Pentacyclic triterpenes modulate farnesoid X receptor expression in colonic epithelial cells: Implications for colonic secretory function.

The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important regulator of intestinal and metabolic function. Previous studies suggest that pentacyclic triterpenes (PCTs), a class of plant-derived bioactive phytochemical, can modulate FXR activity and may therefore offer therapeutic benefits. Here, we investigated the effects of a prototypical PCT, hederagenin (HG), on FXR expression, activity, and antisecretory actions in colonic epithelial cells. T84 cells and murine enteroid-derived monolayers were employed to assess HG effects on FXR expression and activity in colonic epithelia. We measured mRNA levels by qRT-PCR and protein by ELISA and immunoblotting. Transepithelial Cl- secretion was assessed as changes in short circuit current in Ussing chambers. We determined HG treatment (5-10 µM) alone did not induce FXR activation but significantly increased expression of the receptor, both in T84 cells and murine enteroid-derived monolayers. This effect was accompanied by enhanced FXR activity, as assessed by FGF-15/19 induction in response to the synthetic, GW4064, or natural FXR agonist, chenodeoxycholic acid. Effects of HG on FXR expression and activity were mimicked by another PCT, oleanolic acid. Furthermore, we found FXR-induced downregulation of cystic fibrosis transmembrane conductance regulator Cl- channels and inhibition of transepithelial Cl- secretion were enhanced in HG-treated cells. These data demonstrate that dietary PCTs have the capacity to modulate FXR expression, activity, and antisecretory actions in colonic epithelial cells. Based on these data, we propose that plants rich in PCTs, or extracts thereof, have excellent potential for development as a new class of "FXR-targeted nutraceuticals".

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling.

Infections with non-typhoidal Salmonella spp. represent the most burdensome foodborne illnesses worldwide, yet despite their prevalence, the mechanism through which Salmonella elicits diarrhoea is not entirely known. Intestinal ion transporters play important roles in fluid and electrolyte homeostasis in the intestine. We have previously shown that infection with Salmonella caused decreased colonic expression of the chloride/bicarbonate exchanger SLC26A3 (down-regulated in adenoma; DRA) in a mouse model. In this study, we focused on the mechanism of DRA downregulation during Salmonella infection, by using murine epithelial enteroid-derived monolayers (EDMs). The decrease in DRA expression caused by infection was recapitulated in EDMs and accompanied by increased expression of Atonal Homolog 1 (ATOH1), the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. This suggested biased epithelial differentiation towards the secretory, rather than absorptive phenotype. In addition, the downstream Notch effector, Notch intracellular domain (NICD) and Hes1 were decreased following Salmonella infection. The relevance of Notch signalling was further investigated using a γ-secretase inhibitor, which recapitulated the downregulation in Hes1 and DRA as well as upregulation in ATOH1 and Muc2 seen following infection. Our findings suggest that Salmonella infection may result in a shift from absorptive to secretory cell types through Notch inhibition, which explains why there is a decreased capacity for absorption and ultimately the accumulation of diarrhoeal fluid. Our work also shows the value of EDMs as a model to investigate mechanisms that might be targeted for therapy of diarrhoea caused by Salmonella infection. KEY POINTS: Salmonella is a leading foodborne pathogen known to cause high-chloride-content diarrhoea. Salmonella infection of murine enteroid-derived monolayers decreased DRA expression. Salmonella infection resulted in upregulation of the secretory epithelial marker ATOH1, the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. Downregulation of DRA may result from infection-induced Notch inhibition, as reflected by decreased expression of Notch intracellular domain and Hes1, as well as from decreased HNF1α signalling. The imbalance in intestinal epithelial differentiation favouring secretory over absorptive cell types is a possible mechanism by which Salmonella elicits diarrhoea and may be relevant therapeutically.

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Aberrant Epithelial Differentiation Contributes to Pathogenesis in a Murine Model of Congenital Tufting Enteropathy.

BACKGROUND & AIMS: Congenital tufting enteropathy (CTE) is an intractable diarrheal disease of infancy caused by mutations of epithelial cell adhesion molecule (EpCAM). The cellular and molecular basis of CTE pathology has been elusive. We hypothesized that the loss of EpCAM in CTE results in altered lineage differentiation and defects in absorptive enterocytes thereby contributing to CTE pathogenesis. METHODS: Intestine and colon from mice expressing a CTE-associated mutant form of EpCAM (mutant mice) were evaluated for specific markers by quantitative real-time polymerase chain reaction, Western blotting, and immunostaining. Body weight, blood glucose, and intestinal enzyme activity were also investigated. Enteroids derived from mutant mice were used to assess whether the decreased census of major secretory cells could be rescued. RESULTS: Mutant mice exhibited alterations in brush-border ultrastructure, function, disaccharidase activity, and glucose absorption, potentially contributing to nutrient malabsorption and impaired weight gain. Altered cell differentiation in mutant mice led to decreased enteroendocrine cells and increased numbers of nonsecretory cells, though the hypertrophied absorptive enterocytes lacked key features, causing brush border malfunction. Further, treatment with the Notch signaling inhibitor, DAPT, increased the numbers of major secretory cell types in mutant enteroids (graphical abstract 1). CONCLUSIONS: Alterations in intestinal epithelial cell differentiation in mutant mice favor an increase in absorptive cells at the expense of major secretory cells. Although the proportion of absorptive enterocytes is increased, they lack key functional properties. We conclude that these effects underlie pathogenic features of CTE such as malabsorption and diarrhea, and ultimately the failure to thrive seen in patients.

Potentiation of calcium-activated chloride secretion and barrier dysfunction may underlie EGF receptor tyrosine kinase inhibitor-induced diarrhea.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFr TKIs) are first-line therapies for various cancers, and cause dose-limiting severe diarrhea in many patients. We hypothesized that diarrhea caused by EGFr TKIs might reflect actions on epithelial transport, barrier function, or both, which we tested using cell cultures including murine and human enteroid-derived monolayers (EDMs), analyzed using electrophysiological and other relevant methods. EGFr TKIs (such as afatinib, erlotinib, and osimertinib) reversed the acute inhibitory effect of EGF on chloride secretion induced by carbachol (CCh) across T84 human colonic epithelial cells, which correlated with the diarrhea-inducing effect of each agent clinically. EGFr TKIs also reduced transepithelial electrical resistance (TEER), whereas co-treatment with CCh delayed the decrease in TEER compared with that of cells co-treated with EGF. Furthermore, afatinib and erlotinib prevented EGF- or CCh-induced EGFr phosphorylation. EGFr TKIs also suppressed phosphorylation of extracellular signal-regulated kinase (Erk)1/2 in response to EGF, whereas they had weaker effects on CCh-induced Erk1/2 phosphorylation. In human EDMs, EGF potentiated ion transport induced by CCh, whereas afatinib reversed this effect. The ability of EGFr TKIs to reverse the effects of EGF on calcium-dependent chloride secretion could contribute to the diarrheal side effects of these agents, and their disruption of epithelial barrier dysfunction is likely also pathophysiologically significant. CCh-activated Erk1/2 phosphorylation was relatively insensitive to EGFr TKIs and delayed the deleterious effects of EGFr TKIs on barrier function. These findings confirm and extend those of other authors, and may be relevant to designing strategies to overcome the diarrheal side effects of EGFr TKIs.

Congenital Tufting Enteropathy-Associated Mutant of Epithelial Cell Adhesion Molecule Activates the Unfolded Protein Response in a Murine Model of the Disease.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

Epithelial transport in digestive diseases: mice, monolayers, and mechanisms.

The transport of electrolytes and fluid by the intestinal epithelium is critical in health to maintain appropriate levels of fluidity of the intestinal contents. The transport mechanisms that underlie this physiological process are also subject to derangement in various digestive disease states, such as diarrheal illnesses. This article summarizes the 2019 Hans Ussing Lecture of the Epithelial Transport Group of the American Physiological Society and discusses some pathways by which intestinal transport is dysregulated, particularly in the setting of infection with the diarrheal pathogen, Salmonella, and in patients treated with small-molecule inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFr-TKI). The burdensome diarrhea in patients infected with Salmonella may be attributable to decreased expression of the chloride-bicarbonate exchanger downregulated in adenoma (DRA) that participates in electroneutral NaCl absorption. This outcome is possibly secondary to increased epithelial proliferation and/or decreased epithelial differentiation that occurs following infection. Conversely, the diarrheal side effects of cancer treatment with EGFr-TKI may be related to the known ability of EGFr-associated signaling to reduce calcium-dependent chloride secretion. Overall, the findings described may suggest targets for therapeutic intervention in a variety of diarrheal disease states.

Enteroids expressing a disease-associated mutant of EpCAM are a model for congenital tufting enteropathy.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule (EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers (Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors (Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease.NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule (EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

Should We Divide Crohn's Disease Into Ileum-Dominant and Isolated Colonic Diseases?

Crohn's disease (CD) is an inflammatory bowel disease that can involve any region of the gastrointestinal tract. First described in 1932 as terminal ileitis or regional enteritis, it predominately involves the ileum with or without colonic involvement. Isolated colonic CD was first described in 1960 and since then the phenotypic classification of CD has evolved to stratify patients into isolated ileal, ileocolonic, or isolated colonic involvement. In the current review we evaluate the published literature regarding differences in epidemiology, natural history, pathogenesis, response to therapy, and disease monitoring, when stratified by disease location. Based on the available evidence consideration could be given to a new classification for CD, which splits it into ileum dominant (isolated ileal and ileocolonic) and isolated colonic disease. This may allow for a more optimized approach to clinical care and scientific research for CD.

Interleukin 9 Alters Epithelial Barrier and E-cadherin in Eosinophilic Esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic TH2-assocated inflammatory condition accompanied by substantial impairments in epithelial barrier function and increased numbers of interleukin 9 (IL-9) expressing inflammatory cells. While IL-9 is known to affect barrier function in the intestine, the functional effects of IL-9 on the esophagus are unclear. Herein we aimed to understand the expression of the IL-9 receptor and effects of IL-9 on the epithelium in EoE. METHODS: We used esophageal biopsies from pediatric EoE patients with active and inactive disease to analyze the expression of the IL-9 receptor, the adherens junction protein E-cadherin and the tight junction protein claudin-1. We treated primary human esophageal epithelial cells with IL-9 to understand its effects on E-cadherin expression and function. RESULTS: Active EoE subjects had increased epithelial expression of IL-9 receptor mRNA and protein (P < 0.05) and decreased membrane bound E-cadherin (P < 0.01) and claudin-1 (P < 0.05) expression. IL-9 receptor expression and mislocalized claudin-1 positively correlated and while membrane bound E-cadherin expression negatively correlated with the degree of histologic epithelial remodeling (P < 0.05). IL-9 decreased epithelial resistance in stratified primary human esophageal epithelial cells (P < 0.01) and membrane bound E-cadherin in epithelial cell monolayers (P < 0.01). CONCLUSIONS: These data suggest that IL-9, its receptor, and its effects on E-cadherin may be important mechanisms for epithelial barrier disruption in EoE.

The Role of Ion Transporters in the Pathophysiology of Infectious Diarrhea.

Every year, enteric infections and associated diarrhea kill millions of people. The situation is compounded by increases in the number of enteric pathogens that are acquiring resistance to antibiotics, as well as (hitherto) a relative paucity of information on host molecular targets that may contribute to diarrhea. Many forms of diarrheal disease depend on the dysregulation of intestinal ion transporters, and an associated imbalance between secretory and absorptive functions of the intestinal epithelium. A number of major transporters have been implicated in the pathogenesis of diarrheal diseases and thus an understanding of their expression, localization, and regulation after infection with various bacteria, viruses, and protozoa likely will prove critical in designing new therapies. This article surveys our understanding of transporters that are modulated by specific pathogens and the mechanism(s) involved, thereby illuminating targets that might be exploited for new therapeutic approaches.

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera.

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.

Endogenous and exogenous control of gastrointestinal epithelial function: building on the legacy of Bayliss and Starling.

Transport of fluid and electrolytes in the intestine allows for appropriate adjustments in luminal fluidity while reclaiming water used in digesting and absorbing a meal, and is closely regulated. This article discusses various endogenous and exogenous mechanisms whereby transport is controlled in the gut, placing these in the context of the ideas about the neurohumoral control of alimentary physiology that were promulgated by William Bayliss and Ernest Starling. The article considers three themes. First, mechanisms that intrinsically regulate chloride secretion, centred on the epidermal growth factor receptor (EGFr), are discussed. These may be important in ensuring that excessive chloride secretion, with the accompanying loss of fluid, is not normally stimulated by intestinal distension as the meal passes through the gastrointestinal tract. Second, mechanisms whereby probiotic microorganisms can impart beneficial effects on the gut are described, with a focus on targets at the level of the epithelium. These findings imply that the commensal microbiota exert important influences on the epithelium in health and disease. Finally, mechanisms that lead to diarrhoea in patients infected with an invasive pathogen, Salmonella, are considered, based on recent studies in a novel mouse model. Diarrhoea is most likely attributable to reduced expression of absorptive transporters and may not require the influx of neutrophils that accompanies infection. Overall, the goal of the article is to highlight the many ways in which critical functions of the intestinal epithelium are regulated under physiological and pathophysiological conditions, and to suggest possible targets for new therapies for digestive disease states.

Modulation of the microbiota-gut-brain axis by probiotics in a murine model of inflammatory bowel disease.

UNLABELLED: Anxiety, depression, and altered memory are associated with intestinal diseases, including inflammatory bowel disease (IBD). Understanding the link between these behavioral changes and IBD is important clinically since concomitant mood disorders often increase a patient's risk of requiring surgery and developing secondary functional gastrointestinal diseases. Anxiety-like behavior (light/dark box test) and recognition memory (novel object recognition task) were determined at the peak and during resolution of inflammation in the dextran sodium sulfate (DSS) mouse model of acute colitis. DSS (5 days) was administered via drinking water followed by 3 or 9 days of normal drinking water to assess behavior during active or resolving inflammation, respectively. Disease (weight, colon length, and histology) was assessed and the composition of the gut microbiota was characterized by using qPCR on fecal pellet DNA. In a subset of mice, pretreatment with probiotics was started 1 wk prior to commencing DSS. During active inflammation (8 days), mice demonstrated impaired recognition memory and exhibited anxiety-like behavior vs. CONTROLS: These behavioral defects were normalized by 14 days post-DSS. Shifts in the composition of the gut microbiota were evident during active inflammation, notably as decreases in lactobacilli and segmented filamentous bacteria, which were also reversed once the disease had resolved. Administration of probiotics could prevent the behavioral defects seen in acute DSS. Taken together, our findings indicate that changes in mood and behavior are present during acute inflammation in murine IBD and associated with dysbiosis and that these outcomes can be prevented by the administration of probiotics.

Mutation of EpCAM leads to intestinal barrier and ion transport dysfunction.

UNLABELLED: Congenital tufting enteropathy (CTE) is a devastating diarrheal disease seen in infancy that is typically associated with villous changes and the appearance of epithelial tufts. We previously found mutations in epithelial cell adhesion molecule (EpCAM) to be causative in CTE. We developed a knock-down cell model of CTE through transfection of an EpCAM shRNA construct into T84 colonic epithelial cells to elucidate the in vitro role of EpCAM in barrier function and ion transport. Cells with EpCAM deficiency exhibited decreased electrical resistance, increased permeability, and decreased ion transport. Based on mutations in CTE patients, an in vivo mouse model was developed, with tamoxifen-inducible deletion of exon 4 in Epcam resulting in mutant protein with decreased expression. Tamoxifen treatment of Epcam (Δ4/Δ4) mice resulted in pathological features of villous atrophy and epithelial tufts, similar to those in human CTE patients, within 4 days post induction. Epcam (Δ4/Δ4) mice also showed decreased expression of tight junctional proteins, increased permeability, and decreased ion transport in the intestines. Taken together, these findings reveal mechanisms that may underlie disease in CTE. KEY MESSAGES: Knock-down EpCAM cell model of congenital tufting enteropathy was developed. In vivo inducible mouse model was developed resulting in mutant EpCAM protein. Cells with EpCAM deficiency demonstrated barrier and ion transport dysfunction. Tamoxifen-treated Epcam (Δ4/Δ4) mice demonstrated pathological features. Epcam (Δ4/Δ4) mice showed improper barrier function and ion transport.

Probiotics normalize the gut-brain-microbiota axis in immunodeficient mice.

The gut-brain-microbiota axis is increasingly recognized as an important regulator of intestinal physiology. Exposure to psychological stress causes activation of the hypothalamic-pituitary-adrenal (HPA) axis and causes altered intestinal barrier function, intestinal dysbiosis, and behavioral changes. The primary aim of this study was to determine whether the effects of psychological stress on intestinal physiology and behavior, including anxiety and memory, are mediated by the adaptive immune system. Furthermore, we wanted to determine whether treatment with probiotics would normalize these effects. Here we demonstrate that B and T cell-deficient Rag1(-/-) mice displayed altered baseline behaviors, including memory and anxiety, accompanied by an overactive HPA axis, increased intestinal secretory state, dysbiosis, and decreased hippocampal c-Fos expression. Both local (intestinal physiology and microbiota) and central (behavioral and hippocampal c-Fos) changes were normalized by pretreatment with probiotics, indicating an overall benefit on health conferred by changes in the microbiota, independent of lymphocytes. Taken together, these findings indicate a role for adaptive immune cells in maintaining normal intestinal and brain health in mice and show that probiotics can overcome this immune-mediated deficit in the gut-brain-microbiota axis.

Altered expression and localization of ion transporters contribute to diarrhea in mice with Salmonella-induced enteritis.

BACKGROUND & AIMS: Salmonella enterica serovar Typhimurium is an enteropathogen that causes self-limiting diarrhea in healthy individuals, but poses a significant health threat to vulnerable populations. Our understanding of the pathogenesis of Salmonella-induced diarrhea has been hampered by the lack of a suitable mouse model. After a dose of oral kanamycin, Salmonella-infected congenic BALB/c.D2(NrampG169) mice, which carry a wild-type Nramp1 gene, develop clear manifestations of diarrhea. We used this model to elucidate the pathophysiology of Salmonella-induced diarrhea. METHODS: BALB /c.D2(NrampG169) mice were treated with kanamycin and then infected with wild-type or mutant Salmonella by oral gavage. Colon tissues were isolated and Ussing chambers, quantitative polymerase chain reaction, immunoblot, and confocal microscopy analyses were used to study function and expression of ion transporters and cell proliferation. RESULTS: Studies with Ussing chambers demonstrated reduced basal and/or adenosine 3',5'-cyclic monophosphate-mediated electrogenic ion transport in infected colonic tissues, attributable to changes in chloride or sodium transport, depending on the segment studied. The effects of infection were mediated, at least in part, by effector proteins secreted by the bacterial Salmonella pathogenicity island 1- and Salmonella pathogenicity island-2-encoded virulence systems. Infected tissue showed reduced expression of the chloride-bicarbonate exchanger down-regulated in adenoma in surface colonic epithelial cells. Cystic fibrosis transmembrane conductance regulator was internalized in colonic crypt epithelial cells without a change in overall expression levels. Confocal analyses, densitometry, and quantitative polymerase chain reaction revealed that expression of epithelial sodium channel ß was reduced in distal colons of Salmonella-infected mice. The changes in transporter expression, localization, and/or function were accompanied by crypt hyperplasia in Salmonella-infected mice. CONCLUSIONS: Salmonella infection induces diarrhea by altering expression and/or function of transporters that mediate water absorption in the colon, likely reflecting the fact that epithelial cells have less time to differentiate into surface cells when proliferation rates are increased by infection.

Interferon-γ alters downstream signaling originating from epidermal growth factor receptor in intestinal epithelial cells: functional consequences for ion transport.

The epidermal growth factor receptor (EGFr) regulates many cellular functions, such as proliferation, apoptosis, and ion transport. Our aim was to investigate whether long term treatment with interferon-γ (IFN-γ) modulates EGF activation of downstream signaling pathways in intestinal epithelial cells and if this contributes to dysregulation of epithelial ion transport in inflammation. Polarized monolayers of T(84) and HT29/cl.19A colonocytes were preincubated with IFN-γ prior to stimulation with EGF. Basolateral potassium transport was studied in Ussing chambers. We also studied inflamed colonic mucosae from C57BL/6 mice treated with dextran sulfate sodium or mdr1a knock-out mice and controls. IFN-γ increased intestinal epithelial EGFr expression without increasing its phosphorylation. Conversely, IFN-γ caused a significant decrease in EGF-stimulated phosphorylation of specific EGFr tyrosine residues and activation of ERK but not Akt-1. In IFNγ-pretreated cells, the inhibitory effect of EGF on carbachol-stimulated K(+) channel activity was lost. In inflamed colonic tissues, EGFr expression was significantly increased, whereas ERK phosphorylation was reduced. Thus, although it up-regulates EGFr expression, IFN-γ causes defective EGFr activation in colonic epithelial cells via reduced phosphorylation of specific EGFr tyrosine residues. This probably accounts for altered downstream signaling consequences. These observations were corroborated in the setting of colitis. IFN-γ also abrogates the ability of EGF to inhibit carbachol-stimulated basolateral K(+) currents. Our data suggest that, in the setting of inflammation, the biological effect of EGF, including the inhibitory effect of EGF on Ca(2+)-dependent ion transport, is altered, perhaps contributing to diarrheal and other symptoms in vivo.

Apical leptin induces chloride secretion by intestinal epithelial cells and in a rat model of acute chemotherapy-induced colitis.

The purpose of this study was to investigate whether luminal leptin alters ion transport properties of the intestinal epithelium under acute inflammatory conditions. Monolayers of human intestinal T(84) epithelial cells and a rat model of chemotherapy-induced enterocolitis were used. Cells were treated with leptin and mounted in Ussing chambers to measure basal and secretagogue-induced changes in transepithelial short-circuit current (I(sc)). Furthermore, the role of MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating responses to leptin was investigated. Acute colitis in Sprague-Dawley rats was induced by intraperitoneal injection of 40 mg/kg methotrexate. Leptin (100 ng/ml) induced a time-dependent increase in basal I(sc) in T(84) intestinal epithelial cells (P < 0.01). Moreover, pretreatment of T(84) cells with leptin for up to 1 h significantly potentiated carbachol- and forskolin-induced increases in I(sc). Pretreatment with an inhibitor of MAPK abolished the effect of leptin on basal, carbachol- and forskolin-induced chloride secretion (P < 0.05). However, the PI3K inhibitor, wortmannin, only blunted the effect of leptin on forskolin-induced increases in I(sc). Furthermore, leptin treatment evoked both ERK1/2 and Akt1 phosphorylation in T(84) cells. In the rat model, luminal leptin induced significant increases in I(sc) across segments of proximal and, to a lesser extent, distal colon (P < 0.05). We conclude that luminal leptin is likely an intestinal chloride secretagogue, particularly when present at elevated concentrations and/or in the setting of inflammation. Our findings may provide a mechanistic explanation, at least in part, for the clinical condition of secretory diarrhea both in hyperleptinemic obese patients and in patients with chemotherapy-induced intestinal inflammation.