Distribution Of Tight Junction Proteins Research Articles

Hypoxia and heat stress affect epithelial integrity in a Caco-2/HT-29 co-culture

Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers

BackgroundThe blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group’s previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC.MethodsWe used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC’s impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC’s effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5.ResultsOur data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data.ConclusionIn agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

Protective Effects of Baicalin on Peritoneal Tight Junctions in Piglets Challenged with Glaesserella parasuis

Glaesserella parasuis (G. parasuis) causes inflammation and damage to piglets. Whether polyserositis caused by G. parasuis is due to tight junctions damage and the protective effect of baicalin on it have not been examined. Therefore, this study aims to investigate the effects of baicalin on peritoneal tight junctions of piglets challenged with G. parasuis and its underlying molecular mechanisms. Piglets were challenged with G. parasuis and treated with or without baicalin. RT-PCR was performed to examine the expression of peritoneal tight junctions genes. Immunofluorescence was carried out to detect the distribution patterns of tight junctions proteins. Western blot assays were carried out to determine the involved signaling pathways. Our data showed that G. parasuis infection can down-regulate the tight junctions expression and disrupt the distribution of tight junctions proteins. Baicalin can alleviate the down-regulation of tight junctions mRNA in peritoneum, prevent the abnormalities and maintain the continuous organization of tight junctions. Our results provide novel evidence to support that baicalin has the capacity to protect peritoneal tight junctions from G. parasuis-induced inflammation. The protective mechanisms of baicalin could be associated with inhibition of the activation of PKC and MLCK/MLC signaling pathway. Taken together, these data demonstrated that baicalin is a promising natural agent for the prevention and treatment of G. parasuis infection.

Quercetin protects retina external barrier from oxidative stress injury by promoting autophagy

Purpose: This study aimed to investigate the protective effects of quercetin on the tight junction proteins of human retinal pigment epithelial cells (ARPE-19 cells) suffering from oxidative stress injury and explore the possible mechanism. Methods: H2O2 (300 μM) was used to establish an oxidative stress model of ARPE-19 cells. ARPE-19 cells were pretreated with different concentrations (0–80 μM) of quercetin before H2O2 exposure. The expression and distribution of tight junction proteins and autophagy-related proteins were detected by Western blot and immunostaining. ARPE-19 cells were pretreated with 5 mM 3-methyladenine (3-MA). Results: The cell viability weakened in the H2O2 group compared with the control group. However, it was preserved after pretreatment with quercetin. It was observed that the expression levels of occludin, claudin-1 were decreased in the H2O2 group. Quercetin treatment significantly enhanced the expression levels of them as compared to the H2O2 group. H2O2 alone strongly decreased the Zonula occludens protein 1 (ZO-1) expression in the cytomembrane. Quercetin supplementation enhanced the accumulation of ZO-1 in ARPE-19 cells. The expression levels of Beclin-1 and Microtubule associated protein light chain 3 II (LC-3II) increased, and that of P62 decreased in the quercetin protection group. The appearance of LC-3II, which examined by immunofluorescence experiments, enhanced in the quercetin protection group as compared with the control group. The expression levels of beclin-1 and LC-3II increased, and that of P62 increased in the autophagy-inhibited group compared with the quercetin protection group. The levels of occludin and claudin-1 also decreased. Conclusion: Quercetin prevents the loss of tight junction proteins by upregulating autophagy after oxidative stress in ARPE-19 cells.

Sodium houttuyfonate enhances the intestinal barrier and attenuates inflammation induced by Salmonella typhimurium through the NF-κB pathway in mice

Salmonella typhimurium (ST), as an aggressive bacterium, mainly causes intestinal inflammation and diarrhea. Sodium houttuyfonate (SH) is a derivative of houttuynin in the active oil of Houttuynia cordata, which is stable in nature and has anti-inflammatory activity. In this study, we used BALB/c mice infected with ST as experimental subjects and aimed to study the regulatory effect of SH on the intestinal tract and to explain its anti-inflammatory mechanism. Compared with the ST group, SH treatment improved the morphology of jejunum mucosa and alleviated the pathological damage to colon tissue. In addition, SH protected the intestinal barrier by regulating the localization and distribution of tight junction proteins. Meanwhile, SH significantly decreased the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and inflammation-related enzymes (iNOS, COX-2). Moreover, further western blot results suggested that SH inhibited the expression of p-IκBα and p-p65 in intestinal tissues. These results demonstrated that SH maintained the intestinal barrier and attenuated the production of intestinal proinflammatory cytokines by regulating the NF-κB signaling pathway, thereby providing protection for the intestine.

Bacteria Remediate the Effects of Food Additives on Intestinal Function in an in vitro Model of the Gastrointestinal Tract.

As the site of nutrient absorption, the small intestine is continuously exposed to preservatives and additives present in consumed food. While the effects of diet on the lower gastrointestinal tract are widely studied, the effects of food additives on the small intestinal epithelium and microbiota are less clearly understood. The goal of this work was to develop and establish a physiologically relevant model of the upper gastrointestinal tract to study the complex interactions between food additives, individual bacterial species, and intestinal function. To achieve this, an in vitro model incorporating simulated digestion, human intestinal epithelial cells, and the commensal, Gram-positive Lactobacillus rhamnosus, or the opportunistic, Gram-negative Escherichia coli was developed. This model was used to assess intestinal permeability and alkaline phosphatase activity following exposure to high glucose (HG), salt, emulsifier (TWEEN 20), food (milk chocolate candies) or chemical grade titanium dioxide nanoparticles (TiO2-NP), and food (whole wheat bread) or chemical grade gluten. It was found that HG increased intestinal permeability, the presence of bacteria remediated the negative effects of HG on intestinal permeability, and a decrease in permeability and IAP activity was observed with increasing concentration of TWEEN 20 both in the presence and absence of bacteria. While L. rhamnosus influenced the activity of intestinal alkaline phosphatase and tight junction protein distribution, E. coli produced indole to reinstate intestinal permeability. The source of TiO2 and gluten led to altered impacts on permeability and IAP activity. The growth of E. coli and L. rhamnosus was found to depend on the type of food additive used. Overall, the presence of bacteria in the in vitro model influenced the effects of food additives on intestinal function, suggesting a complex association between diet and upper GI microbiota. This model provides a method to study small intestinal function and host-microbe interactions in vitro in both healthy and diseased conditions.

Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles

Cognitive impairment is a well-known complication of diabetes mellitus (DM). Microvascular compromise was described one DM complication. Recently we showed blood brain barrier (BBB) permeability and memory loss are associated with diminution of tight junctions (TJ) in brain endothelium and pericyte coverage and inflammation in cerebral microvessels and brain tissue paralleling hyperglycemia in mice of both DM types. The current study demonstrates that exposure of brain microvessels to hyperglycemic conditions or advanced glycation end products (AGEs) ex vivo resulted in significant abnormalities in membranous distribution of TJ proteins. We found significant increase in the amount of extracellular vesicles (EVs) isolated from DM mice and enhanced presence of TJ proteins, occludin and claudin-5, on EVs. Exposure of BMVECs to high glucose and AGEs led to significant augmentation of ICAM and VCAM expression, elevated leukocyte adhesion to and migration across BMVEC monolayers, and increased BBB permeability in vitro. Pericytes exposed to hyperglycemia and AGEs displayed diminished expression of integrin α1, PDGF-R1β and connexin-43. Our findings indicate BBB compromise in DM ex vivo, in vitro and in vivo models in association with BMVEC/pericyte dysfunction and inflammation. Prevention of BBB injury may be a new therapeutic approach to avert cognitive demise in DM.

In Vitro Evaluation of the Protective Role of Lactobacillus StrainsAgainst Inorganic Arsenic Toxicity.

Inorganic arsenic [iAs, As(III)+ As(V)] is considered a human carcinogen. Recent studies show that it has also toxic effects on the intestinal epithelium which might partly explain its systemic toxicity. The aim of this study is to evaluate the protective role of lactic acid bacteria (LAB) against the toxic effects of iAs on the intestinal epithelium. For this purpose, the human colonic cells Caco-2 were exposed to As(III) in the presence of various LAB strains or their conditioned medium. Results showed that some strains and their conditioned media partially revert the oxidative stress, the production of pro-inflammatory cytokines, the alterations of the distribution of tight junction proteins, and the cell permeability increases caused by As(III). These results show that both soluble factors secreted or resulting from LAB metabolism and cell-cell interactions are possibly involved in the beneficial effects. Therefore, some LAB strains have potential as protective agents against iAs intestinal barrier disruption.

EPA and DHA attenuate deoxynivalenol-induced intestinal porcine epithelial cell injury and protect barrier function integrity by inhibiting necroptosis signaling pathway.

Deoxynivalenol (DON) is one of the most common mycotoxins that contaminates food or feed and cause intestinal damage. Long-chain n-3 polyunsaturated fatty acids (PUFA) such as EPA and DHA exert beneficial effects on intestinal integrity in animal models and clinical trials. Necroptosis signaling pathway plays a critical role in intestinal cell injury. This study tested the hypothesis that EPA and DHA could alleviate DON-induced injury to intestinal porcine epithelial cells through modulation of the necroptosis signaling pathway. Intestinal porcine epithelial cell 1 (IPEC-1) cells were cultured with or without EPA or DHA (6.25-25μg/mL) in the presence or absence of 0.5μg/mL DON for indicated time points. Cell viability, cell number, lactate dehydrogenase (LDH) activity, cell necrosis, transepithelial electrical resistance (TEER), fluorescein isothiocyanate-labeled dextran4kDa (FD4) flux, tight junction protein distribution, and protein abundance of necroptosis related signals were determined. EPA and DHA promoted cell growth indicated by higher cell viability and cell number, and inhibited cell injury indicated by lower LDH activity in the media. EPA and DHA also improved intestinal barrier function, indicated by higher TEER and lower permeability of FD4 flux as well as increased proportions of tight junction proteins located in the plasma membrane. Moreover, EPA and DHA decreased cell necrosis demonstrated by live cell imaging and transmission electron microscopy. Finally, EPA and DHA downregulated protein expressions of necroptosis related signals including tumor necrosis factor receptor (TNFR1), receptor interacting protein kinase 1 (RIP1), RIP3, phosphorylated mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase family 5 (PGAM5), dynamin-related protein 1 (Drp1), and high mobility group box-1 protein (HMGB1). EPA and DHA also inhibited protein expression of caspase-3 and caspase-8. These results suggest that EPA and DHA prevent DON-induced intestinal cell injury and enhance barrier function, which is associated with inhibition of the necroptosis signaling pathway.

Protective Effect of Naringin on In Vitro Gut-Vascular Barrier Disruption of Intestinal Microvascular Endothelial Cells Induced by TNF-α.

Naringin is a polymethoxylated flavonoid commonly found in citrus species and has therapeutic potential in intestinal disorders. However, the effect and mechanism of naringin on gut-vascular barrier disruption has not yet been reported. This study aimed to investigate the distinguishing and selectively protective effects of naringin on tumor necrosis factor (TNF)-α-induced gut-vascular barrier disruption and elucidate the potential mechanism. In the present study, an in vitro gut-vascular barrier model composed of rat intestinal microvascular endothelial cells (RIMVECs) was studied. Evans blue-albumin efflux assay showed that naringin (50 μM) evidently protected the integrity of RIMVEC monolayer barriers against TNF-α-induced disruption. Naringin maintained the expression and distribution of tight junction proteins including zona occludin-1, occludin, claudin-1, and claudin-2. Additionally, naringin protected RIMVECs from TNF-α-induced apoptosis and cell migration suppression (41.1 ± 2.2 vs 51.1 ± 3.5%; 61.0 ± 5.1 vs 72.2 ± 6.2%). Our results indicate that naringin effectively ameliorates gut-vascular barrier disruption.

Time-dependent alteration to the tight junction structure of distal intestinal epithelia in type 2 prediabetic mice

AimHigh-fat diet (HFD) intake has been associated with changes in intestinal microbiota composition, increased intestinal permeability, and onset of type 2 diabetes mellitus (T2DM). The aim of this work was twofold: 1) to investigate the structural and functional alterations of the tight junction (TJ)-mediated intestinal epithelial barrier of ileum and colon, that concentrate most of the microbiota, after exposure to a HFD for 15, 30 and 60 days, and 2) to assess the effect of in vitro exposure to free fatty acids (FFAs), one of the components of HFD, on paracellular barrier of colon-derived Caco-2 cells. Methods/key findingsHFD exposure induced progressive metabolic changes in male mice that culminated in prediabetes after 60d. Morphological analysis of ileum and colon mucosa showed no signs of epithelial rupture or local inflammation but changes in the junctional content/distribution and/or cellular content of TJ-associated proteins (claudins-1, -2, -3, and occludin) in intestinal epithelia were seen mainly after a prediabetes state has been established. This impairment in TJ structure was not associated with significant changes in intestinal permeability to FITC-dextran. Exposure of Caco-2 monolayers to palmitic or linoleic acids seems to induce a reinforcement of TJ structure while treatment with oleic acid had a more diverse effect on TJ protein distribution. SignificanceTJ structure in distal intestinal epithelia can be specifically impaired by HFD intake at early stage of T2DM, but not by FFAs in vitro. Since the TJ change in ileum/colon was marginal, probably it does not contribute to the disease onset.

Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction

BackgroundEnteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers.ResultsHere we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level.ConclusionThis study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

Creating an Intestinal Epithelial Cell Culture Model to Screen Novel Phytochemical Therapies for Postmenopausal Women (FS09-05-19)

ObjectivesThe intestinal epithelium serves as barrier maintained by tight junction (TJ) proteins. TJ integrity is compromised by release of matrix-metalloproteases (MMPs). Degradation of TJ proteins may lead to increased permeability of the small intestine. Previous research has shown that MMPs are up-regulated with estrogen deficiency. In postmenopausal women, there may be a causal relationship between intestinal permeability and inflammation-related disease. The purpose of this project is to create an in vitro cell culture model, representative of the estrogen-deficient small intestine in vivo, to screen phytochemicals that may attenuate increased intestinal permeability observed with a decline in estrogen levels. MethodsHuman colorectal adenocarcinoma (CaCo-2) cells seeded on 12 mm Transwell® inserts were co-cultured with 3T3 Swiss fibroblasts, a major source of MMPs in the intestines, in 12-well plates. Cells were pre-treated with 10 μM universal estrogen receptor inhibitor (ICI 181,780; ABCAM, Boston, MA) for 1 hour, then treated with 10 nM estrogen (17 β-Estradiol; Sigma-Aldrich, St. Louis, MO) with or without MMP-9 Inhibitor (MMP-9 Inhibitor I; Millipore Sigma, Burlington, MA). Transepithelial electrical resistance (TER) was measured at 24, 48, and 72 hours following treatment. Confocal immunocytochemistry was used to determine the expression and distribution of TJ proteins. Differences between treatment groups were analyzed via ANOVA using SAS 9.3. ResultsInhibition of the estrogen receptors resulted in downregulation of TJ proteins in this model, suggesting a decline in intestine epithelial barrier integrity. MMP 9 inhibition concomitant with suppression of estrogen signaling resulted in increased TER values. ConclusionsMMP 9 inhibition when estrogen signaling is blocked enhances the intestinal epithelial barrier integrity. Future work will use this model to identify food components, especially those known to inhibit MMP 9, that may attenuate the decline in intestine epithelial barrier integrity observed with estrogen deficiency. Our long-term goal is to identify nutrition-related therapeutic targets to decrease the various inflammation-related comorbidities associated with estrogen deficiency, thereby improving the health of postmenopausal women. Funding SourcesCollege of Allied Health Student Research and Creativity Grant.

Cranberry Juice Phytochemicals Attenuated Ethanol-Induce Epithelial Barrier Dysfunction on Caco-2 Monolayers (P06-094-19)

ObjectivesThe objective of this study was to determine whether cranberry juice phytochemicals protect intestinal barrier using Caco-2 monolayer models, and to elucidate the underlying mechanisms of cranberry juice phytochemicals on ethanol-induced barrier dysfunction. MethodsCranberry juice phytochemicals were extracted using Amberlite FPX66 resin and phytochemicals composition were analyzed on HPLC. Caco-2 monolayers were incubated with cranberry juice phytochemicals at different concentrations for 2 hours before treatment with 4.5% ethanol. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-labeled dextran (FITC) was used to evaluate paracellular permeability. Epithelial integrity and distribution of tight junction proteins (Occludin and ZO-1) in Caco-2 monolayers were determined by immunofluorescence labeling method. Protein levels of ZO-1, Occludin and Claudin-1 were determined by immunoblotting. ResultsCranberry juice phytochemicals consisted of malvidin 3,5-diglucoside, petunidin 3-glucoside, chlorogenic acid and caffeic acid. Ethanol induced decrease of TEER while pretreatment of cranberry juice phytochemicals protected Caco-2 monolayer integrity in a dose-dependent manner. Monolayers treated with 7.5, 15, and 30 μg/mL of cranberry juice phytochemicals had TEER value 11.6%, 22.7% and 30.5% higher than the ethanol-treatment monolayers, respectively. This was accompanied by 40%, 55%, and 50% decreases of the paracellular permeability compared to ethanol-treated monolayers. Immunofluorescent analysis showed that occludin was localized tightly around cell membrane and colocalize with ZO-1 in the normal Caco-2 monolayers. Exposure to 4.5% ethanol disrupted both occludin and ZO-1 networks and resulted in gap formation among the cells. Cranberry juice phytochemicals attenuated these changes induced by ethanol exposure. Ethanol exposure significantly reduced the protein levels of occludin and ZO-1. Pretreatment with cranberry juice phytochemicals increased the protein levels of occludin dose dependently but did not affect ZO-1 level. ConclusionsCurrent study suggested that cranberry juices phytochemicals attenuate ethanol-induced intestinal barrier dysfunction in part by enhancing the tight junction assemblies. Funding SourcesNA.

CCAT1 lncRNA Promotes Inflammatory Bowel Disease Malignancy by Destroying Intestinal Barrier via Downregulating miR-185-3p.

The long noncoding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) has been reported to play a vital role in the development of cancer. Although the link between inflammation and cancer initiation is well established, whether CCAT1 is involved in inflammation and promotes inflammatory bowel disease (IBD) malignancy remains undetermined. We aimed to investigate the expression of CCAT1 in IBD and the effect of CCAT1 overexpression on intestinal epithelial barrier function. The relationship between CCAT1 and the inflammation-related pathway was analyzed in both colorectal cancer (CRC) and IBD patients. Gene expression was detected by real-time polymerase chain reaction and Western blot. Transepithelial electrical resistance (TEER) and FD-4 flux measurement were used to test the effect of CCAT1 and miR-185-3p on intestinal epithelial barrier function. Luciferase assay was performed to validate the target site of miR-185-3p on 3'-UTR of MLCK mRNA. Gene set enrichment analysis revealed that several inflammation-related genes were enriched in the CCAT1 high-expressed group of CRC patients. The relationship between CCAT1 and inflammation activation in IBD patients was further confirmed. CCAT1 expression positively correlated with MLCK, which acts as a protein kinase to phosphorylate myosin light chain and induces tight junction protein distribution, whereas it was negatively correlated with miR-185-3p in IBD tissues. We also determined that CCAT1 overexpression increased Caco-2 monolayer permeability and upregulated MLCK. Furthermore, CCAT1-induced MLCK overexpression and IBD disease progression were significantly attenuated by miR-185-3p. The CCAT1/miR-185-3p/MLCK signaling pathway is strongly activated to destroy barrier function and promotes the pathogenesis of IBD.

Abstract 2902: Targeting the tight junction protein claudin-1 in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery, but the prognosis of patients with metastatic disease remains very poor. The current treatment of metastatic CRC (mCRC) relies on therapy combining chemotherapy and targeted therapies. However, relapses are observed in most cases due to the occurrence of drug resistance. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies. We previously showed that Claudin-1 (CLDN1), a major constituent of tight junctions, is overexpressed at the membrane of CRC cells which makes it a good target for antibodies. Besides, CLDN1 is differentially expressed in the new CRC molecular subtypes. We have then developed a monoclonal antibody (mAb) against CLDN1, called 6F6, which targets the extracellular part of CLDN1. Results showed that the 6F6 mAb significantly decreased cell growth, survival, and migration in vitro as well as in vivo. Furthermore, we demonstrated the cytostatic effect of the 6F6 mAb through proliferation assays. We are now deciphering the role of CLDN1 on 3D spheroids, a model that reflects the physiopathology. Using immunofluorescence staining on frozen sections we are exploring the impact of CLDN1 on spheroids proliferation. We are also investigating on the distribution of tight junction proteins implicated on cell proliferation in the presence or absence of CLDN1. CLDN1 is a new promising therapeutic target in CRC, it is now interesting to elucidate its role in colorectal cancer cell proliferation. Keywords: Colorectal, Cancer, Claudin-1, monoclonal antibodies, spheroids, proliferation Citation Format: Sara Cherradi, Adeline Ayrolles-Torro, Nadia Vezzo-Vié, Eve Combes, Lucile Canterel-Thouennon, Fabien Gava, Valérie Lobjois, Bernard Ducommun, Celine Gongora, Maguy Del Rio. Targeting the tight junction protein claudin-1 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2902.

Lactobacillus plantarum ameliorates tumour necrosis factor-induced bacterial translocation in Caco-2 cells by regulation of TLR4 expression.

Purpose. Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis. In inflammatory conditions, commensal bacteria exploit transcytotic pathways to cross the intestinal epithelium in a TLR4-dependent manner. The aim of this study was to test the hypothesis that Lactobacillus plantarum ameliorates tumour necrosis factor-induced bacterial translocation by regulation of Toll-like receptor-4 expression.Methodology. L. plantarum strains were investigated to determine their capacity to inhibit the initial adhesion of Escherichia coli B5 to Caco-2 cells. The inhibitory effects of L. plantarum on TNF-α-induced E. coli B5 translocation across Caco-2 cells were studied. Barrier function and integrity were simultaneously assessed by transepithelial electrical resistance, HRP permeability, LDH release and distribution of tight junctional proteins. Expression of TLR4 was assessed by RT-PCR.Results/Key findings. Pretreatment of monolayers with L. plantarum L2 led to a significant decrease in E. coli B5 adhesion and cell internalization (P<0.01). Exposure to TNF-α for six hours caused a significant increase in E. coli B5 translocation across Caco-2 cells, which was uncoupled from increases in paracellular permeability and disruption of tight junction proteins. Manipulations that induced bacterial translocation were associated with a marked increase in TLR4 mRNA expression and IL-8 secretion. L. plantarum L2 significantly abrogated TNF-α-induced bacterial translocation of E. coli B5, and also downregulated expression of TLR4 and IL-8 in intestinal epithelial cells.Conclusion. Live L. plantarum L2 can inhibit TNF-α-induced transcellular bacterial translocation via regulation of TLR4 expression.

Brain-derived neurotrophic factor modulates intestinal barrier by inhibiting intestinal epithelial cells apoptosis in mice.

We aimed to investigate the effects of brain-derived neurotrophic factor (BDNF) on apoptosis of intestinal epithelial cells (IECs) and alterations of intestinal barrier integrity using BDNF knock-out mice model. Colonic tissues from BDNF(+/+) mice and BDNF(+/-) mice were prepared for this study. The integrity of colonic mucosa was evaluated by measuring trans-mucosa electrical resistance and tissue conductance in Ussing chamber. The colonic epithelial structure was analyzed by transmission electron microscopy. Apoptosis involvement was determined with TUNEL staining, active caspase-3 immunostaining and Western blotting for the protein expression of active caspase-3, Bax and Bcl-2. The expression levels and distribution of tight junction proteins were evaluated by immunohistochemistry or Western blots. Compared with BDNF(+/+) mice, BDNF(+/-) mice displayed impaired integrity and ultrastructure alterations in their colonic mucosa, which was characterized by diminished microvilli, mitochondrial swelling and epithelial cells apoptosis. Altered intestinal barrier function was linked to excessive apoptosis of IECs demonstrated by the higher proportion of TUNEL-positive apoptotic cells and enhanced caspase activities in BDNF(+/-) mice. Increased expression of Bax and claudin-2 proteins and reduced Bcl-2 and tight junction proteins (occludin, ZO-1 and claudin-1) expression were also detected in the colonic mucosa of BDNF(+/-) mice. BDNF may play a role in the maintenance of intestinal barrier integrity via its anti-apoptotic properties.

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A.

Shuganyin decoction improves the intestinal barrier function in a rat model of irritable bowel syndrome induced by water-avoidance stress

BackgroundTo determine the effect of Shuganyin decoction (SGD) on the intestinal barrier function in an irritable bowel syndrome (IBS) rat model induced by water-avoidance stress.MethodsForty male Wistar rats were divided into control, water-avoidance stress (WAS) group, WAS plus Shuganyin decoction (SGD) group and WAS plus dicetel (Dicetel) group. IBS was induced in rats by subjecting them to water-avoidance stress for 7 days. On day 4 of the WAS protocol, the rats were treated for 7 consecutive days (days 4–11) with SGD, dicetel or a negative control (saline). The number of feces granules, histopathological changes of the intestine and mast cell (MC) morphometry were determined. Intestinal permeability was approximated by measuring the absorption of FITC-dextran 4400 (FD-4) from the lumen into the bloodstream in vivo and in vitro experiments. Also, the expression of protease active receptor-2 (PAR-2) and tumor necrosis factor-α (TNF-α) was estimated using immunohistochemical staining and ELISA, respectively. Tight junction (TJ) protein abundance was measured following a quantitative immunofluorescent analysis of intestinal sections and western blotting.ResultsIn vivo, WAS elicited a significantly increase in the transfer of FD-4 from the intestine to blood about threefold in 30 min compared with control group. After treated with SGD, the intestinal permeability to FD-4 of WAS-induced rats was significantly attenuated (P < 0.05). In vitro, the permeability coefficient (Papp) values were measured for FD-4 absorption across the excised intestine. WAS was shown to increase the intestinal permeability to (4.695 ± 0.3629) × 10−7 cm/s in 120 min, which was 2.6-fold higher than the control group. Rats treated with SGD showed a significant decrease in Papp values of FD-4 as compared to WAS group (P < 0.05). Furthermore, by immunofluorescent detection we found that WAS elicited the irregular distribution of TJ proteins. Using the quantitative analysis software of the medical image, the average optical density and protein abundance of TJ proteins was shown to be lower in the WAS group as compared to control group, (P < 0.05). SGD could attenuate this response and improve TJ distribution (P < 0.05). Western blot analysis confirmed that TJ protein abundance was significantly decreased in WAS group and that they could be returned to control levels following an SGD treatment. WAS also induced an increase in number of MCs, their area and diameter as compared to controls. These observations were attenuated with an SGD or dicetel treatment. Similarly, the expression of PAR-2 and TNF-α exceeded control values in the WAS group and were shown to be successfully attenuated with an SGD treatment.ConclusionWAS-induced IBS rat model exhibited intestinal barrier dysfunction, which was manifested as tight junction damage and structural rearrangements that increased the intestinal permeability. Under these conditions, MCs were activated and degranulated in the intestinal mucosa leading to the activation of PAR-2. Our data showed that SGD could inhibit the activation of MCs and down-regulate the expression of both PAR-2 and TNF-α. In turn, this was shown to improve the expression and structural arrangement of TJ proteins in the intestinal mucosa, thereby regulating the intestinal permeability. It was concluded that Shuganyin could protect the intestinal barrier.