Human Colonic T84 Cells Research Articles

ADS024, a single-strain live biotherapeutic product of Bacillus velezensis alleviates dextran sulfate-mediated colitis in mice, protects human colonic epithelial cells against apoptosis, and maintains epithelial barrier function.

Epithelial cell apoptosis and compromised gut barrier function are features of inflammatory bowel disease. ADS024 is a single-strain live biotherapeutic product (LBP) of Bacillus velezensis under development for treating ulcerative colitis (UC). The cytoprotective effects of the sterile filtrate of ADS024's secreted products on UC patient-derived colonic tissues, human primary colonic epithelial cells (HPEC), and human colonic epithelial T84 cells were evaluated. ADS024 filtrate significantly inhibited apoptosis and inflammation with reduced Bcl-2 Associated X-protein (BAX) and tumor necrosis factor (TNF) mRNA expression in fresh colonic explants from UC patients. Exposure to UC patient-derived serum exosomes (UCSE) induced apoptosis with increased cleaved caspase 3 protein expression in HPECs. ADS024 filtrate diminished the UCSE-mediated apoptosis by inhibiting cleaved caspase 3. TNFα and interferon-gamma (IFNγ) damaged epithelial barrier integrity with reduced transepithelial electrical resistance (TEER). ADS024 filtrate partially attenuated the TEER reduction and restored tight junction protein 1 (TJP1) expression. Oral live ADS024 treatment reduced weight loss, disease activity, colonic mucosal injury, and colonic expression of interleukin 6 (IL-6) and TNFα in dextran sodium sulfate (DSS)-treated mice with colitis. Thus, ADS024 may protect the colonic epithelial barrier in UC via anti-inflammatory, anti-apoptotic, and tight-junction protection mechanisms.

Probiotics Play a Key Role in Alleviating Bile Acid (BA)-Induced Cytokine Release and Tight Junction (TJ) Dysfunction in Human Colonic T84 Cells

High colonic BAs play a role in pathogenesis of diarrheal diseases in ~1% of the population. Probiotic supplements are often used to help alleviate the symptoms. We have previously reported that the primary BA, chenodeoxycholic acid (CDCA; 500μM), alters the pore and leak functions of TJs and disrupts barrier integrity in T84 cells, while its derivative, lithocholic acid (LCA; 50μM), did not; CDCA action involved reactive oxygen species, the proinflammatory cytokine IL-8, and apoptosis. Further, probiotics±LCA ameliorated CDCA-induced apoptosis and oxidative stress (FASEB J, 36, R5817, 2022). In the present study, we hypothesize that probiotics could ameliorate CDCA-induced cytokine release, barrier disruption and increases in paracellular permeability in T84 cells.Probiotics in Up & UpTM extra strength supplement ( B. bacterium and L. bacillus strains; 30 billion CFUs) were grown in T84 cell culture media±BA under anaerobic conditions at 37°C and sterile filtered to obtain conditioned media (CM), which was used in subsequent experiments. T84 cells grown to confluency in 12-well plates or Transwells to a Trans Epithelial Resistance (TER) of >1KΩ.cm2 were treated with CM±BA. Following 0.5, 1, 2, 3, 4, 6 and 18 hours (h) incubation: IL-8 released in the media was measured with a sandwich ELISA (pg/ml); TJ pore function measured as TER (Ω.cm2) and TJ leak function measured as FITC-10 kDa dextran flux (F10D; μg). *, p≤0.05 different from control (CTL); #, p≤0.05 different from treatment with only CM.CDCA increased IL-8 release over time, with significant increases starting at 6h (pg/ml; CTL:320±20; CDCA:1524±180*; n=4) and being maintained up to 18h (CTL:700±40; CDCA:3760±350*; +ve CTL: TNFα (100 ng/ml):2600±170*; n≥6; other time points not shown). As previously shown, LCA reduced basal and CDCA-induced IL-8 release (18h, LCA:348±50*; CDCA+LCA:752±30*; n≥4). Probiotic CM±LCA also significantly decreased basal and CDCA-induced IL-8 release (18h, CM:335±50*; CM+CDCA:810±43#; CM+LCA:155±15#; CM+CDCA+LCA:280±102#; n≥4).In terms of TJ pore function, CDCA caused a dramatic reduction in TER in 1 hr and LCA had no effect (% decrease vs CTL;1h, CDCA:88±9*; LCA:2±3; CDCA+LCA:88±5*; n=3). Similarly, probiotic CM did not alter CDCA±LCA-induced decreases in TER (CM+CDCA: 83±8*; CM+LCA: 3±7, CM+CDCA+LCA: 85±9*; n=4). In contrast, probiotic CM reduced the effects of CDCA on the leak function by 60% (18h, F10D flux, μg: CTL:5±1; CDCA:97±5*; CM:6±1; CM+CDCA:36±7#; n=3). LCA decreased CDCA’s effect by 40%, which was further attenuated by CM (18h (μg): LCA:11±7; CM+LCA:8±1; CDCA+LCA:51±3#; CM+CDCA+LCA:18±3#; n≥3). In summary, probiotics and LCA mitigate CDCA-induced inflammation by decreasing IL-8 release and TJ dysfunction by decreasing leak, but not pore function in T84 cells. Understanding the mechanism by which probiotics restore barrier integrity will help identify novel therapeutic strategies to target symptoms in patients with BA associated diarrhea. JS & MP, Institutional funds, Benedictine Univ; HD, EK, IN, FA, & YN: NSSRP, Benedictine Univ. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Probiotics Play a Role in Alleviating Bile Acid (BA)‐induced Apoptosis and Oxidative Stress in Human Colonic T84 Cells

BA‐malabsorption diarrhea (BAD) has been identified in one‐third of patients with inflammatory bowel disease (IBD) and affects >200 million people worldwide. Probiotic supplements are used to promote gut health, restoring microbiome and bile acid composition in these patients. We have previously reported the yin and yang in bile acid (BA) action, wherein the primary BA, chenodeoxycholic acid (CDCA; 500μM), altered tight junctions (TJ) and increased paracellular permeability in T84 cells, while its secondary BA, lithocholic acid (LCA; 50μM), did not. High colonic concentrations (>500μM) of CDCA have been reported in patients with IBD which can modify the gut microflora. Here, we studied the effect of a probiotic supplement on BA action in T84 cells and hypothesized that probiotics could ameliorate BA‐induced apoptosis, oxidative stress and barrier disruption.To study the crosstalk between BA and microflora, probiotic strains in Up & Up extra strength supplement (30 billion CFUs) were grown in T84 epithelial cell culture media ± BA under anaerobic conditions at 37°C. Bacteria grew well in control media (CFU x 108: 6 ± 1) after 48h with Lactobacillus paracasei identified as the predominant species by 16S rRNA sequencing. Further, microbial growth was modified by BAs. 500μM CDCA caused a significant reduction in the growth rate (CFU x 108: 0.07± 0.3; p<0.001, n=4) with L. paracasei remaining predominant. 50μM LCA alone did not alter growth but reversed the effect of CDCA on bacterial growth (CFU x 108: LCA: 6 ± 0.3; CDCA+LCA: 6 ± 1, n=4). Interestingly, LCA ± CDCA changed the bacterial composition with L. rhamnosus becoming the prevalent species. Culture media ± BA was sterile filtered to remove bacteria and the conditioned media (CM) obtained was used in subsequent experiments.T84 cells were grown to confluency in 6‐well plates and treated overnight with CM± BA. Apoptosis (Annexin V, flow cytometry, fluorescence microscopy), cell death (lactate dehydrogenase (LDH) assay, colorimetry) and mitochondrial ROS (mROS, CellRox, flow cytometry) were measured.Flow cytometry and LDH assay revealed that CM caused a dose‐dependent increase in T84 cell apoptosis with 100% CM causing significant (35‐fold) T84 cell death (n=3). The optimal CM dilution with minimal cell death was identified to be 1:8. As seen previously, CDCA (500μM) caused a small increase in apoptosis, while LCA (50μM) did not; CM± LCA reversed CDCA’s effect on cell death (% cell death, CTRL: 10±3 vs CM: 9±4; LCA: 9±3 vs CM+LCA: 10±2; CDCA: 19±4 vs CM+CDCA: 6±3; CDCA+LCA: 16±6 vs CM+CDCA+LCA: 8±3, p<0.05, n=5). Similarly, CDCA increased mROS and this was attenuated by CM (CellRox+ cells, in %, DMSO: 8±1; CDCA: 18±1; CM: 9±1; CM+CDCA: 9±2, n≥3). Though LCA alone did not have an effect, CM+LCA attenuated CDCA‐induced mROS release (LCA: 9±1; CDCA+LCA: 15±1 CM+LCA: 11±1, CM+CDCA+LCA: 9±2; n=4). We demonstrate that BA altered bacterial growth and probiotic CM±LCA reverses CDCA‐induced apoptosis and oxidative stress in T84 cells. We are currently studying the effect of CM on BA‐induced increases in paracellular permeability and TJ dysfunction. Delineating the role played by probiotics in alleviating BAD and understanding the mechanism of action could lead to novel therapeutic strategies for inflammation‐associated diarrheas.

Limosilactobacillus reuteri ATCC 6475 metabolites upregulate the serotonin transporter in the intestinal epithelium.

The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.

Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel.(CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl−) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl− secretion in different regions of native intestine activated by the Ca2+-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl− gradient, strongly supporting TMEM16A as primarily a luminal Cl− channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl− channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl− secretion.

Repurposing calcium-sensing receptor agonist cinacalcet for treatment of CFTR-mediated secretory diarrheas.

Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures, such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca2+. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl– secretion by greater than 80%. Cinacalcet also reduced Cl– secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) Cl– secretion following activation of CaSR and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed loop models of cholera and traveler’s diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3–mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas, including cholera, traveler’s diarrhea, and VIPoma.

Bifidobacterium dentium-derived y-glutamylcysteine suppresses ER-mediated goblet cell stress and reduces TNBS-driven colonic inflammation

ABSTRACT Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds.

Cucumis sativus extract elicits chloride secretion by stimulation of the intestinal TMEM16A ion channel

Context Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. Objective This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. Materials and methods CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. Results CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. Conclusion A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.

Intestinal TMEM16A function as a luminal chloride channel

There has been a high level of uncertainty whether TMEM16A channels are truly expressed on the apical membranes of enterocytes and contribute to luminal Cl− secretion. Here, we describe a series of independent experimental approaches including qPCR, western blot, immuno‐staining and electrophysiology using Tmem 16a+/− mouse intestinal tissue, human colonic tissue and T84 cells to characterize the expression, localization and physiological function of TMEM16A and its regulation by the multi‐PDZ domain‐containing protein NHERF1.qPCR analysis revealed that Tmem16a transcripts are differentially expressed across the intestinal epithelial tissue with highest expression in the colon. Tmem16a mRNA expression did not correlate with TMEM16A protein expression. Western blot analysis of TMEM16A in mouse intestinal tissue revealed that one (Boster Bio PA2290) out of five commercially available polyclonal antibodies appreciably recognized a band at the expected size of below 100 kDa monomeric protein in jejunum, ileum, proximal and distal colon, which was absent in Tmem16a+/− mice. Ussing chamber experiments confirmed that TMEM16A protein abundance correlated with Cl− secretion in different regions of native intestine activated by the Ca2+‐dependent agonist carbachol (CCH). In Tmem16a+/− colon, basal and CCH stimulated Isc was largely reduced. Immunostaining of intestinal tissue with PA2290 antibody confirmed luminal expression of TMEM16A proteins compared to Tmem16a+/− mice. Luminal but not serosal application of MONNA, a TMEM16A specific inhibitor, resulted in significant reduction of CCH stimulated Isc, indicating that TMEM16A is primarily expressed and functions as a luminal Cl− channel. Luminal expression of TMEM16A in human colon was confirmed by confocal microscopy. Localization of TMEM16A to the luminal membrane may be facilitated through interactions with the scaffold protein NHERF1 which we found immunoprecipitated TMEM16A. Our results indicate that TMEM16A is prominently expressed in different segments of native mouse intestine as a luminal membrane protein and in human colonic tissue. TMEM16A also interacts with NHERF1. These data are consistent with TMEM16A localizing to the luminal membrane via the actin cytoskeleton and NHERF1 to influence CCH stimulated luminal Clsecretion.Support or Funding InformationUniversity Grant Commission, and Indian Council of Medical Research, Govt. of India

Fluoresceinamine‐tagged Chenodeoxycholic Acid (CDCA‐FA) causes Epithelial Barrier Dysfunction and Moves Paracellularly in Human Colonic T84 Cells

Bile acid (BA)‐induced diarrhea affects one‐third of patients with chronic diarrhea, but the underlying mechanism is not clear. We recently reported that exposure to CDCA (500μM) altered tight junction (TJ) pore function by decreasing transepithelial resistance (TER) and leak function by increasing paracellular permeability across T84 monolayers; this involved release of reactive oxygen species (ROS) and inflammatory cytokines. The secondary BA lithocholic acid (LCA) and ROS inhibitors attenuate leak, but not pore, TJ function (Physiol Reports, ‘17, 5: e13294). We also showed that CDCA alters TJs only when added to the apical side (AS) of the monolayer and increases Cl− secretion only when added to the basolateral side (BLS) (AJP Cell, 2013, 305: C447‐56). We hypothesize that luminal BAs alter TJ permeability and travel paracellularly to reach the BLS.To test this, we synthesized a novel fluorescent BA, CDCA‐FA, by converting the carboxylic acid of CDCA to an acid chloride and then adding the FA tag. This was purified via column chromatography and the structure, molecular formula and mass (C45 H53N O7; 719.38) confirmed by NMR and mass spectroscopy. Functional studies were performed on confluent T84 cells grown on Transwells (TER >1000Ωcm2), treated AS with DMSO (CTRL), 500μM CDCA or CDCA‐FA ± 50μM LCA for 0.5–18 hours (H). Cell viability was measured by propidium iodide staining, fluorescence microscopy and Image J analysis. CDCA‐FA effects on TJs were assessed by examining: i) pore function measured as TER; ii) leak function measured as apparent permeability of CDCA‐FA (PappCDCA‐FA) compared to CDCA‐induced Fluorescein Isothiocyanate‐10 kDa dextran (F10D) flux across the monolayer. The effect of ROS scavenger, N‐acetyl cysteine (NAC; 1mM), and LCA (50μM) on CDCA‐FA's effects on TJ functions were also assessed.Overnight exposure to 500μM CDCA‐FA, like CDCA, caused a small (~18%) decrease in cell viability that was not altered by LCA (n=6). Again, similar to CDCA, CDCA‐FA showed a time‐dependent decrease in TER (in Ωcm2; t=0 H: CTRL: 927 ± 130; CDCA‐FA: 926 ± 154; t =0.5 H: CTRL: 836 ± 54; CDCA‐FA: 222 ± 28*; t=18 H; CTRL: 731 ± 10; CDCA‐FA: 107 ± 18*; n=4; *p<0.05). Furthermore, CDCA‐FA's effect on TER was neither altered by NAC nor LCA (% decrease vs. CTRL; 18 H CDCA‐FA: 85±2; CDCA‐FA+NAC: 88±5; CDCA‐FA+LCA: 87±3; CDCA‐FA+LCA+NAC: 85±6, n=3). In contrast, CDCA‐FA permeability increased over time; this increase was attenuated by LCA and further decreased by NAC (18 H, Papp CDCA‐FA, cm/sec, CDCA‐FA: 57±2; CDCA‐FA+LCA: 24±3; CDCA‐FA+NAC: 20±4; CDCA‐FA+LCA+NAC: 8±2; p<0.05, n≥3). This action of CDCA‐FA on the leak function is similar to that of CDCA on F10D flux (18 H, Papp F10D, cm/sec: CTRL: 8±1; CDCA: 165±12; p<0.05, n=6). Thus, using a novel fluorescent BA probe, CDCA‐FA, we demonstrate that, like CDCA, it increases TJ pore and leak functions in T84 cells. We conclude that CDCA from the apical side traverses to the basolateral side by a ROS‐dependent increase in paracellular permeability and that LCA limits the movement of CDCA through the TJs. Identifying ways to prevent BA's access to the BLS may be an effective therapeutic tool for the treatment of BA‐induced diarrhea.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Institutional Funds, Ben. U. to JS and DMR; Institutional Funds, UIC to MCRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence that Zinc Deficiency Impairs Gut Epithelial Barrier and intestinal Immunity

There is growing interest in dietary factors, in particular micronutrients, from the perspective of disease pathogenesis and potential for treatment. Diarrheal disease in infant results in abnormally low concentrations of serum zinc. Zinc has been tested for its ability to treat and prevent diarrheal diseases in many large field trials over a period of over 4 decades and has generally been found effective. The mechanism by which zinc deficiency causes diarrhea is not known. The present study investigated the impact of zinc deficiency on the host, particularly of intestinal tight junction (TJ) integrity and proteins that are involved in intestinal absorption and secretion along with the pathogenesis of enteric infection. We have used human colonic T84 cells to study barrier function and Shigella to test susceptibility of intestinal infection due to zinc deficiency. T84 grown onto transwell inserts in zinc deficient media showed a low transepithelial electrical resistance (TEER) compare to zinc sufficient media (1014 ± 165 Ω.cm2 vs 3663 ± 293 Ω.cm2) in confluent T84 monolayers. We furthermore analysed the correlation between TEER and paracellular ionic conductance. We found that zinc deficiency altered paracellular ionic conductance. Bacterial transmigration studies showed a significant increase in apico‐basolateral transmigration of Shigella (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) which peaked at 6hrs post infection. Transmission electron microscopy data showed widened TJ complex due to few membrane fusion proteins and distorted TJ morphology, whereas the desmosomes were still intact. Electrophysiological studies with Ussing chamber and qPCR quantification demonstrated reduced cAMP dependent electrogenic Cl− secretion (34.5 ± 1.25 μA/cm2 vs 17.75 ± 2.3 μA/cm2) along with reduced expression of CFTR in zinc deficient cells, resulting dehydrated lumen thus aiding in bacterial colonization. Our results suggest that zinc deficiency caused (1) perturbed barrier function and integrity (2) altered Cl− secretion leading to susceptibility of infection. We conclude that adequate zinc is required to maintain intestinal barrier health to avoid risk against intestinal infection.Support or Funding InformationGovt of India National Funding AgencyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Yin and Yang of bile acid action on tight junctions in a model colonic epithelium.

Gastrointestinal epithelial barrier loss due to tight junction (TJ) dysfunction and bile acid‐induced diarrhea are common in patients with inflammatory diseases. Although excess colonic bile acids are known to alter mucosal permeability, few studies have compared the effects of specific bile acids on TJ function. We report that the primary bile acid, chenodeoxycholic acid (CDCA), and its 7α‐dehydroxylated derivative, lithocholic acid (LCA) have opposite effects on epithelial integrity in human colonic T84 cells. CDCA decreased transepithelial barrier resistance (pore) and increased paracellular 10 kDa dextran permeability (leak), effects that were enhanced by proinflammatory cytokines (PiC [ng/mL]: TNF α[10] + IL‐1ß[10] + IFN γ[30]). CDCA reversed the cation selectivity of the monolayer and decreased intercellular adhesion. In contrast, LCA alone did not alter any of these parameters, but attenuated the effects of CDCA ± PiC on paracellular permeability. CDCA, but not PiC, decreased occludin and not claudin‐2 protein expression; CDCA also decreased occludin localization. LCA ± CDCA had no effects on occludin or claudin expression/localization. While PiC and CDCA increased IL‐8 production, LCA reduced both basal and PiC ± CDCA‐induced IL‐8 production. TNF α + IL1ß increased IFN γ, which was enhanced by CDCA and attenuated by LCA. CDCA±PiC increased production of reactive oxygen species (ROS) that was attenuated by LCA. Finally, scavenging ROS attenuated CDCA's leak, but not pore actions, and LCA enhanced this effect. Thus, in T84 cells, CDCA plays a role in the inflammatory response causing barrier dysfunction, while LCA restores barrier integrity. Understanding the interplay of LCA, CDCA, and PiC could lead to innovative therapeutic strategies for inflammatory and diarrheal diseases.

Fungus‐Derived Zearalenone as an Inhibitor of cAMP‐Dependent Chloride Secretion in Human Colonic Epithelial T84 Cells

Secretory diarrhea remains a major health problem caused by either intestinal infection or side‐effect of many drugs. An elevation in intracellular cAMP activates transepithelial Cl− secretion, which provides the driving force for secretory diarrhea. Therefore, inhibition of cAMP‐activated transepithelial Cl− secretion in intestinal mucosa is regarded as a potential therapeutic strategy for secretory diarrhea. This study aimed to identify fungus‐derived compounds that inhibit cAMP‐activated transepithelial Cl− secretion and investigate its underlying mechanism(s). From the screening of compounds derived from fungi, zearalenone (β‐resorcyclic acid lactone produced by Fusarium fungi) was found to inhibit cAMP‐activated Cl− secretion across human intestinal epithelial (T84) cell monolayers as measured by short‐circuit current (Isc) analysis with half maximal inhibitory concentration (IC50) of ~ 0.5 μM. This effect was observed only when zearalenone was added into apical side, but not basolateral side, suggesting that zearalenone affects mainly apical transport processes. We further demonstrated that zearalenone reversibly inhibited cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated apical Cl− transport with IC50 ~ 0.8 μM, indicating that zearalenone inhibits cAMP‐acivated Cl− secretion in T84 cell, at least in part, by inhibiting CFTR Cl− channel activity. The mechanisms of zearalenone‐induced CFTR inhibition involved neither phosphodiesterase‐mediated cAMP degradation, multi‐drug resistance protein 4‐mediated cAMP efflux nor protein phosphatase‐mediated CFTR dephosphorylation. In addition, the CFTR inhibitory effect of zearalenone was not affected by co‐treatment with β‐estradiol, indicating that the CFTR inhibition by this compound did not result from its binding to estrogen binding site on the CFTR. Zearalenone at concentrations up to 50 μM did not produce the cytotoxic effect on T84 cells. This study revealed that zearalenone represents the first class of CFTR inhibitors derived from fungi, which may be useful in the treatment of secretory diarrhea resulting from overactivation of cAMP‐dependent chloride secretion.Support or Funding InformationThis work was supported by the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation and the National Science and Technology Development Agency and BRG5890008 grant from the Thailand Research Fund and Mahidol University

Probiotic Bacteria and their Supernatants Protect Enterocyte Cell Lines from Enteroinvasive Escherichia coli (EIEC) Invasion.

Probiotic microorganisms have attracted a growing interest for prevention and therapy of gastrointestinal disorders. Many probiotic strains have been shown to inhibit growth and metabolic activity of enteropathogenic bacteria as well as their adhesion and invasion to intestinal cells. In the present study, we evaluated the interference of bacteria-free supernatants (BFS) of cultures belonging to sixteen strains of lactobacilli and bifidobacteria, with invasion of enteroinvasive Escherichia coli (EIEC) strain, using human colonic adenocarcinoma cell lines, T84 and Caco2 cells. To assess invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted. In addition, three different invasion inhibition assays were designed; namely co-incubation, pre-incubation and treatment with the BFS of probiotics. Data obtained and theoretical calculation showed that the most effective assay in the prevention of pathogen invasion was treatment with BFS. Besides, co-incubation assay was more valid than pre-incubation assay in invasion prevention. The obtained results suggest that probiotics may produce some metabolites that strongly prevent invasion of enteroinvasive E.coli into the small and large intestine. Also, probiotics are able to compete with or exclude pathogen invasion.

Chenodeoxycholic acid requires activation of EGFR, EPAC, and Ca2+ to stimulate CFTR-dependent Cl- secretion in human colonic T84 cells.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl- secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+ In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+ were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.

Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.

Phorbol Dibutyrate (PDB) Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) May Involve Down‐Regulation of MicroRNAs (miRs)

In colonic epithelia, the concerted action of apical membrane Cl− channels and basolateral membrane Na+/K+‐ATPase, Na+‐K+‐2Cl− cotransporters and K+ channels, account for net Cl− secretion. CFTR, the major apical Cl− channel, is regulated by protein kinases (PK) A and C. We had previously showed in human colonic T84 cells that the cAMP activator, prostaglandin E1 (PGE1,10nM), stimulated Cl− secretion via CFTR, measured as short circuit current (ΔISc, μA/cm2) and this was attenuated by both acute and prolonged pretreatment with the PKC activator, phorbol dibutyrate (PDB, 100nM). Further, 2h of pretreatment with PDB significantly reduced CFTR mRNA. Since microRNAs (miRs) are known to modulate proteins by either degrading mRNA, or by inhibiting translation, we initially tested whether 6 specific CFTR related miRs: −145, −331, −384, −494, −1246 and 1290 were regulated by PDB. However, the expressions of these miRs were not significantly altered by PDB (FASEB 29:855.5,’15). In this study, we further explored the molecular basis of the attenuation in Cl− secretion. We first confirmed, by Western blotting, that CFTR protein expression is down regulated at 2h PDB treatment (n=2). In addition, the mRNA (60%, n=3) and protein expression for NKCC was also decreased. Since CFTR 3′ UTR contains almost 500 recognized miR target sites, we next undertook a detailed algorithm screening process involving TargetScan, miRDB, EMBL‐EBI and Diana to identify further possible CFTR miR regulators. We hypothesized that the miRs identified through algorithmic screening would be up‐regulated after PDB treatment, leading to a decrease in CFTR expression. We specifically screened miRs: −362, −433, −509, −600, −607, −619, −631, and −944, which were identified by at least 3 algorithms. After treating T84 cells with ±100nM PDB for 2h, 6h, and 24h, RNA was isolated using TriZol Reagent and cDNA was synthesized using an Invitrogen cDNA synthesis kit. We compared the expression levels of these miRs using an Invitrogen miRNA qRT‐PCR Kit with U6 snRNA as an internal control. Based on three separate experiments each (p<0.05), the results show that miR −607 (Control: 1.37±0.18, 2h: 0.51±0.08, 6h: 0.77±0.09, 24h: 0.55±0.18), miR‐619 (Control: 2.04±0.92, 2h: 0.58±0.05, 6h: 0.42±0.06, 24h: 0.44±0.04), and miR‐944 (Control: 1.50±0.30, 2h: 0.46±0.07, 6h: 0.36±0.08, 24h: 0.40±0.13) expressions were significantly down‐regulated at all measured time‐points. miR‐631 (Control: 1.24±0.18) expression was significantly down‐regulated at 2h (0.60±0.05) and 6h (0.58±0.04) but not at 24h (0.78±0.22). The results with miR‐607 are in contrast to studies in Caco2 cells, where an increase in miR‐607 leads to a decrease in CFTR expression (Biochem J. 438, p.25–32 2011). The other miRs tested did not show significant alteration of expression at any measured time‐point. Thus, PDB treatment reduces CFTR and NKCC expression and down regulates specific miRs. We speculate that perhaps there is an intermediary protein, the target of miRs: −607, −619, and −944, that alters CFTR/NKCC expression.Support or Funding InformationAmerican Physiological Society Undergraduate Research Excellence Fellowship (UGREF)

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (J H +) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J H + (~63%), without altering basal pHi (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and J H + was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J H + was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.

Phorbol Dibutyrate (PDB) Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Does Not Involve MicroRNAs (miRNAs)

PDB is a tumor promotor and activator of protein kinase C. In human colonic T84 cells, pretreatment with PDB attenuated PGE1- stimulated Cl- secretion as measured by short circuit current (ΔISc, μA/cm2). MiRNAs are known to modulate a variety of proteins by degrading their mRNA or by inhibiting their translation. Since CFTR is the major Cl- channel in T84 cells, and CFTR-specific miRNAs have been identified, we examined if the PDB-attenuation of Cl- secretion involves miRNAs. Acute treatment with PDB significantly reduced 10μM PGE1-stimulated ISc (PGE1: 36.7±7.3; +200nM PDB, 20 min: 8.9±2.1; n蠅6). A 2 hour PDB exposure caused a similar reduction in PGE1-stimulated ISc (PGE1: 36.7±7.3; + 100nM PDB, 2h: 10 ±2.5; n=3). The 2hr treatment followed by an acute dose (100nM PDB 2h + 200nM PDB 20 min) still reduced the PGE1 prosecretory response (9± 4; n=3). This is possibly due to a reduction in the expression of CFTR since CFTR mRNA is also significantly reduced after 2h pretreatment with PDB (relative expression: Control: 3.56±0.7; PDB: 1.38±0.32; n>4). However this attenuation of CFTR function was lost with chronic treatment (100nM PDB, 24h: 18.7 ±8.4; 48h: 45.3 ±7.1; 72h: 32.7 ±13.8; n蠅3). Based on algorithm screening, we identified 6 miRNAs that may regulate CFTR: miR-145, -331, -384, -494, -1246 and -1290. Using a miRNA qRT-PCR Kit we compared the expression of these miRNAs in T84 cells treated with +100nM PDB for 2h. U6 snRNA was used as internal control. Although these miRNAs were expressed in T84 cells, PDB did not significantly alter their expression. Thus, it is unlikely that the common “CFTR-miRNAs” are involved in the PDB-induced attenuation of CFTR expression and function.

Bile Acid (BA) Stimulation of Cl ‐ Secretion Involves Intricate Crosstalk Cascades in Human Colonic T84 Cells

We recently showed that the BA chenodeoxycholate (CDCA) stimulates CFTR-mediated Cl- secretion in T84 cells via protein kinase A (PKA; AJP 305:C447-56, '13). We now demonstrate that CDCA signaling is far more complex, involving additional pathways. It is unlikely that BA specific receptors, TGR5 and FXR, are involved since their specific agonists do not increase Cl- secretion as measured by short circuit current (ΔISc, µA/cm2; TGR5: 20µM Ciprofloxacin: -3.4±4.8; FXR: 5µM GW4064: 1.5±1.5; n蠅3). However, the EGF receptor (EGFR) inhibitor AG1478 attenuates CDCA's increase in ISc (FASEB 28:908.4,'14) and decreases CDCA-induced EGFR phosphorylation by 50%. Although EGFR activation is necessary, EGF alone does not stimulate Cl- secretion (n=3). Downstream of EGFR, while CDCA increases p38 MAPK and ERK phosphorylation, their respective inhibitors alone or in combination do not alter CDCA-stimulated ISc (n蠅3). In contrast, inhibition of PI3K decreases CDCA's effect on ISc (CDCA: 14.5±2.8; + 0.5µM Wortmannin: 6.3±1.7; n蠅4; p<0.05) independent of AKT (CDCA: 14.3±1.7; + 1µM MK2206: 13.1±3; n蠅3). CDCA significantly increases IP3 production (µmoles/well: DMSO: 0.94±0.1; CDCA: 1.67±0.1; n蠅3) and the IP3 receptor inhibitor decreases CDCA-induced Isc (CDCA: 15.5±2.9; + 50µM 2-APB: 6.3±0.4; n=3). Also, Ca2+ chelation inhibits CDCA action (CDCA: 15.7±2.6; + 20µM BAPTA: 3.3±0.2; n=4). We conclude that CDCA-stimulated Cl- secretion does not involve TGR5, FXR, EGF, p38, ERK, or AKT but utilizes an EGFR-PI3K mechanism and involves intracellular Ca2+. How PKA is involved in this crosstalk remains to be determined.