Serotonin Transporter Deficiency Induces Metabolic Alterations in the Ileal Mucosa.

Serotonin transporter (SERT) deficiency has been implicated in metabolic syndrome, intestinal inflammation, and microbial dysbiosis. Interestingly, changes in microbiome metabolic capacity and several alterations in host gene expression, including lipid metabolism, were previously observed in SERT-/- mice ileal mucosa. However, the precise host or microbial metabolites altered by SERT deficiency that may contribute to the pleiotropic phenotype of SERT KO mice are not yet understood. This study investigated the hypothesis that SERT deficiency impacts lipid and microbial metabolite abundances in the ileal mucosa, where SERT is highly expressed. Ileal mucosal metabolomics was performed by Metabolon on wild-type (WT) and homozygous SERT knockout (KO) mice. Fluorescent-activated cell sorting (FACS) was utilized to measure immune cell populations in ileal lamina propria to assess immunomodulatory effects caused by SERT deficiency. SERT KO mice exhibited a unique ileal mucosal metabolomic signature, with the most differentially altered metabolites being lipids. Such changes included increased diacylglycerols and decreased monoacylglycerols in the ileal mucosa of SERT KO mice compared to WT mice. Further, the ileal mucosa of SERT KO mice exhibited several changes in microbial-related metabolites known to play roles in intestinal inflammation and insulin resistance. SERT KO mice also had a significant reduction in the abundance of ileal group 3 innate lymphoid cells (ILC3). In conclusion, SERT deficiency induces complex alterations in the ileal mucosal environment, indicating potential links between serotonergic signaling, gut microbiota, mucosal immunity, intestinal inflammation, and metabolic syndrome.

Potential Dietary and Therapeutic Strategies Involving Indole-3-Carbinole in Preclinical Models of Intestinal Inflammation.

Diet-microbiota interactions are emerging as important contributors in the pathogenesis of inflammatory bowel diseases (IBD), characterized by chronic inflammation of the GI tract. The aryl hydrocarbon receptor (AhR) transcription factor regulates xenobiotic metabolism and is activated by exogenous ligands, including indole-3-carbinole (I3C), which is found in cruciferous vegetables. However, studies investigating the impact of dietary I3C and AhR in preclinical models resembling human IBD are lacking. Mice (WT or AhR KO in IECs, 6-8 weeks) or SAMP/YitFC and AKR/J control (4 weeks, m/f) were fed an AhR ligand-depleted or I3C (200 ppm)-supplemented diet. There were increased levels of LPS and exacerbated inflammation, resulting in increased mortality in AhRΔIEC mice fed the AhR ligand-depleted diet in response to chronic DSS. The mechanisms underlying the protective effects of I3C supplementation during colonic colitis involved amelioration of intestinal inflammation and restoration of the altered gut microbiota, particularly the families of clostridicae and lachnospriaceae. Furthermore, the AhR-depleted diet led to the emergence of pathobiont Parvibacter caecicola in WT mice. SAMP/YitFc mice with spontaneous ileitis showed significant recovery in epithelial abnormalities when fed dietary I3C. These data demonstrate the critical role of AhR and the mechanisms of dietary I3C in maintaining epithelial homeostasis and ameliorating inflammation.

Serum metabolomic analysis in cirrhotic alcohol-associated liver disease patients identified differentially altered microbial metabolites and novel potential biomarkers for disease severity.

BACKGROUND: Alcohol-Associated Liver Disease (ALD) is a leading cause of liver mortality. Mechanisms responsible for severe ALD and the roles of gut microbiota are not fully understood. Multi-omics tools have enabled a better understanding of metabolic alterations and can aid in identifying metabolites as biomarkers for severe ALD. AIMS: Examine differences between cirrhotic and non-cirrhotic ALD, investigate microbial contributions to such changes, and identify potential diagnostic and prognostic metabolites for severe ALD. METHODS: Untargeted metabolomics were performed on the serum of 11 non-cirrhotic and 11 cirrhotic ALD patients. Data were analyzed using MetOrigin and Metaboanalyst to identify enriched pathways. RESULTS: Increased methylated nucleotides, gamma-glutamyl amino acids, bile acids, and specific metabolites kynurenine and campesterol were increased in ALD cirrhosis, whereas branched-chain amino acids, serotonin, and xanthurenate were decreased. Microbial contributions included increases in the short-chain fatty acid indolebutyrate and methionine sulfoxide in ALD cirrhosis. The analysis also identified the potential for serum levels of 3-ureidopropionate, cis-3,3-methyleneheptanoylglycine, retinol, and valine to be used as biomarkers for clinical assessment of alcohol-associated cirrhosis. CONCLUSION: We have identified a set of metabolites that are differentially altered in cirrhotic compared to non-cirrhotic ALD that can potentially be used as biomarkers for the severity of the disease.

Mucosal Metabolomic Signatures in Chronic Colitis: Novel Insights into the Pathophysiology of Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) involve complex interactions among genetic factors, aberrant immune activation, and gut microbial dysbiosis. While metabolomic studies have focused on feces and serum, fewer investigations have examined the intestinal mucosa despite its crucial role in metabolite absorption and transport. The goals of this study were twofold: to test the hypothesis that gut microbial dysbiosis from chronic intestinal inflammation leads to mucosal metabolic alterations suitable for therapeutic targeting, and to address gaps in metabolomic studies of intestinal inflammation that have overlooked the mucosal metabolome. The chronic DSS colitis was induced for five weeks in 7-9-week-old wild-type C57BL/6J male mice followed by microbial profiling with targeted 16srRNA sequencing service. Mucosal metabolite measurements were performed by Metabolon (Morrisville, NC). The data were analyzed using the bioinformatic tools Pathview, MetOrigin, and Metaboanalyst. The novel findings demonstrated increases in several host- and microbe-derived purine, pyrimidine, endocannabinoid, and ceramide metabolites in colitis. Origin analysis revealed that microbial-related tryptophan metabolites kynurenine, anthranilate, 5-hydroxyindoleacetate, and C-glycosyltryptophan were significantly increased in colon mucosa during chronic inflammation and strongly correlated with disease activity. These findings offer new insights into the pathophysiology of IBD and provide novel potential targets for microbial-based therapeutics.

Bile acids as inflammatory mediators and modulators of intestinal permeability.

Bile acids are critical for the digestion and absorption of lipids and fat-soluble vitamins; however, evidence continues to emerge supporting additional roles for bile acids as signaling molecules. After they are synthesized from cholesterol in the liver, primary bile acids are modified into secondary bile acids by gut flora contributing to a diverse pool and making the composition of bile acids highly sensitive to alterations in gut microbiota. Disturbances in bile acid homeostasis have been observed in patients with Inflammatory Bowel Diseases (IBD). In fact, a decrease in secondary bile acids was shown to occur because of IBD-associated dysbiosis. Further, the increase in luminal bile acids due to malabsorption in Crohn's ileitis and ileal resection has been implicated in the induction of diarrhea and the exacerbation of inflammation. A causal link between bile acid signaling and intestinal inflammation has been recently suggested. With respect to potential mechanisms related to bile acids and IBD, several studies have provided strong evidence for direct effects of bile acids on intestinal permeability in porcine and rodent models as well as in humans. Interestingly, different bile acids were shown to exert distinct effects on the inflammatory response and intestinal permeability that require careful consideration. Such findings revealed a potential effect for changes in the relative abundance of different bile acids on the induction of inflammation by bile acids and the development of IBD. This review summarizes current knowledge about the roles for bile acids as inflammatory mediators and modulators of intestinal permeability mainly in the context of inflammatory bowel diseases.

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

High-throughput screening and selection of mammalian cells for enhanced protein production.

The production of recombinant proteins for biotherapeutic use is a multibillion dollar industry, which has seen continual growth in recent years. In order to produce the best protein with minimal cost and time, selection methods are utilized during the cell line development process in order to select for the most desirable clonal cell line from a heterogeneous transfectant pool. Today, there is a vast array of potential selection methods available, which vary in cost, complexity and efficacy. This review aims to highlight cell line selection methods that exist for the isolation of high-producing clones, and also reviews techniques that can be used to predict, at a small scale, the performance of clones at large, industrially-relevant scales.