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1.
Gastroenterology ; 2024 May 11.
Article in English | MEDLINE | ID: mdl-38735402

ABSTRACT

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.

2.
3.
Cell Mol Gastroenterol Hepatol ; 15(4): 903-919, 2023.
Article in English | MEDLINE | ID: mdl-36535508

ABSTRACT

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.


Subject(s)
Colitis, Ulcerative , Interleukin-33 , Animals , Mice , Interleukin-33/metabolism , Immunity, Innate , CD4-Positive T-Lymphocytes , Sulfate Transporters/genetics , Sulfate Transporters/metabolism , Antiporters/metabolism
4.
Am J Physiol Cell Physiol ; 323(6): C1720-C1727, 2022 12 01.
Article in English | MEDLINE | ID: mdl-36189974

ABSTRACT

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.


Subject(s)
MicroRNAs , Sodium-Hydrogen Exchanger 3 , Humans , Caco-2 Cells , Down-Regulation , Epithelial Cells/metabolism , MicroRNAs/genetics , Sodium-Hydrogen Exchanger 3/genetics
5.
Am J Physiol Gastrointest Liver Physiol ; 321(2): G223-G231, 2021 08 01.
Article in English | MEDLINE | ID: mdl-34231393

ABSTRACT

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.


Subject(s)
Intestinal Mucosa/metabolism , MicroRNAs/metabolism , Monocarboxylic Acid Transporters/genetics , Caco-2 Cells , Humans , MicroRNAs/genetics , Monocarboxylic Acid Transporters/metabolism
6.
Cell Microbiol ; 23(4): e13298, 2021 04.
Article in English | MEDLINE | ID: mdl-33237610

ABSTRACT

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.


Subject(s)
Autophagy , Cryptosporidium parvum/pathogenicity , Epithelial Cells/pathology , Epithelial Cells/parasitology , Host-Parasite Interactions , Caco-2 Cells , Humans , Intestinal Mucosa/parasitology , Tight Junction Proteins/metabolism
7.
Gastroenterology ; 160(4): 1240-1255.e3, 2021 03.
Article in English | MEDLINE | ID: mdl-33189700

ABSTRACT

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.


Subject(s)
Antiporters/deficiency , Chloride-Bicarbonate Antiporters/deficiency , Dysbiosis/immunology , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/pathology , Sulfate Transporters/deficiency , Animals , Antiporters/genetics , CELF1 Protein/metabolism , Caco-2 Cells , Cadherins/metabolism , Chloride-Bicarbonate Antiporters/genetics , Disease Models, Animal , Dysbiosis/microbiology , Dysbiosis/pathology , Gene Knockdown Techniques , Humans , Inflammatory Bowel Diseases/immunology , Inflammatory Bowel Diseases/microbiology , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Male , Mice , Mice, Knockout , Occludin/metabolism , Permeability , Sulfate Transporters/genetics , Tight Junctions/pathology
8.
Gene Rep ; 182020 Mar.
Article in English | MEDLINE | ID: mdl-32864506

ABSTRACT

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.

9.
Nanomedicine (Lond) ; 15(25): 2459-2474, 2020 10.
Article in English | MEDLINE | ID: mdl-32975467

ABSTRACT

Aim: To evaluate the efficacy of locally delivered nanomedicine, vasoactive intestinal peptide in sterically stabilized micelles (VIP-SSM) to the colon and conduct in vitro release studies of a potential oral formulation. Materials & methods: Intracolonic instillation of VIP-SSM was tested in a mouse model of dextran sulfate sodium-induced colitis. Based on the effective mouse dose, human equivalent dose containing nanomedicine powder was filled into enteric coated capsules for in vitro release testing. Results: Colonic delivery of VIP-SSM significantly alleviated colitis. VIP-SSM containing capsules completely dissolved at colonic pH allowing micelles to reform with active VIP. Capsule formulations exhibited reproducible release profiles when stored up to 6 weeks demonstrating stability. Conclusion: VIP-SSM is an effective nanomedicine formulation which appears to have potential for oral treatment of colitis in humans. [Formula: see text].


Subject(s)
Colitis , Nanomedicine , Animals , Capsules , Colitis/drug therapy , Male , Mice , Mice, Inbred C57BL , Vasoactive Intestinal Peptide
10.
Am J Physiol Cell Physiol ; 318(3): C502-C513, 2020 03 01.
Article in English | MEDLINE | ID: mdl-31913697

ABSTRACT

Olfactory receptor-78 (Olfr-78) is a recently identified G protein-coupled receptor activated by short-chain fatty acids acetate and propionate. A suggested role for this receptor exists in the prostate where it may influence chronic inflammatory response leading to intraepithelial neoplasia. Olfr-78 has also been shown to be expressed in mouse colon. Short-chain fatty acids and their receptors are well known to modulate inflammation in the gut. Considering this possibility, we first explored if colitis regulated Olfr-78 expression in the gut, where we observed a significant reduction in the expression of Olfr-78 transcript in mouse models of dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. To more directly test this, mice deficient in Olfr-78 were administered with DSS in water for 7 days and were found to have increased expression of IL-1ß and inflammatory signs in colon compared with control mice. Next, we explored the expression of its human counterpart olfactory receptor family 51, subfamily E, member 2 (OR51E2) in human intestinal samples and observed that it was in fact also expressed in human colon samples. RNA sequence analysis revealed significant changes in the genes involved in infection, immunity, inflammation, and colorectal cancer between wild-type and Olfr-78 knockout mice. Collectively, our findings show that Olfr-78 is highly expressed in colon and downregulated in DSS- and TNBS-induced colitis, and DSS-treated Olfr-78 null mice had increased colonic expression of cytokine RNA levels, suggesting a potential role for this receptor in intestinal inflammation. Future investigations are needed to understand how Olfr-78/OR51E2 in both mouse and human intestine modulates gastrointestinal pathophysiology.


Subject(s)
Colitis/metabolism , Intestinal Mucosa/metabolism , Neoplasm Proteins/biosynthesis , Receptors, Odorant/biosynthesis , Animals , Colitis/genetics , Colitis/pathology , Female , HT29 Cells , Humans , Intestinal Mucosa/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasm Proteins/genetics , Receptors, Odorant/genetics
11.
Acta Physiol (Oxf) ; 228(1): e13332, 2020 01.
Article in English | MEDLINE | ID: mdl-31177627

ABSTRACT

AIM: P-glycoprotein (Pgp/MDR1) plays a major role in intestinal homeostasis. Decrease in Pgp function and expression has been implicated in the pathogenesis of IBD. However, inhibitory mechanisms involved in the decrease of Pgp in inflammation are not fully understood. Angiotensin II (Ang II), a peptide hormone predominantly expressed in the epithelial cells of the crypt-villus junction of the intestine, has been shown to exert pro-inflammatory effects in the gut. It is increased in IBD patients and animals with experimental colitis. Whether Ang II directly influences Pgp is not known. METHODS: Pgp activity was measured as verapamil-sensitive 3 H-digoxin flux. Pgp surface expression and exocytosis were measured by cell surface biotinylation studies. Signalling pathways were elucidated by Western blot analysis and pharmacological approaches. RESULTS: Ang II (10 nM) significantly inhibited Pgp activity at 60 minutes. Ang II-mediated effects on Pgp function were receptor-mediated as the Ang II receptor 1 (ATR1) antagonist, losartan, blocked Pgp inhibition. Ang II effects on Pgp activity appeared to be mediated via PI3 kinase, p38 MAPK and Akt signalling. Ang II-mediated inhibition of Pgp activity was associated with a decrease in the surface membrane expression of Pgp protein via decreased exocytosis and was found to be dependent on the Akt pathway. Short-term treatment of Ang II (2 mg/kg b.wt., 2 hours) to mice also decreased the membrane expression of Pgp protein levels in ileum and colon. CONCLUSION: Our findings provide novel insights into the role of Ang II and ATR1 in decreasing Pgp expression in intestinal inflammation.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Angiotensin II/pharmacology , Epithelial Cells/metabolism , Intestinal Mucosa/cytology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Angiotensin II/administration & dosage , Animals , Caco-2 Cells , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Gene Expression Regulation/drug effects , Humans , Mice , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism , Receptor, Angiotensin, Type 2/genetics , Receptor, Angiotensin, Type 2/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
12.
Inflamm Bowel Dis ; 26(4): 534-545, 2020 03 04.
Article in English | MEDLINE | ID: mdl-31634391

ABSTRACT

BACKGROUND: Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. AIMS: Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. METHODS: Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. RESULTS: All-trans retinoic acid (10 µM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1ß and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. CONCLUSION: Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.


Subject(s)
Antiporters/metabolism , Chloride-Bicarbonate Antiporters/metabolism , Colitis/drug therapy , Intestinal Mucosa/drug effects , Sulfate Transporters/metabolism , Tretinoin/pharmacology , Animals , Antiporters/genetics , Caco-2 Cells , Chloride-Bicarbonate Antiporters/genetics , Colon/metabolism , Dextran Sulfate/toxicity , Diarrhea/drug therapy , Disease Models, Animal , Epithelial Cells/metabolism , Humans , Inflammation/metabolism , Interferon-gamma/drug effects , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Male , Mice , Mice, Inbred C57BL , RNA, Messenger/genetics , Sulfate Transporters/genetics , Up-Regulation , Weight Loss/drug effects
13.
Am J Physiol Cell Physiol ; 317(6): C1205-C1212, 2019 12 01.
Article in English | MEDLINE | ID: mdl-31483700

ABSTRACT

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.


Subject(s)
Antiporters/genetics , Chloride-Bicarbonate Antiporters/genetics , Cryptosporidiosis/genetics , Cryptosporidium parvum/pathogenicity , Gene Expression Regulation/genetics , Intestinal Mucosa/metabolism , Sulfate Transporters/genetics , Animals , Antibodies, Neutralizing/pharmacology , Antiporters/antagonists & inhibitors , Antiporters/metabolism , Caco-2 Cells , Chloride-Bicarbonate Antiporters/antagonists & inhibitors , Chloride-Bicarbonate Antiporters/metabolism , Chlorides/metabolism , Cryptosporidiosis/metabolism , Cryptosporidiosis/parasitology , Cryptosporidium parvum/physiology , Host-Parasite Interactions/genetics , Humans , Ileum/metabolism , Ileum/parasitology , Intestinal Mucosa/parasitology , Ion Transport , Janus Kinases/genetics , Janus Kinases/metabolism , Mice , Mice, Inbred C57BL , Organoids/metabolism , Organoids/parasitology , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Sulfate Transporters/antagonists & inhibitors , Sulfate Transporters/metabolism
14.
Am J Physiol Cell Physiol ; 317(2): C200-C208, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31042422

ABSTRACT

Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl-/ HCO3- exchanger, also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed, Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore, we investigated the regulation of PAT-1 expression by microRNAs (miRNA), as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3'-untranslated region (UTR) revealed potential binding sites for several miRNAs, suggesting the role of miRNAs in modulating PAT1 expression. miRNAs showing highest context scores (125a-5p, 339-5p, 423-5p, 485-5p, and 501-3p) were selected as candidates for their effects on the activity of a 263-bp PAT-1 3'-untranslated region (UTR) fragment cloned into pmirGLO vector upstream of luciferase. The 3'-UTR activity was measured by dual luciferase reporter assay in Caco-2, T-84, HT-29, and SK-CO15 cells. Transient transfection of PAT-1 3'-UTR significantly decreased the relative luciferase activity compared with the empty vector suggesting binding of potential miRNA(s) to the PAT-1 3'-UTR. Among all the selected candidates, cotransfection with miRNA mimics 125a-5p and 423-5p further decreased PAT-1 3'-UTR activity. Furthermore, increasing miR-125a-5p abundance via mimic transfection in Caco-2 cells decreased both mRNA and protein levels of PAT-1. Our results demonstrate a novel regulatory mechanism of intestinal PAT-1 expression via miR-125a-5p that could be of therapeutic importance in disorders associated with decreased PAT-1 expression and function.


Subject(s)
Colon/metabolism , Intestinal Mucosa/metabolism , MicroRNAs/metabolism , Oxalic Acid/metabolism , Sulfate Transporters/metabolism , 3' Untranslated Regions , Binding Sites , Caco-2 Cells , Down-Regulation , HT29 Cells , Humans , MicroRNAs/genetics , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sulfate Transporters/genetics
15.
Tissue Barriers ; 6(2): e1463897, 2018.
Article in English | MEDLINE | ID: mdl-29737913

ABSTRACT

Inflammatory bowel diseases broadly categorized into Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract with increasing prevalence worldwide. The etiology of the disease is complex and involves a combination of genetic, environmental, immunological and gut microbial factors. Recurring and bloody diarrhea is the most prevalent and debilitating symptom in IBD. The pathogenesis of IBD-associated diarrhea is multifactorial and is essentially an outcome of mucosal damage caused by persistent inflammation resulting in dysregulated intestinal ion transport, impaired epithelial barrier function and increased accessibility of the pathogens to the intestinal mucosa. Altered expression and/or function of epithelial ion transporters and channels is the principle cause of electrolyte retention and water accumulation in the intestinal lumen leading to diarrhea in IBD. Aberrant barrier function further contributes to diarrhea via leak-flux mechanism. Mucosal penetration of enteric pathogens promotes dysbiosis and exacerbates the underlying immune system further perpetuating IBD associated-tissue damage and diarrhea. Here, we review the mechanisms of impaired ion transport and loss of epithelial barrier function contributing to diarrhea associated with IBD.


Subject(s)
Diarrhea/etiology , Diarrhea/physiopathology , Inflammatory Bowel Diseases/complications , Inflammatory Bowel Diseases/physiopathology , Humans
16.
Am J Physiol Gastrointest Liver Physiol ; 315(1): G43-G52, 2018 07 01.
Article in English | MEDLINE | ID: mdl-29597352

ABSTRACT

Clostridium difficile infection (CDI) is the primary cause of nosocomial diarrhea in the United States. Although 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 a decrease in both NHE3 (Na+/H+ exchanger) and DRA (downregulated in adenoma, Cl-/[Formula: see text] exchanger), resulting in decreased electrolyte absorption, is implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and downregulated in patients with CDI, but the role of DRA in CDI 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 demonstrated that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA, as NHE3 and PAT-1 (putative anion transporter 1) protein levels were unaffected by TcdA and TcdB. Additionally, purified TcdA and TcdA + TcdB, but not TcdB, resulted in a decrease in colonic DRA protein levels in a toxigenic mouse model of CDI. Finally, patients with recurrent CDI also exhibited significantly reduced expression of colonic DRA protein. Together, these findings indicate that C. difficile toxins markedly downregulate intestinal expression of DRA which may contribute to the diarrheal phenotype of CDI. NEW & NOTEWORTHY Our studies demonstrate, for the first time, that C. difficile toxins reduce DRA protein, but not mRNA, levels in intestinal epithelial cells. These findings suggest that a downregulation of DRA may be a critical factor in C. difficile infection-associated diarrhea.


Subject(s)
Antiporters/metabolism , Bacterial Toxins/metabolism , Chloride-Bicarbonate Antiporters/metabolism , Clostridioides difficile/physiology , Enterocolitis, Pseudomembranous , Sulfate Transporters/metabolism , Animals , Disease Models, Animal , Enterocolitis, Pseudomembranous/metabolism , Enterocolitis, Pseudomembranous/microbiology , Enterocolitis, Pseudomembranous/pathology , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Mice , RNA, Messenger/metabolism , Sodium-Hydrogen Exchangers , Transcription Factors/metabolism
17.
Cell Microbiol ; 20(6): e12830, 2018 06.
Article in English | MEDLINE | ID: mdl-29444370

ABSTRACT

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 106  oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and ß-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.


Subject(s)
Adherens Junctions/parasitology , Cell Adhesion Molecules/antagonists & inhibitors , Cryptosporidiosis/pathology , Cryptosporidium parvum/growth & development , Down-Regulation , Host-Pathogen Interactions , Tight Junctions/parasitology , Animals , Caco-2 Cells , Disease Models, Animal , Gene Expression Profiling , Humans , Intestinal Mucosa/parasitology , Intestinal Mucosa/pathology , Mice , Mice, Inbred C57BL , Models, Biological , Permeability
18.
Am J Physiol Gastrointest Liver Physiol ; 314(3): G309-G318, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29167115

ABSTRACT

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 µM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.


Subject(s)
Colon/metabolism , DNA Methylation , Epigenesis, Genetic , Ileum/metabolism , Sodium-Hydrogen Exchanger 3/genetics , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Azacitidine/pharmacology , Caco-2 Cells , Colon/drug effects , DNA Methylation/drug effects , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/metabolism , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic/drug effects , Gene Expression Regulation , Humans , Ileum/drug effects , Male , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , RNA Interference , Sodium-Hydrogen Exchanger 3/metabolism
19.
J Vis Exp ; (121)2017 03 16.
Article in English | MEDLINE | ID: mdl-28362399

ABSTRACT

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 in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.


Subject(s)
Carrier Proteins/metabolism , Intestinal Mucosa/metabolism , Biological Transport , Caco-2 Cells/metabolism , Cell Culture Techniques , Cell Membrane/metabolism , Cell Membrane Permeability , Epithelial Cells/metabolism , Epithelium/metabolism , Humans , Intestinal Mucosa/cytology , Intestines/cytology
20.
Nanomedicine ; 13(2): 659-665, 2017 02.
Article in English | MEDLINE | ID: mdl-27553076

ABSTRACT

The gut hormone, glucagon like peptide-1 (GLP-1) exerts anti-inflammatory effects. However, its clinical use is limited by its short half-life. Previously, we have shown that GLP-1 as a nanomedicine (GLP-1 in sterically stabilized phospholipid micelles, GLP-1-SSM) has increased in vivo stability. The current study was aimed at testing the efficacy of this GLP-1 nanomedicine in alleviating colonic inflammation and associated diarrhea in dextran sodium sulfate (DSS) induced mouse colitis model. Our results show that GLP-1-SSM treatment markedly alleviated the colitis phenotype by reducing the expression of pro-inflammatory cytokine IL-1ß, increasing goblet cells and preserving intestinal epithelial architecture in colitis model. Further, GLP-1-SSM alleviated diarrhea (as assessed by luminal fluid) by increasing protein expression of intestinal chloride transporter DRA (down regulated in adenoma). Our results indicate that GLP-1 nanomedicine may act as a novel therapeutic tool in alleviating gut inflammation and associated diarrhea in inflammatory bowel disease (IBD).


Subject(s)
Colitis/drug therapy , Glucagon-Like Peptide 1/administration & dosage , Inflammation , Nanomedicine , Animals , Dextran Sulfate/therapeutic use , Diarrhea/drug therapy , Diarrhea/etiology , Disease Models, Animal , Glucagon-Like Peptide 1/therapeutic use , Mice
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