Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

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Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine

PLoS ONE ◽

10.1371/journal.pone.0256143 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0256143

Author(s):

Pascal Hoffmann ◽

Nadine Schnepel ◽

Marion Langeheine ◽

Katrin Künnemann ◽

Guntram A. Grassl ◽

...

Keyword(s):

Infectious Diseases ◽

Cholera Toxin ◽

Short Circuit ◽

Animal Health ◽

Animal Experiments ◽

Chloride Secretion ◽

Farm Animals ◽

Laboratory Animals ◽

Species Specific

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

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Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

Infection and Immunity ◽

10.1128/iai.73.6.3627-3635.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3627-3635 ◽

Cited By ~ 17

Author(s):

Juliette K. Tinker ◽

Jarrod L. Erbe ◽

Randall K. Holmes

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Fusion Proteins ◽

Cell Biology ◽

Fluorescent Protein ◽

Cultured Cells ◽

Secretory Diarrhea ◽

E Coli ◽

Twin Arginine Translocation ◽

Heat Labile

ABSTRACT Cholera toxin (CT) is an AB5 toxin responsible for the profuse secretory diarrhea resulting from Vibrio cholerae infection. CT consists of a pentameric, receptor-binding B subunit (CTB) and a monomeric A subunit (CTA) that has latent enzymatic activity. In addition to its enterotoxicity, CT has potent mucosal adjuvant activity and can also function as a carrier molecule with many potential applications in cell biology. In earlier studies, the toxic CTA1 domain was replaced by several other antigenic protein domains to produce holotoxin-like chimeras for use as potential mucosal vaccines. In the present study we utilized the twin arginine translocation (tat) system to produce fluorescent CT chimeras, as well as fluorescent chimeras of Escherichia coli heat-labile toxins LTI and LTIIb. Fusion proteins containing either green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP) and the A2 domain of CT, LTI, or LTIIb were transported to the periplasm of E. coli by the tat system, and the corresponding B polypeptides of CT, LTI, and LTIIb were transported to the periplasm by the sec system. The fluorescent fusion proteins were shown to assemble spontaneously and efficiently with the corresponding B polypeptides in the periplasm to form chimeric holotoxin-like molecules, and these chimeras bound to and entered cultured cells in a manner similar to native CT, LTI, or LTIIb. The GFP and mRFP derivatives of CT, LT, and LTIIb developed here are useful tools for studies on the cell biology of trafficking of the CT/LT family of bacterial enterotoxins. In addition, these constructs provide proof in principle for the development of novel chimeric CT-like or LT-like vaccine candidates containing CTA2 fusion proteins that cannot be delivered to the periplasm of E. coli by use of the sec secretion pathway.

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Plasmid Vectors Encoding Cholera Toxin or the Heat-Labile Enterotoxin from Escherichia coli Are Strong Adjuvants for DNA Vaccines

Journal of Virology ◽

10.1128/jvi.76.9.4536-4546.2002 ◽

2002 ◽

Vol 76 (9) ◽

pp. 4536-4546 ◽

Cited By ~ 58

Author(s):

Joshua Arrington ◽

Ralph P. Braun ◽

Lichun Dong ◽

Deborah H. Fuller ◽

Michael D. Macklin ◽

...

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Dna Vaccines ◽

Biological Effects ◽

Laboratory Animals ◽

Plasmid Vectors ◽

Cytokine Responses ◽

Heat Labile ◽

B Subunits ◽

Adjuvant Effects

ABSTRACT Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-γ]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00320.2009 ◽

2010 ◽

Vol 298 (5) ◽

pp. G714-G721 ◽

Cited By ~ 13

Author(s):

Raschid M. Hoda ◽

Michael Scharl ◽

Stephen J. Keely ◽

Declan F. McCole ◽

Kim E. Barrett

Keyword(s):

Epithelial Cells ◽

Rat Model ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Short Circuit ◽

Chloride Secretion ◽

Secretory Diarrhea ◽

Intestinal Epithelial ◽

Sprague Dawley ◽

Ussing Chambers

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 T84 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 ( Isc). 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 Isc in T84 intestinal epithelial cells ( P < 0.01). Moreover, pretreatment of T84 cells with leptin for up to 1 h significantly potentiated carbachol- and forskolin-induced increases in Isc. 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 Isc. Furthermore, leptin treatment evoked both ERK1/2 and Akt1 phosphorylation in T84 cells. In the rat model, luminal leptin induced significant increases in Isc 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.

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Phosphatidylinositol 3-Kinase-dependent Pathways Oppose Fas-induced Apoptosis and Limit Chloride Secretion in Human Intestinal Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m106226200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47563-47574 ◽

Cited By ~ 16

Author(s):

Maria T. Abreu ◽

Elizabeth T. Arnold ◽

Jimmy Y. C. Chow ◽

Kim E. Barrett

Keyword(s):

Epithelial Cells ◽

Barrier Function ◽

Intestinal Inflammation ◽

Short Circuit ◽

Chloride Secretion ◽

Pi3 Kinase ◽

Secretory Diarrhea ◽

Cross Linking ◽

Barrier Dysfunction ◽

Kinase Inhibition

The epithelial lining of the intestine serves as a barrier to lumenal bacteria and can be compromised by pathologic Fas-mediated epithelial apoptosis. Phosphatidylinositol (PI)3-kinase signaling has been described to limit apoptosis in other systems. We hypothesized that PI3-kinase-dependent pathways regulate Fas-mediated apoptosis and barrier function in intestiynal epithelial cells (IEC). IEC lines (HT-29 and T84) were exposed to agonist anti-Fas antibody in the presence or absence of chemical inhibitors of PI3-kinase (LY294002 and wortmannin). Apoptosis, barrier function, changes in short circuit current (ΔIsc), and expression of adhesion molecules were assessed. Inhibition of PI3-kinase strongly sensitized IEC to Fas-mediated apoptosis. Expression of constitutively active Akt, a principal downstream effector of the PI3-kinase pathway, protected against Fas-mediated apoptosis to an extent that was comparable with expression of a genetic caspase inhibitor, p35. PI3-kinase inhibition sensitized to apoptosis by increasing and accelerating Fas-mediated caspase activation. Inhibition of PI3-kinase combined with cross-linking Fas was associated with increased permeability to molecules that were <400 Da but not those that were >3,000 Da. Inhibition of PI3-kinase resulted in chloride secretion that was augmented by cross-linking Fas. Confocal analyses revealed polymerization of actin and maintenance of epithelial cell adhesion molecule-mediated interactions in monolayers exposed to anti-Fas antibody in the context of PI3-kinase inhibition. PI3-kinase-dependent pathways, especially Akt, protect IEC against Fas-mediated apoptosis. Inhibition of PI3-kinase in the context of Fas signaling results in increased chloride secretion and barrier dysfunction. These findings suggest that agonists of PI3-kinase such as growth factors may have a dual effect on intestinal inflammation by protecting epithelial cells against immune-mediated apoptosis and limiting chloride secretory diarrhea.

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Adenylyl Cyclase 6 Expression Is Essential for Cholera Toxin–Induced Diarrhea

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz013 ◽

2019 ◽

Vol 220 (11) ◽

pp. 1719-1728 ◽

Cited By ~ 4

Author(s):

Robert A Fenton ◽

Sathish K Murali ◽

Izumi Kaji ◽

Yasutada Akiba ◽

Jonathan D Kaunitz ◽

...

Keyword(s):

Epithelial Cells ◽

Adenylyl Cyclase ◽

Cholera Toxin ◽

Knockout Mice ◽

Intestinal Epithelial Cells ◽

Short Circuit ◽

Secretory Diarrhea ◽

Intestinal Epithelial ◽

Α Subunit ◽

Small Intestinal

AbstractBackgroundCholera toxin (CT)–induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)–mediated active Cl– secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown.MethodsWe generated intestinal epithelial cell–specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea.ResultsAC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin.ConclusionsAC6 is a novel target for the treatment of CT-induced diarrhea.

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Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions

Journal of Experimental Medicine ◽

10.1084/jem.20050421 ◽

2005 ◽

Vol 202 (7) ◽

pp. 975-986 ◽

Cited By ~ 115

Author(s):

Chunying Li ◽

Keanna S. Dandridge ◽

Anke Di ◽

Kevin L. Marrs ◽

Erica L. Harris ◽

...

Keyword(s):

Epithelial Cells ◽

Cholera Toxin ◽

Lysophosphatidic Acid ◽

Protein Interactions ◽

Wound Repair ◽

Short Circuit ◽

Fluid Secretion ◽

Secretory Diarrhea ◽

Macromolecular Complex ◽

Apical Surface

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor–2 and CFTR through a PSD95/Dlg/ZO-1–based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

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Endothelin stimulates chloride secretion across canine tracheal epithelium

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1991.261.2.l188 ◽

1991 ◽

Vol 261 (2) ◽

pp. L188-L194 ◽

Cited By ~ 2

Author(s):

P. I. Plews ◽

Z. A. Abdel-Malek ◽

C. A. Doupnik ◽

G. D. Leikauf

Keyword(s):

Epithelial Cells ◽

Short Circuit ◽

Second Messengers ◽

Short Circuit Current ◽

Chloride Secretion ◽

Tracheal Epithelium ◽

Membrane Phospholipids ◽

Cl Secretion ◽

Tracheal Epithelial Cells ◽

Tracheal Epithelial

The endothelins (ET) are a group of isopeptides produced by a number of cells, including canine tracheal epithelial cells. Because these compounds are endogenous peptides that may activate eicosanoid metabolism, we investigated the effects of ET on Cl secretion in canine tracheal epithelium. Endothelin 1 (ET-1) was found to produce a dose-dependent change in short-circuit current (Isc) that increased slowly and reached a maximal value within 10-15 min. When isopeptides of ET were compared, 300 nM ET-1 and ET-2 produced comparable maximal increases in Isc, whereas ET-3 produced smaller changes in Isc (half-maximal concentrations of 2.2, 7.2, and 10.4 nM, respectively). Ionic substitution of Cl with nontransported anions, iodide and gluconate, reduced ET-1-induced changes in Isc. Furthermore, the response was inhibited by the NaCl cotransport inhibitor, furosemide. In paired tissues, ET-1 significantly increased mucosal net 36Cl flux without significant effect on 22Na flux. The increase in Isc induced by ET was diminished by pretreatment with indomethacin. The second messengers mediating the increase in Isc were investigated in cultured canine tracheal epithelial cells. ET-1 stimulated the release of [3H]arachidonate from membrane phospholipids, increased intracellular Ca2+ (occasionally producing oscillations), and increased adenosine 3',5'-cyclic monophosphate accumulation. The latter was diminished by indomethacin. Thus ET is a potent agonist of Cl secretion (with the isopeptides having the following potency: ET-1 greater than or equal to ET-2 greater than ET-3) and acts, in part, through a cyclooxygenase-dependent mechanism.

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Chloride secretion and conductance of teleost opercular membrane: effects of prolactin

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1982.242.3.r380 ◽

1982 ◽

Vol 242 (3) ◽

pp. R380-R389 ◽

Cited By ~ 5

Author(s):

J. K. Foskett ◽

T. E. Machen ◽

H. A. Bern

Keyword(s):

Active Transport ◽

Short Circuit ◽

Chloride Secretion ◽

Alternative Interpretation ◽

Chloride Cells ◽

Dependent Manner ◽

Transport Pathway ◽

Accurate Analysis ◽

Equivalent Circuit Analysis ◽

Fish Yield

Effects of prolactin on transport properties of opercular membranes from seawater-adapted tilapia, Sarotherodon mossambicus, have been examined. These membranes are high conductance (average Gt approximately 4 mS.cm-2) tissues with short-circuit currents (I) equal to net chloride secretion. Despite high Gt, nonlinear current-voltage relationships suggest that opercular membranes cannot be classified as "leaky" tissues. Variability among membranes is reflected in a linear relationship between I and Gt with a slope equal to 26 mV and the zero-current Gt intercept equal to 0.45 mS.cm-2. Prolactin injections decrease I and Gt in a dose-dependent manner. Phosphodiesterase inhibition, without effect on I in untreated fish, often partially reverses these prolactin effects. Gt-I data from prolactin-treated fish yield a slope of 18 mV and a Gt intercept of 0.10 mS.cm-2. The effects of prolactin are discussed in terms of conventional equivalent circuit analysis. Discrepancies between predictions based on this model and the actual data indicate that an alternative interpretation, based on a heterogeneous cell population, is more accurate. Analysis of this circuit suggests that the ratio of paracellular to active transport pathway conductances associated with chloride cells is constant and that differences in Gt and I are due to parallel changes in these conductances. Prolactin may effectively "remove" chloride cells from these membranes as well as inhibit (reversible by elevated cellular cAMP levels) active transport pathway conductance of remaining cells.

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