Human T84 Research Articles

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Calcium-sensing receptor activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl− channels, and basolateral membrane K+ channels. 30 μM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Campylobacter jejuni Modulates Reactive Oxygen Species Production and NADPH Oxidase 1 Expression in Human Intestinal Epithelial Cells

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX1), a key ROS-generating enzyme in IECs and antioxidant defence genes CAT and SOD1. Furthermore, inhibition of NOX1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade, and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery.

Potentially probiotic Limosilactobacillus reuteri from human milk strengthens the gut barrier in T84 cells and a murine enteroid model.

At conception, the infant gut barrier is immature, gradually developing with regular intake of maternal milk. This study addressed whether the barrier-strengthening effect of breast feeding might be attributable, at least in part, to autochthonous beneficial human milk bacteria. Twelve bacterial strains from the breast milk of Pakistani mothers who underwent cesarean delivery (NPL-88, NPL-157, NPL-179, NPL-181, NPL-388 (Limosilactobacillus reuteri), NPL-76, NPL-495, NPL-504 (Limosilactobacillus fermentum), NPL-415 (Lactobacillus pentosus), NPL-412, NPL-416 (Lactiplantibacilllus plantarum) and NPL-374 (Bifidobacterium longum) were shortlisted based on their tolerance to acidic pH (2.8-4.2) and bile (0.1-0.3%). The effect of these bacteria on gut barrier function in the presence and absence of pathogens was assessed as changes in transepithelial electrical resistance (TEER) in the human T84 colonic epithelial cell line and in murine enteroid-derived monolayers (EDMs). The TEER of T84 cells monolayers rose in the presence of most of the human milk strains, being most pronounced in case of L. reuteri NPL-88 (34% within five h), exceeding the effect of the well-known probiotic L. acidophilus (20%). qRT-PCR, western blot and immunofluorescent staining associated the increase in TEER with enhanced expression of tight junction proteins. Pretreatment of murine EDMs with NPL-88 also largely prevented the ability of the pathogen, Salmonella, to decrease TEER (87±1.50%; P<0.0001, n=4). Human milk lactic acid bacteria are potential probiotics that can strengthen gut barrier function and protect breastfed neonates against enteric infections.

Investigation of TLR4 Antagonists for Prevention of Intestinal Inflammation

Activation of toll-like receptor 4 (TLR4) has been shown to be a major influence on the inflammatory signalling pathways in intestinal mucositis (IM), as demonstrated by TLR4 knock-out mice. Pharmacological TLR4 inhibition has thus been postulated as a potential new therapeutic approach for the treatment of IM but specific TLR4 inhibitors have yet to be investigated. As such, we aimed to determine whether direct TLR4 antagonism prevents inflammation in pre-clinical experimental models of IM. The non-competitive and competitive TLR4 inhibitors, TAK-242 (10 µM) and IAXO-102 (10 µM), respectively, or vehicle were added to human T84, HT-29, and U937 cell lines and mouse colonic explants 1 h before the addition of lipopolysaccharide (LPS) (in vitro: 100 µg/mL; ex vivo: 10 µg/mL), SN-38 (in vitro: 1 µM or 1 nM; ex vivo: 2 µM), and/or tumour necrosis factor-alpha (TNF-α) (5 µg/mL). Supernatant was collected for human IL-8 and mouse IL-6 enzyme-linked immunosorbent assays (ELISAs), as a measure of inflammatory signalling. Cell viability was measured using XTT assays. Explant tissue was used in histopathological and RT-PCR analysis for genes of interest: TLR4, MD2, CD14, MyD88, IL-6, IL-6R, CXCL2, CXCR1, CXCR2. SN-38 increased cytostasis compared to vehicle (P < 0.0001). However, this was not prevented by either antagonist (P > 0.05) in any of the 3 cell lines. Quantitative histological assessment scores showed no differences between vehicle and treatment groups (P > 0.05). There were no differences in in vitro IL-8 (P > 0.05, in all 3 cells lines) and ex vivo IL-6 (P > 0.05) concentrations between vehicle and treatment groups. Transcript expression of all genes was similar across vehicle and treatment groups (P > 0.05). TLR4 antagonism using specific inhibitors TAK-242 and IAXO-102 was not effective at blocking IM in these pre-clinical models of mucositis. This work indicates that specific epithelial inhibition of TLR4 with these compounds is insufficient to manage mucositis-related inflammation. Rather, TLR4 signalling through immune cells may be a more important target to prevent IM.

Lubiprostone is non-selective activator of cAMP-gated ion channels and Clc-2 has a minor role in its prosecretory effect in intestinal epithelial cells.

Loss of prosecretory Cl- channel CFTR activity is considered as the key cause of gastrointestinal disorders in cystic fibrosis including constipation and meconium ileus. Clc-2 is proposed as an alternative Cl- channel in intestinal epithelia that can compensate for CFTR loss-of-function. Lubiprostone is an FDA-approved drug with Clc-2 activation as its presumed mechanism of action. However, relative contribution of Clc-2 in intestinal Cl- secretion and the mechanism of action of lubiprostone remain controversial due to lack of selective Clc-2 inhibitors. Using recently identified selective Clc-2 inhibitor AK-42, we characterized the roles of Clc-2 in Cl- secretion in human intestinal epithelial T84 cells. Clc-2 inhibitor AK-42 had minimal (15%) inhibitory effect on secretory short-circuit current (Isc) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTRinh-172) caused 2-3 fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory Isc by 20%, whereas CFTRinh-172 caused 2-3 fold greater inhibition. In addition to increasing CFTR and Clc-2-mediated apical Cl- conductance, lubiprostone increased basolateral membrane K+ conductance, which was completely reversed by cAMP-activated K+ channel inhibitor BaCl2 All components of lubiprostone-induced secretion (Clc-2, CFTR and K+ channels) were inhibited by ~65% with the extracellular Ca2+-sensing receptor (CaSR) activator cinacalcet that stimulates cAMP hydrolysis. Lastly, EP4 prostaglandin receptor inhibitor GW627368 pretreatment inhibited lubiprostone-induced secretion by 40% without any effect on forskolin response. Our findings suggest that Clc-2 has minor role in cAMP-induced intestinal Cl- secretion; and lubiprostone is not a selective Clc-2 activator, but general activator of cAMP-gated ion channels in human intestinal epithelial cells. Significance Statement Cl- channel Clc-2 activation is the proposed mechanism of action of the FDA-approved constipation drug lubiprostone. Using first-in-class selective Clc-2 inhibitor AK-42, we showed that Clc-2 has minor contribution in intestinal Cl- secretion induced by lubiprostone and cAMP agonists. We also found that lubiprostone is a general activator of cAMP-gated ion channels in human intestinal epithelial cells (via EP4 receptors). Our findings clarify the roles of Clc-2 in intestinal Cl- secretion and elucidate the mechanism of action of approved-drug lubiprostone.

A55 INTERLEUKIN-10 ELICITS CYTOPROTECTION VIA MITOCHONDRIAL SIGNAL TRANSDUCER AND ACTIVATORS OF TRANSCRIPTION 3 (MTSTAT3) TO PREVENT BACTERIAL PATHOBIONT EVOKED MITOCHONDRIAL FRAGMENTATION IN GUT EPITHELIA

BackgroundAberrant mitochondrial function is increasingly reported in inflammatory bowel disease (IBD). We recently reported that the IBD-associated pathobiont, adherent-invasive E. coli (AIEC) (strain LF82)-evoked epithelial mitochondrial fragmentation and mitochondrial depolarization. Remarkably, the transcription factor, signal transducer and activator of transcription (STAT)-3, can migrate to mitochondria (mtSTAT3) to regulate functions such as reactive oxygen species and ATP production. This non-canonical activity is dependent on STAT3 phosphorylation at serine727. IL-10 is an important regulator of enteric homeostasis that signals via STAT-3, however whether IL-10 affects mitochondrial dynamics is unknown.AimsTo determine (1) if IL-10 preserves mitochondrial function in E. coli-LF82-infected epithelia through mtSTAT3, and if so, (2) to identify the mechanism by which mtSTAT3 restores mitochondrial functions.MethodsThe human colon-derived T84 epithelial cells or human organoids were exposed to E. coli LF82 (108 cfu/ml, 4h) ± co-treatment or an 18h pre-treatment with IL-10 (10 ng/ml). The effect of IL-10 on bacterial growth and its invasion of T84 cells were assessed by growth curve analysis and bacterial internalisation assays. We assessed mitochondrial network morphology using mito-tracker red and confocal microscopy, mitochondrial membrane potential with the fluorescent-dye TMRE and flow cytometry and fluorescence microscopy, and oxygen consumption rate (OCR) and ATP levels. STAT3 phosphorylation was analysed in whole cell protein extracts by western blot. Pharmacological inhibitors of JAK and Erk1/2 were used to examine the pathway of mtSTAT3 activation.ResultsIL-10 affected neither E. coli-LF82 growth nor invasion of T84 epithelia, but substantially reduced the AIEC-induced mitochondrial fragmentation in T84 cells and organoids. IL-10 preservation of the mitochondrial network was accompanied by increased mitochondrial membrane potential, OCR, and ATP levels in E. coli-LF82 infected T84 cells. IL-10 + E. coli-LF82-treated epithelia displayed increased phospho-S727 STAT3 that was reduced by inhibition of Erk1/2 but not JAK activity.ConclusionsMitochondria are a potential drug target in IBD, and the E. coli-LF82 pathobiont can be added to a growing list of microbial pathogens that evoke epithelial mitochondrial dysfunction. We have uncovered that IL-10 can stabilize mitochondrial function in E. coli-LF82 infected cells, possibly through mtSTAT-3. Our results underscore the role of IL-10/STAT3 signaling in preserving mitochondrial functions that could result in mitochondria-targeted therapeutics in IBD and other bacteria-driven enteropathies.Funding AgenciesCIHRAIHS

Navigating Through Cell-Based In vitro Models Available for Prediction of Intestinal Permeability and Metabolism: Are We Ready for 3D?

.Traditionally, in vitro studies to quantify the intestinal permeability of drugs have relied on two-dimensional cell culture models using human colorectal carcinoma cell lines, namely Caco-2, HT 29 and T84 cells. Although these models have been commonly used for high-throughput screening of xenobiotics in preclinical studies, they do not fully recapitulate the morphology and functionality of enterocytes found in the human intestine in vivo. Efforts to improve the physiological and functional relevance of in vitro intestinal models have led to the development of enteroids/intestinal organoids and microphysiological systems. These models leverage advances in three-dimensional cell culture techniques and stem cell technology (in addition to microfluidics for microphysiological systems), to mimic the architecture and microenvironment of the in vivo intestine more accurately. In this commentary, we will discuss the advantages and limitations of these established and emerging intestinal models, as well as their current and potential future applications for the pre-clinical assessment of oral therapies.Graphic abstract

2′-Fucosyllactose impacts the expression of mucus-related genes in goblet cells and maintains barrier function of gut epithelial cells

Cost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown. Here, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells. Our findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells.

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

ABSTRACT Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn’s disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin.

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivoIMPORTANCEC. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

Activation of the PKA signaling pathway stimulates oxalate transport by human intestinal Caco2-BBE cells.

Most kidney stones are composed of calcium oxalate, and small increases in urine oxalate enhance the stone risk. The mammalian intestine plays a crucial role in oxalate homeostasis, and we had recently reported that Oxalobacter-derived factors stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation. We therefore evaluated whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, PKA was activated with forskolin and IBMX (F/I). F/I significantly stimulated (3.7-fold) [14C]oxalate transport by C2 cells [≥49% of which is mediated by the oxalate transporter SLC26A6 (A6)], an effect completely blocked by the PKA inhibitor H89, indicating that it is PKA dependent. PKA stimulation of intestinal oxalate transport is not cell line specific, since F/I similarly stimulated oxalate transport by the human intestinal T84 cells. F/I significantly increased (2.5-fold) A6 surface protein expression by use of immunocytochemistry. Assessing [14C]oxalate transport as a function of increasing [14C]oxalate concentration in the flux medium showed that the observed stimulation is due to a F/I-induced increase (1.8-fold) in Vmax and reduction (2-fold) in Km. siRNA knockdown studies showed that significant components of the observed stimulation are mediated by A6 and SLC26A2 (A2). Besides enhancing A6 surface protein expression, it is also possible that the observed stimulation is due to PKA-induced enhanced A6 and/or A2 transport activity in view of the reduced Km. We conclude that PKA activation positively regulates oxalate transport by intestinal epithelial cells and that PKA agonists might therapeutically impact hyperoxalemia, hyperoxaluria, and related kidney stones.

Dimethylfumarate Inhibits Colorectal Carcinoma Cell Proliferation: Evidence for Cell Cycle Arrest, Apoptosis and Autophagy.

Recent studies have proven that Dimethylfumarate (DMF) has a marked anti-proliferative impact on diverse cancer entities e.g., on malignant melanoma. To explore its anti-tumorigenic potential, we examined the effects of DMF on human colon carcinoma cell lines and the underlying mechanisms of action. Human colon cancer cell line HT-29 and human colorectal carcinoma cell line T84 were treated with or without DMF. Effects of DMF on proliferation, cell cycle progression, and apoptosis were analyzed mainly by Bromodeoxyuridine (BrdU)- and Lactatdehydrogenase (LDH)-assays, caspase activation, flowcytometry, immunofluorescence, and immunoblotting. In addition, combinational treatments with radiation and chemotherapy were performed. DMF inhibits cell proliferation in both cell lines. It was shown that DMF induces a cell cycle arrest in G0/G1 phase, which is accompanied by upregulation of p21 and downregulation of cyclin D1 and Cyclin dependent kinase (CDK)4. Furthermore, upregulation of autophagy associated proteins suggests that autophagy is involved. In addition, the activation of apoptotic markers provides evidence that apoptosis is involved. Our results show that DMF supports the action of oxaliplatin in a synergetic manner and failed synergy with radiation. We demonstrated that DMF has distinct anti-tumorigenic, cell dependent effects on colon cancer cells by arresting cell cycle in G0/G1 phase as well as activating both the autophagic and apoptotic pathways and synergizes with chemotherapy.

Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Bile acid diarrhea (BAD) is common with ileal resection, Crohn's disease, and diarrhea-predominant irritable bowel syndrome. Here, we demonstrate the efficacy of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (R)-benzopyrimido-pyrrolo-oxazine-dione-27 (BPO-27) in reducing bile acid-induced fluid and electrolyte secretion in colon. Short-circuit current measurements in human T84 colonic epithelial cells and planar colonic enteroid cultures showed a robust secretory response following mucosal but not serosal addition of chenodeoxycholic acid (CDCA) or its taurine conjugate, which was fully blocked by CFTR inhibitors, including (R)-BPO-27. (R)-BPO-27 also fully blocked CDCA-induced secretory current in murine colon. CFTR activation by CDCA primarily involved Ca2+ signaling. In closed colonic loops in vivo, luminal CDCA produced a robust secretory response, which was reduced by ∼70% by (R)-BPO-27 or in CFTR-deficient mice. In a rat model of BAD produced by intracolonic infusion of CDCA, (R)-BPO-27 reduced the elevation in stool water content by >55%. These results implicate CFTR activation in the colon as a major prosecretory mechanism of CDCA, a bile acid implicated in BAD, and support the potential therapeutic efficacy of CFTR inhibition in bile acid-associated diarrheas.-Duan, T., Cil, O., Tse, C. M., Sarker, R., Lin, R., Donowitz, M., Verkman, A. S. Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Reactive Nitrogen and Oxygen Species (RNS and ROS) Play a Role in Bile Acid (BA)‐Induced Barrier Dysfunction and Proinflammatory Cytokines (PiC) Release in Human Colon Carcinoma T84 Cells

Dysfunction of mucosal immune response and tight junctions (TJ) play an important role in the pathogenesis of inflammatory bowel diseases (IBD) and diarrhea. We reported that excess BA, namely chenodeoxycholic acid (CDCA; 500μM) induced PiC release and altered T84 TJ by increasing ROS. The effect on leak function of TJ was attenuated by CDCA's derivative, lithocholic acid (LCA, 50 μM) and ROS inhibitors (Physiol Rep, ′17, 5: e13294). In patients with IBD, PiC upregulate inducible nitric oxide synthase (iNOS). Thus, we hypothesize a role for RNS in mucosal damage and studied the involvement of oxidative/nitrosative stress in BA‐induced TJ dysfunction and cytokine release in T84 cells.We previously described the synthesis of fluorescein amine‐tagged CDCA (CDCA‐FA) and its use to track transepithelial (TE) BA movement. Confluent T84 cells (TE Resistance; TER &gt;1000Ωcm2) were treated apically with DMSO, 500μM CDCA‐FA, 50μM LCA, CDCA+LCA, ± PiC ([ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), ± 50μM L‐NAME ( L‐NG‐Nitroarginine methyl ester, NOS inhibitor), ± 1mM NAC, (N‐acetyl cysteine, ROS scavenger), for 0.5–18 H. [NO2/NO3] was measured by the Griess assay. We examined the role of RNS/ROS in BA action by studying the effect of BAs±L‐NAME± NAC on: a. Apoptosis (Annexin V, Flow cytometry); b. Paracellular permeability (Pore function as TER; Leak function as TE CDCA‐FA movement); and c. IL‐8 release (ELISA).CDCA (18 H), but not LCA, increased [NO2/NO3] 3‐fold and this was enhanced by PiC (μmol/mg protein; DMSO: 12±1; CDCA: 36±1; LCA: 16± 4, PiC: 18±1; PiC+CDCA: 55±2, n=3). LCA decreased CDCA±PiC‐induced [NO2/NO3] (CDCA+LCA: 25±1; PiC+LCA: 13±2, PiC+CDCA+LCA: 27±4; p&lt;0.05). Inhibiting RNS did not alter CDCA‐induced apoptosis. In pore function, L‐NAME reduced the initial rate of CDCA‐FA‐induced decrease in TER (Ωcm2/sec; 1H CDCA‐FA: 12±1; CDCA‐FA+L‐NAME: 7±1, n&gt;3; p&lt;0.05 ), with no statistical difference at 4 and 18H. L‐NAME altered leak function, reducing CDCA‐FA flux by ~30% at 18 H, (Apparent permeability: Papp ×10−9 cm/sec: CDCA‐FA: 57±2; CDCA‐FA+L‐NAME: 30±1; p&lt;0.05, n=3). LCA decreased CDCA‐FA flux by ~58%, and LCA+L‐NAME completely attenuated it (CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA+L‐NAME: 3±1; p&lt;0.05, n&gt;3). NAC+LCA caused only a ~85% decrease in CDCA‐FA flux (CDCA‐FA+LCA+NAC: 8±2).Like CDCA, CDCA‐FA±PiC stimulated IL‐8 release, which was decreased by LCA. Inhibiting RNS, but not ROS, caused ~40% reduction in CDCA‐induced IL‐8 release (ng/ml; CDCA: 4.6±0.5; CDCA+L‐NAME: 2.7±0.1; CDCA+NAC: 4.2±0.3; n=3), suggesting a role for NO in BA‐induced inflammatory process.We demonstrate a novel role for RNS in CDCA‐induced TJ dysfunction in T84 cells. Inhibiting RNS in the presence of LCA reverses CDCA action on leak function and reduces PiC release. Equally important, its roles overlap with but are distinct from ROS. Understanding the role of ROS/RNS in BA action can lead to novel therapeutic strategies for IBD.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Formononetin promotes apoptosis of colorectal cancer cells via activation of mitochondria-dependent MAPK pathway

Purpose: To investigate whether formononetin exhibits antitumor activity in colorectal cancer cell lines via the mitochondria-dependent mitogen-activated protein kinase (MAPK) pathway. Methods: Human colorectal cells were treated with various doses of formononetin for 24 h, followed by Cell Counting Kit-8 (CCK-8) assay and western blot. Human colorectal cells were incubated with equivalent vehicle (DMSO) or 100 μM formononetin for 24 h, followed by nuclear staining with propidium iodide (PI) and diamidino-2-phenylindole (DAPI) for analyses of apoptosis. Human colorectal cells were incubated with equivalent vehicle or 100 μM formononetin for 24 h followed by analysis of cell migration and invasion. Human colorectal cells were incubated with equivalent vehicle (DMSO) or 100 μM formononetin for various duration (3, 6, 12, and 24 h), followed by detection of intracellular reactive oxygen species (ROS) level and measurement of mitochondrial membrane potential (Δψm) to monitor mitochondria functionality. Results: In human colorectal cancer cell lines SW1463 and T84, formononetin (> 20 μM) significantly inhibited cell growth (p < 0.05) in a dose-dependent manner, noticeably induced apoptosis, and suppressed cell migration and invasion. Western blot analysis revealed that formononetin treatment caused significantly increased levels of proapoptotic proteins, and suppression of cell proliferationrelated protein and matrix metallopeptidases (MMP) levels. Formononetin also induced mitochondrial depolarization and ROS generation in a time-dependent manner, indicating that formononetin mediates human colorectal cancer cell apoptosis via activation of MAPK pathway in a dose-dependent manner. Conclusion: Formononetin induces human colorectal cancer cell apoptosis via mitochondriadependent MAPK pathway, thus lending experimental support for the clinical application of formononetin for colorectal cancer therapy. Keywords: Formononetin, Colorectal cancer, Mitochondria, Reactive oxygen species, Cytochrome C, Mitogen-activated protein kinase

Structure and biological activities of a hexosamine-rich cell wall polysaccharide isolated from the probiotic Lactobacillus farciminis.

Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [→6αGlcpNAc1 → 4βManpNAc1 → 4βGlcpNAc1→] highly substituted with single residues of αGlcp, αGalp and αGlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-α/IFN-γ cytokine-mediated epithelium impairment.

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Manipulate Host Autophagy by Impairing SUMOylation.

The intestinal mucosa of Crohn’s disease (CD) patients is abnormally colonized with adherent-invasive Escherichia coli (AIEC) that are able to adhere to and to invade intestinal epithelial cells (IECs), to survive in macrophages, and to induce a pro-inflammatory response. AIEC persist in the intestine, and induce inflammation in CEABAC10 transgenic mice expressing human CAECAM6, the receptor for AIEC. SUMOylation is a eukaryotic-reversible post-translational modification, in which SUMO, an ubiquitin-like polypeptide, is covalently linked to target proteins. Here, we investigated the role of SUMOylation in host responses to AIEC infection. We found that infection with the AIEC LF82 reference strain markedly decreased the levels of SUMO-conjugated proteins in human intestinal epithelial T84 cells. This was also observed in IECs from LF82-infected CEABAC10 transgenic mice. LF82-induced deSUMOylation in IECs was due in part to increased level of microRNA (miR)-18, which targets PIAS3 mRNA encoding a protein involved in SUMOylation. Over-expression of SUMOs in T84 cells induced autophagy, leading to a significant decrease in the number of intracellular LF82. Consistently, a decreased expression of UBC9, a protein necessary for SUMOylation, was accompanied with a decrease of LF82-induced autophagy, increasing bacterial intracellular proliferation and inflammation. Finally, the inhibition of miR-18 significantly decreased the number of intracellular LF82. In conclusion, our results suggest that AIEC inhibits the autophagy response to replicate intracellularly by manipulating host SUMOylation.

Involvement of CYP1B1 in interferon γ-induced alterations of epithelial barrier integrity.

CYP1B1 and CYP1A1 are important extra-hepatic cytochromes, expressed in the colon and involved in the metabolism of dietary constituents and exogenous compounds. CYP1B1 expression is increased by pro-inflammatory cytokines, and it has been recently implicated in regulation of blood brain barrier function. We investigated its involvement in the increased permeability of the intestinal epithelial barrier observed in inflammatory conditions. Epithelial monolayers formed by human T84 colon carcinoma cells cultured on transwells, were disrupted by incubation with IFNγ (10ng·mL-1 ). Monolayer integrity was measured using transepithelial electrical resistance. CYP1A1 and CYP1B1 inhibitors or inducers were applied apically. Potential mechanisms of action were investigated using RT-qPCR. IFNγ disrupts the barrier integrity of the T84 monolayers and increases CYP1B1 and HIF1α mRNA expression. CYP1B1 induction is inhibited by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (100μM) but not by the HIF1α inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (50μM). Inhibition of CYP1B1 with the selective inhibitor 2,4,3',5'-tetramethoxystilbene (100nM) partly reverses the effects of IFNγ on epithelial permeability. These data suggest that increased expression of CYP1B1 is involved in the effects of IFNγ on epithelial permeability. Inhibition of CYP1B1 counteracts the alterations of epithelial barrier integrity induced by IFNγ and could thus have a therapeutic potential in disorders of intestinal permeability associated with inflammation.