human intestinal epithelial Caco-2 cells examined the efficacy of LA or its CS in counteracting the inhibitory effects of interferon-γ (IFN-γ) on Cl(-)/HCO3 (-) exchange activity. Pretreatment of cells with LA or LA-CS for 1 h followed by coincubation with IFN-γ significantly alleviated the inhibitory effects of IFN-γ on Cl(-)/HCO3 (-) exchange activity. In t...
Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable t... more Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable toxicity in the kidney. Histological studies have indicated that mitochondria, microsomes, lysosomes and plasma membranes of renal proximal convoluted tubules in particular are major GM targets. Despite numerous investigations, the biochemical/cellular basis of GM nephrotoxicity is not well understood. Recently reactive oxygen species (ROS) are considered to be important mediators of GM-induced nephrotoxicity. We hypothesize that GM causes damage to intracellular organelles and affects their structural integrity and alters metabolic and other functional capabilities. To address above hypothesis a long-term, time-dependent effect of GM has been studied on blood/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM) and basolateral (BLM), lysosomes and oxidative stress in renal tissues. A nephrotoxic dose of GM (80 mg/kg body weight) was administered to rats daily...
<jats:title>Abstract</jats:title> <jats:p>Inflammatory bowel diseases (IBDs) ar... more <jats:title>Abstract</jats:title> <jats:p>Inflammatory bowel diseases (IBDs) are chronic inflammatory multifactorial diseases caused by genetic, immune, and environmental factors. A decrease in intestinal serotonin transporter (SERT), which controls the extracellular availability of serotonin (5-HT) has been implicated in IBD. We previously showed that SERT deletion in mice altered gut bacterial community structure. The gut microbiota-derived metabolites are functional intermediaries between the microbiota and host. Here, we investigated the impact of SERT deficiency on gut metabolites under basal conditions and chronic colitis mimicking human IBD. Methods: Global metabolic profiles were analyzed by "Metabolon" (Durham, NC) on fecal samples from wild type littermates (WT) and SERT KO mice given water or chronic DSS (2.5% DSS for 5 weeks, n=7–9/group). Data were analyzed by ANOVA contrasts (difference between groups) and by two-way ANOVA (P&amp;lt;0.05, q&amp;lt;0.01). Results: SERT KO exhibited more severe colitis vs WT as assessed by histological score, myeloperoxidase activity and colon length. There was more pronounced decrease in the mRNA of tight junction proteins (TJs) occludin-1 and ZO-1 in SERT KO DSS vs WT DSS intestine. Metabolic profiling revealed that SERT deficiency alone resulted in extremely low levels of fecal ectoine, a bacterial derived solute that maintains TJ proteins expression. DSS treatment of WT (but not SERT KO) resulted in a significant increase in microbial derived metabolites including phenylalanine, N-acetylphenylalanine, tyrosine derivatives, glutamate, glutamine and benzoate derivatives. An increase in Trimethylamine N-oxide (TMAO), the short chain fatty acids butyrate/isobutyrate, TCA cycle metabolites; and a decrease in several metabolites including spermidine and various primary and secondary bile acids occurred in DSS treated WT and SERT KO to varying degrees; suggesting that these pathways may contribute to the colitis severity in SERT KO mice. Several secondary bile acids, ketone bodies, the metabolites of pterin, riboflavin pathway (FAD and FMN) and fatty acid metabolism pathways were increased in SERT KO (basal and DSS) suggesting genotype related differences in microbial community. We recently showed an impairment of Aryl hydrocarbon receptor (AhR), an IBD susceptible gene, in SERT KO mice, which could be partly due to altered availability of ligands. Indeed, the bacterial derived AhR ligand tryptamine was extremely low in SERT KO (basal and DSS). DSS increased the host derived AhR ligand, kynurenine in WT, but not in SERT KO.Conclusion:These data highlight the impact of serotonergic machinery and SERT inhibition on host physiology and pathophysiology of IBD. The results provide unique insights into gut bacteria derived metabolites and may aid in the development of novel treatment for disorders with altered SERT and 5-HT availability (Supported by CCFA and NIH).</jats:p>
American journal of physiology. Gastrointestinal and liver physiology, Jan 29, 2018
Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States.... more Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States. While C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile associated diarrhea remains poorly understood. Studies have shown that both NHE3 (Na/H exchanger) and DRA (Down Regulated in Adenoma, Cl/HCO exchanger) resulting in decreased electrolyte absorption are implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and down-regulated in patients with CDI, but the role of DRA in C. difficile infection remains unknown. In the current studies, we examined the effects of C. difficile toxins TcdA and TcdB on DRA protein and mRNA levels in intestinal epithelial cells (IECs). Our data demonstrates that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA,...
Journal of visualized experiments : JoVE, Mar 16, 2017
The intestinal epithelium has important transport and barrier functions that play key roles in no... more The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used i...
American journal of physiology. Gastrointestinal and liver physiology, Jun 14, 2016
SLC26A3 or Down-regulated in adenoma (DRA) is the major Cl(-)/HCO3 (-)exchanger involved in elect... more SLC26A3 or Down-regulated in adenoma (DRA) is the major Cl(-)/HCO3 (-)exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that up-regulate DRA activity may serve as potential anti-diarrheals. In this regard, Sphingosine-1-Phosphate (S1P), a member of bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, current studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-)exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-)exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promo...
American journal of physiology. Gastrointestinal and liver physiology, Jan 15, 2014
Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials... more Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus acidophilus (LA) and its culture supernatant (CS) stimulated Cl(-)/HCO3 (-) exchange activity, acutely via an increase in the surface levels of downregulated in adenoma (DRA, SLC26A3) and in long-term treatments via increasing its expression involving transcriptional mechanisms. However, the role of LA in modulating DRA activity under inflammatory conditions is not known. Current in vitro studies using human intestinal epithelial Caco-2 cells examined the efficacy of LA or its CS in counteracting the inhibitory effects of interferon-γ (IFN-γ) on Cl(-)/HCO3 (-) exchange activity. Pretreatment of cells with LA or LA-CS for 1 h followed by coincubation with IFN-γ significantly alleviated the inhibitory effects of IFN-γ on Cl(-)/HCO3 (-) exchange activity. In t...
Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable t... more Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable toxicity in the kidney. Histological studies have indicated that mitochondria, microsomes, lysosomes and plasma membranes of renal proximal convoluted tubules in particular are major GM targets. Despite numerous investigations, the biochemical/cellular basis of GM nephrotoxicity is not well understood. Recently reactive oxygen species (ROS) are considered to be important mediators of GM-induced nephrotoxicity. We hypothesize that GM causes damage to intracellular organelles and affects their structural integrity and alters metabolic and other functional capabilities. To address above hypothesis a long-term, time-dependent effect of GM has been studied on blood/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM) and basolateral (BLM), lysosomes and oxidative stress in renal tissues. A nephrotoxic dose of GM (80 mg/kg body weight) was administered to rats daily...
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