A Direct Link Implicating Loss of SLC26A6 to Gut Microbial Dysbiosis, Compromised Barrier Integrity, and Inflammation.

BACKGROUND & AIMS: Putative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel disease are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut-barrier integrity and susceptibility to colitis is currently elusive. METHODS: Age-matched PKO and wild-type littermates were administered 3.5% dextran sulfate sodium in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. Messenger RNA and protein expression of tight junction proteins were determined by reverse transcription polymerase chain reaction and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression, and H&E staining. Gut microbiome and associated metabolome were analyzed by 16S ribosomal RNA sequencing and mass spectrometry, respectively. RESULTS: PKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to dextran sulfate sodium treatment. In addition, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared with controls. Co-housing wild-type and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of tight junction proteins in the colons of wild-type mice. CONCLUSIONS: Our data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut-barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut-barrier integrity, and its deficiency appears to contribute to the pathogenesis of inflammatory bowel diseases.

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.

Ion Transport Basis of Diarrhea, Paneth Cell Metaplasia, and Upregulation of Mechanosensory Pathway in Anti-CD40 Colitis Mice.

BACKGROUND: Anti-Cluster of differentiation (CD)-40-induced colitis, driven by innate inflammatory responses in the intestine, is a potent animal model exhibiting IBD pathophysiology including diarrhea. However, the ion transport basis of diarrhea and some key mucosal pathways (Paneth cells, stem cell niche, and mechanosensory) in this model have not been investigated. METHODS: Mucosal scrapings and intestinal tissue from control and CD40 antibody (150 µg) treated Rag2-/- mice were examined for gut inflammation, Paneth cell numbers, expression of key transporters, tight/adherens junction proteins, stem cell niche, and mechanosensory pathway via hematoxylin and eosin staining, quantitative polymerase chain reaction, and western blotting. RESULTS: Compared with control, anti-CD40 antibody treatment resulted in a significant loss of body weight (P < .05) and diarrhea at day 3 postinjection. Distal colonic tissues of anti-CD40 mice exhibited increased inflammatory infiltrates, higher claudin-2 expression, and appearance of Paneth cell-like structures indicative of Paneth cell metaplasia. Significantly reduced expression (P < .005) of downregulated in adenoma (key Cl- transporter), P-glycoprotein/multidrug resistantance-1 (MDR1, xenobiotic transporter), and adherens junction protein E-cadherin (~2-fold P < .05) was also observed in the colon of anti-CD40 colitis mice. Interestingly, there were also marked alterations in the stem cell markers and upregulation of the mechanosensory YAP-TAZ pathway, suggesting the activation of alternate regeneration pathway post-tissue injury in this model. CONCLUSION: Our data demonstrate that the anti-CD40 colitis model shows key features of IBD observed in the human disease, hence making it a suitable model to investigate the pathophysiology of ulcerative colitis (UC).

Our studies demonstrate the ion transport basis of diarrhea, downregulation of MDR1 and E-cadherin, Paneth cell metaplasia, and induction of claudin-2 and mechanosensory pathway in anti-CD40 colitis (innate immune-based model of IBD), similar to the human disease.

Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans: report of an expert meeting.

The present report summarizes the United States Department of Veterans Affairs (VA) field-based meeting titled "Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans." Our Veteran patient population experiences a high incidence of service-related chronic physical and mental health problems, such as infection, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), various forms of hematological and non-hematological malignancies, neurologic conditions, end-stage organ failure, requiring transplantation, and posttraumatic stress disorder (PTSD). We report the views of a group of scientists who focus on the current state of scientific knowledge elucidating the mechanisms underlying the aforementioned disorders, novel therapeutic targets, and development of new approaches for clinical intervention. In conclusion, we dovetailed on four research areas of interest: 1) microbiome interaction with immune cells after hematopoietic cell and/or solid organ transplantation, graft-versus-host disease (GVHD) and graft rejection, 2) intestinal inflammation and its modification in IBD and cancer, 3) microbiome-neuron-immunity interplay in mental and physical health, and 4) microbiome-micronutrient-immune interactions during homeostasis and infectious diseases. At this VA field-based meeting, we proposed to explore a multi-disciplinary, multi-institutional, collaborative strategy to initiate a roadmap, specifically focusing on host microbiome-immune interactions among those with service-related chronic diseases to potentially identify novel and translatable therapeutic targets.

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.

Pathophysiology of Enteropathogenic Escherichia coli-induced Diarrhea.

Enteropathogenic Escherichia coli (EPEC) are important diarrheal pathogens of infants and young children. Since the availability of molecular diagnosis methods, we now have new insights into the incidence and prevalence of these infections. Recent epidemiological studies indicate that atypical EPEC (aEPEC) are seen more frequently than typical EPEC (tEPEC) worldwide, including in both endemic diarrhea and diarrhea outbreaks. Therefore, it is important to further characterize the pathogenicity of these emerging strains. The virulence mechanisms and pathophysiology of the attaching and effacing lesion (A/E) and the type-three-secretion-system (T3SS) are complex but well-studied. A/E strains use their pool of locus of enterocyte effacement (LEE)-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host. However, the exact mechanisms of diarrhea in EPEC infection are not completely understood. From the clinical perspective, there is a need for fast, easy, and inexpensive diagnostic methods to define optimal treatment and prevention for children in endemic areas. In this article, we present a review of the classification of EPEC, epidemiology, pathogenesis of the disease caused by these bacteria, determinants of virulence, alterations in signaling, determinants of colonization vs. those of disease, and the limited information we have on the pathophysiology of EPEC-induced diarrhea. This article combines peer-reviewed evidence from our own studies and the results of an extensive literature search in the databases PubMed, EMBASE, and Scopus.

Increased intestinal permeability and downregulation of absorptive ion transporters Nhe3, Dra, and Sglt1 contribute to diarrhea during Clostridioides difficile infection.

BACKGROUND & AIM: Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced by C. difficile are the major determinants of disease. However, the pathophysiological causes of diarrhea during CDI are not well understood. Here, we investigated the effects of C. difficile toxins on paracellular permeability and apical ion transporters in the context of an acute physiological infection. METHODS: We studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure paracellular permeability and ion transporter function across the intestinal tract. Infected intestinal tissues were analyzed by immunofluorescence microscopy and RNA-sequencing to uncover mechanisms of transporter dysregulation. RESULTS: Intestinal permeability was increased through the size-selective leak pathway in vivo during acute CDI in a 2-day-post infection model. Chloride secretory activity was reduced in the cecum and distal colon during infection by decreased CaCC and CFTR function, respectively. SGLT1 activity was significantly reduced in the cecum and colon, accompanied by ablated SGLT1 expression in colonocytes and increased luminal glucose concentrations. SGLT1 and DRA expression was ablated by either TcdA or TcdB during acute infection, but NHE3 was decreased in a TcdB-dependent manner. The localization of key proteins that link filamentous actin to the ion transporters in the apical plasma membrane was unchanged. However, Sglt1, Nhe3, and Dra were drastically reduced at the transcript level, implicating downregulation of ion transporters in the mechanism of diarrhea during CDI. CONCLUSIONS: CDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination likely leads to dysfunctional water and solute absorption in the large bowel, causing osmotic diarrhea. These findings provide insights into the pathophysiological mechanisms underlying diarrhea and may open novel avenues for attenuating CDI-associated diarrhea.

Loss of SLC26A3 Results in Colonic Mucosal Immune Dysregulation via Epithelial-Immune Cell Crosstalk.

BACKGROUND & AIMS: Down-regulation of chloride transporter SLC26A3 or down-regulated in adenoma (DRA) in colonocytes has recently been linked to the pathogenesis of ulcerative colitis (UC). Because exaggerated immune responses are one of the hallmarks of UC, these current studies were undertaken to define the mechanisms by which loss of DRA relays signals to immune cells to increase susceptibility to inflammation. METHODS: NanoString Immunology Panel, fluorescence assisted cell sorting, immunoblotting, immunofluorescence, and quantitative real-time polymerase chain reaction assays were used in wild-type and DRA knockout (KO) mice. Interleukin (IL)-33 blocking was used to determine specific changes in immune cells and co-housing/broad spectrum antibiotics administration, and ex vivo studies in colonoids were conducted to rule out the involvement of microbiota. Colonoid-derived monolayers from healthy and UC patient biopsies were analyzed for translatability. RESULTS: There was a marked induction of Th2 (>2-fold), CD4+ Th2 cells (∼8-fold), RORγt+ Th17, and FOXP3+ regulatory T cells (Tregs). DRA KO colons also exhibited a robust induction of IL-33 (>8-fold). In vivo studies using blocking of IL-33 established that T2 immune dysregulation (alterations in ILC2, Th2, and GATA3+ iTregs) in response to loss of DRA was due to altered epithelial-immune cell crosstalk via IL-33. CONCLUSIONS: Loss of DRA in colonocytes triggers the release of IL-33 to drive a type 2 immune response. These observations emphasize the critical importance of DRA in mucosal immune homeostasis and its implications in the pathogenesis of UC.

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.

Downregulation of NHE-3 (SLC9A3) expression by MicroRNAs in intestinal epithelial cells.

Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.

Enteroviral Infections in Infants.

Enteroviruses (EVs) are major pathogens in young infants. These viruses were traditionally classified into the following four subgenera: polio, coxsackie A and B, and echoviruses. Now that poliomyelitis seems to be controlled in most parts of the world, coxsackie and echoviruses are gaining more attention because (i) the structural and pathophysiological similarities and (ii) the consequent possibilities in translational medicine. Enteroviruses are transmitted mainly by oral and fecal-oral routes; the clinical manifestations include a viral prodrome including fever, feeding intolerance, and lethargy, which may be followed by exanthema; aseptic meningitis and encephalitis; pleurodynia; myopericarditis; and multi-system organ failure. Laboratory diagnosis is largely based on reverse transcriptase-polymerase chain reaction, cell culture, and serology. Prevention and treatment can be achieved using vaccination, and administration of immunoglobulins and antiviral drugs. In this article, we have reviewed the properties of these viruses, their clinical manifestations, and currently available methods of detection, treatment, and prognosis.

Lactobacillus acidophilus Mitigates Osteoarthritis-Associated Pain, Cartilage Disintegration and Gut Microbiota Dysbiosis in an Experimental Murine OA Model.

To test probiotic therapy for osteoarthritis (OA), we administered Lactobacillus acidophilus (LA) by oral gavage (2×/week) after induction of OA by partial medial meniscectomy (PMM). Pain was assessed by von Frey filament and hot plate testing. Joint pathology and pain markers were comprehensively analyzed in knee joints, spinal cords, dorsal root ganglia and distal colon by Safranin O/fast green staining, immunofluorescence microscopy and RT-qPCR. LA acutely reduced inflammatory knee joint pain and prevented further OA progression. The therapeutic efficacy of LA was supported by a significant reduction of cartilage-degrading enzymes, pain markers and inflammatory factors in the tissues we examined. This finding suggests a likely clinical effect of LA on OA. The effect of LA treatment on the fecal microbiome was assessed by 16S rRNA gene amplicon sequencing analysis. LA significantly altered the fecal microbiota compared to vehicle-treated mice (PERMANOVA p < 0.009). Our pre-clinical OA animal model revealed significant OA disease modifying effects of LA as reflected by rapid joint pain reduction, cartilage protection, and reversal of dysbiosis. Our findings suggest that LA treatment has beneficial systemic effects that can potentially be developed as a safe OA disease-modifying drug (OADMD).

α-Bisabolol Mitigates Colon Inflammation by Stimulating Colon PPAR-γ Transcription Factor: In Vivo and In Vitro Study.

The incidence and prevalence of inflammatory bowel disease (IBD, Crohn's disease, and ulcerative colitis) are increasing worldwide. The etiology of IBD is multifactorial, including genetic predisposition, dysregulated immune response, microbial dysbiosis, and environmental factors. However, many of the existing therapies are associated with marked side effects. Therefore, the development of new drugs for IBD treatment is an important area of investigation. Here, we investigated the anti-inflammatory effects of α-bisabolol, a naturally occurring monocyclic sesquiterpene alcohol present in many aromatic plants, in colonic inflammation. To address this, we used molecular docking and dynamic studies to understand how α-bisabolol interacts with PPAR-γ, which is highly expressed in the colonic epithelium: in vivo (mice) and in vitro (RAW264.7 macrophages and HT-29 colonic adenocarcinoma cells) models. The molecular docking and dynamic analysis revealed that α-bisabolol interacts with PPAR-γ, a nuclear receptor protein that is highly expressed in the colon epithelium. Treatment with α-bisabolol in DSS-administered mice significantly reduced Disease Activity Index (DAI), myeloperoxidase (MPO) activity, and colonic length and protected the microarchitecture of the colon. α-Bisabolol treatment also reduced the expression of proinflammatory cytokines (IL-6, IL1ß, TNF-α, and IL-17A) at the protein and mRNA levels. The expression of COX-2 and iNOS inflammatory mediators were reduced along with tissue nitrite levels. Furthermore, α-bisabolol decreased the phosphorylation of activated mitogen-activated protein kinase (MAPK) signaling and nuclear factor kappa B (NFκB) proteins and enhanced colon epithelial PPAR-γ transcription factor expression. However, the PPAR-α and ß/δ expression was not altered, indicating α-bisabolol is a specific stimulator of PPAR-γ. α-Bisabolol also increased the PPAR-γ transcription factor expression but not PPAR-α and ß/δ in pretreated in LPS-stimulated RAW264.7 macrophages. α-Bisabolol significantly decreased the expression of proinflammatory chemokines (CXCL-1 and IL-8) mRNA in HT-29 cells treated with TNF-α and HT-29 PPAR-γ promoter activity. These results demonstrate that α-bisabolol mitigates colonic inflammation by inhibiting MAPK signaling and stimulating PPAR-γ expression.

miR-29a, b, and c regulate SLC5A8 expression in intestinal epithelial cells.

Short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber exert myriad of beneficial effects including the amelioration of inflammation. SCFAs exist as anions at luminal pH; their entry into the cells depends on the expression and function of monocarboxylate transporters. In this regard, sodium-coupled monocarboxylate transporter-1 (SMCT-1) is one of the major proteins involved in the absorption of SCFA in the mammalian colon. However, very little is known about the mechanisms of regulation of SMCT-1 expression in health and disease. MicroRNAs (miRs) are known to play a key role in modulating gene expression. In silico analysis showed miR-29a, b, and c with highest context score and its binding region was conserved among mammals. The 3'-untranslated region (UTR) of human SMCT-1 gene was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with miR-29a, b, and c mimics into Caco-2 and/or T-84 cells. The presence of UTR of this gene significantly decreased luciferase activity compared with empty vector. Cotransfection with miR-29a, b, or c resulted in further decrease in 3'-UTR activity of SMCT-1 luciferase constructs. Mimic transfection significantly decreased SMCT-1 protein expression without altering mRNA expression. Furthermore, the expression of miR-29a and c were significantly lower in mouse colon compared with small intestine, consistent with higher levels of SMCT-1 protein in the colon. Our studies demonstrated a novel finding in which miR-29a, b, and c downregulate SMCT-1 expression in colonic epithelial cells and may partly explain the differential expression of these transporters along the length of the gastrointestinal (GI) tract.NEW & NOTEWORTHY Our study for the first time reports the posttranscriptional regulation of SMCT-1 by miR-29a, b, and c in colonic epithelial cells. We also demonstrate that the expression of these microRNAs is lower in the mouse proximal and distal colon which partially explains the higher expression level of SMCT-1 in the colon compared with small intestine.

A Simple Method for Creating a High-Content Microscope for Imaging Multiplexed Tissue Microarrays.

High-throughput, high-content imaging technologies and multiplex slide scanning have become widely used. Advantages of these approaches include the ability to archive digital copies of slides, review slides as teams using virtual microscopy software, and standardize analytical approaches. The cost and hardware and software inflexibility of dedicated slide scanning devices can, however, complicate implementation. Here, we describe a simple method that allows any microscope to be used for slide scanning. The only requirements are that the microscope be equipped with a motorized filter turret or wheels (for multi-channel fluorescence) and a motorized xyz stage. This example uses MetaMorph software, but the same principles can be used with any microscope control software that includes a few standard functions and allows programming of simple command routines, or journals. The series of journals that implement the method perform key functions, including assistance in defining an unlimited number of regions of interest (ROI) and imaging parameters. Fully automated acquisition is rapid, taking less than 3 hr to image fifty 2.5-mm ROIs in four channels. Following acquisition, images can be easily stitched and displayed using open-source or commercial image-processing and virtual microscope applications. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Hardware and software configuration Basic Protocol 2: Create a preliminary scan Basic Protocol 3: Select, save, and position ROIs Basic Protocol 4: Determine and set autofocus parameters Basic Protocol 5: Acquire tiled images Basic Protocol 6: Review the scans Basic Protocol 7: Reimage ROIs as needed Basic Protocol 8: Stitch, stack, and assemble images Basic Protocol 9: Repeat scanning for multiplex immunostaining or background subtraction.

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.

Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells.

Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.

A Novel Role of SLC26A3 in the Maintenance of Intestinal Epithelial Barrier Integrity.

BACKGROUND & AIMS: The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. METHODS: Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. RESULTS: DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction. CONCLUSIONS: To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis.

Citrobacter rodentium Infection Inhibits Colonic P-glycoprotein Expression.

P-glycoprotein (Pgp/MDR1) serves as a biological barrier that protects intestinal epithelial cells (IECs) by transporting out xenobiotics and bacterial toxins. Decreased Pgp function and expression has been seen in mouse models of inflammatory colitis and also in patients with IBD. Pgp knockout mice spontaneously develop severe colitis, which is also seen in human patients with ulcerative colitis. However, whether Pgp is also altered in infectious colitis is not known. Citrobacter rodentium (CR), a murine pathogen has been shown to cause colonic hyperplasia and colitis in mice by attaching to IECs. The current study investigated the direct effects of Citrobacter rodentium infection on intestinal Pgp expression in mice. Mice were challenged with a single dose of C. rodentium (1 × 109 CFU) by oral gavage for 9 days and Pgp expression in the ileum and colon was measured by real time qRT-PCR and immunofluorescence studies. Our results showed that C. rodentium infection significantly decreased Pgp mRNA and protein expression in the colon, although no significant change was observed in the ileum of mice. These findings suggest that inhibition of the efflux protein, Pgp by C. rodentium can cause perturbations in the intestinal epithelial integrity, which could further lead to the pathogenesis of intestinal inflammation as observed in infectious colitis.

Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells.

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1ß, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.