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At the blood–brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.

The mechanisms underlying diarrhea-predominant irritable bowel syndrome (IBS-D) are poorly understood, but increased intestinal permeability is thought to contribute to symptoms. A recent clinical trial of gluten-free diet (GFD) demonstrated symptomatic improvement, relative to gluten-containing diet (GCD), which was associated with reduced intestinal permeability in non-celiac disease IBS-D patients. The aim of this study was to characterize intestinal epithelial tight junction composition in IBS-D before and after dietary gluten challenge. Biopsies from 27 IBS-D patients (13 GFD and 14 GCD) were examined by H&E staining and semiquantitative immunohistochemistry for phosphorylated myosin II regulatory light chain (MLC), MLC kinase, claudin-2, claudin-8 and claudin-15. Diet-induced changes were assessed and correlated with urinary mannitol excretion (after oral administration). In the small intestine, epithelial MLC phosphorylation was increased or decreased by GCD or GFD, respectively, and this correlated with increased intestinal permeability (P<0.03). Colonocyte expression of the paracellular Na+ channel claudin-15 was also markedly augmented following GCD challenge (P<0.05). Conversely, colonic claudin-2 expression correlated with reduced intestinal permeability (P<0.03). Claudin-8 expression was not affected by dietary challenge. These data show that alterations in MLC phosphorylation and claudin-15 and claudin-2 expression are associated with gluten-induced symptomatology and intestinal permeability changes in IBS-D. The results provide new insight into IBS-D mechanisms and can explain permeability responses to gluten challenge in these patients.

Disruptions in the intestinal epithelial barrier can result in devastating consequences and a multitude of disease syndromes, particularly among preterm neonates. The association between barrier dysfunction and intestinal dysbiosis suggests that the intestinal barrier function is interactive with specific gut commensals and pathogenic microbes. In vitro and in vivo studies demonstrate that probiotic supplementation promotes significant upregulation and relocalization of interepithelial tight junction proteins, which form the microscopic scaffolds of the intestinal barrier. Probiotics facilitate some of these effects through the ligand-mediated stimulation of several toll-like receptors that are expressed by the intestinal epithelium. In particular, bacterial-mediated stimulation of toll-like receptor-2 modulates the expression and localization of specific protein constituents of intestinal tight junctions. Given that ingested prebiotics are robust modulators of the intestinal microbiota, prebiotic supplementation has been similarly investigated as a potential, indirect mechanism of barrier preservation. Emerging evidence suggests that prebiotics may additionally exert a direct effect on intestinal barrier function through mechanisms independent of the gut microbiota. In this review, we summarize current views on the effects of pro- and prebiotics on the intestinal epithelial barrier as well as on non-epithelial cell barrier constituents, such as the enteric glial cell network. Through continued investigation of these bioactive compounds, we can maximize their therapeutic potential for preventing and treating gastrointestinal diseases associated with impaired intestinal barrier function and dysbiosis.

Background Airway epithelial barrier function is maintained by the formation of tight junctions (TJs) and adherens junctions (AJs). Inhalation of cigarette smoke causes airway epithelial barrier dysfunction and may contribute to the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). We assessed the effects of cigarette smoke on barrier function and expression of multiple TJ and AJ proteins in the bronchial epithelium. We also examined whether treatment with glucocorticosteroids (GCSs), long-acting β 2 -agonists (LABAs), and human cathelicidin LL-37 can protect against cigarette smoke extract (CSE)-induced barrier dysfunction. Methods Calu-3 cells cultured at the air-liquid interface were pretreated with or without GCSs, LABAs, GCSs plus LABAs, or LL-37, and subsequently exposed to CSE. Barrier function was assessed by transepithelial electronic resistance (TEER) measurements. Gene and protein expression levels of TJ and AJ proteins were analyzed by quantitative PCR and western blotting, respectively. Immunofluorescence staining of TJ and AJ proteins was performed. Results CSE decreased TEER and increased permeability in a concentration-dependent manner. CSE suppressed gene expression of claudin-1, claudin-3, claudin-4, claudin-7, claudin-15, occludin, E-cadherin, junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1) within 12 h post-CSE exposure, while suppressed protein expression levels of occludin at 12 h. CSE-treated cells exhibited discontinuous or attenuated immunostaining for claudin-1, claudin-3, claudin-4, occludin, ZO-1, and E-cadherin compared with untreated cells. GCS treatment partially restored CSE-induced TEER reduction, while LABA treatment had no effect. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction and gene suppression of TJ and AJ proteins. Human cathelicidin LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. LL-37 also attenuated CSE-induced decreases in gene and protein expression levels of occludin. Conclusions CSE caused airway epithelial barrier dysfunction and simultaneously downregulated multiple TJ and AJ proteins. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction. LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. Use of LL-37 to counteract airway epithelial barrier dysfunction may have significant benefits for respiratory diseases such as asthma and COPD.

Microplastics, which are tiny plastic particles less than 5 mm in diameter, are widely present in the environment, have become a serious threat to aquatic life and human health, potentially causing ecosystem disorders and health problems. The present study aimed to investigate the effects of microplastics, specifically microplastics-polystyrene (MPs-PS), on the structural integrity, gene expression related to tight junctions, and gut microbiota in mice. A total of 24 Kunming mice aged 30 days were randomly assigned into four groups: control male (CM), control female (CF), PS-exposed male (PSM), and PS-exposed female (PSF)(n = 6). There were significant differences in villus height, width, intestinal surface area, and villus height to crypt depth ratio (V/C) between the PS group and the control group(C) (p <0.05). Gene expression analysis demonstrated the downregulation of Claudin-1 , Claudin-2 , Claudin-15 , and Occludin , in both duodenum and jejunum of the PS group (p < 0.05). Analysis of microbial species using 16S rRNA sequencing indicated decreased diversity in the PSF group, as well as reduced diversity in the PSM group at various taxonomic levels. Beta diversity analysis showed a significant difference in gut microbiota distribution between the PS-exposed and C groups (R2 = 0.113, p<0.01), with this difference being more pronounced among females exposed to MPs-PS. KEGG analysis revealed enrichment of differential microbiota mainly involved in seven signaling pathways, such as nucleotide metabolism(p<0.05). The relative abundance ratio of transcriptional pathways was significantly increased for the PSF group (p<0.01), while excretory system pathways were for PSM group(p<0.05). Overall findings suggest that MPs-PS exhibit a notable sex-dependent impact on mouse gut microbiota, with a stronger effect observed among females; reduced expression of tight junction genes may be associated with dysbiosis, particularly elevated levels of Prevotellaceae .

Blood-brain barrier (BBB) dysfunction is associated with worse epilepsy outcomes however the underlying molecular mechanisms of BBB dysfunction remain to be elucidated. Tight junction proteins are important regulators of BBB integrity and in particular, the tight junction protein claudin-5 is the most enriched in brain endothelial cells and regulates size-selectivity at the BBB. Additionally, disruption of claudin-5 expression has been implicated in numerous disorders including schizophrenia, depression and traumatic brain injury, yet its role in epilepsy has not been fully deciphered. Here we report that claudin-5 protein levels are significantly diminished in surgically resected brain tissue from patients with treatment-resistant epilepsy. Concomitantly, dynamic contrast-enhanced MRI in these patients showed widespread BBB disruption. We show that targeted disruption of claudin-5 in the hippocampus or genetic heterozygosity of claudin-5 in mice exacerbates kainic acid-induced seizures and BBB disruption. Additionally, inducible knockdown of claudin-5 in mice leads to spontaneous recurrent seizures, severe neuroinflammation, and mortality. Finally, we identify that RepSox, a regulator of claudin-5 expression, can prevent seizure activity in experimental epilepsy. Altogether, we propose that BBB stabilizing drugs could represent a new generation of agents to prevent seizure activity in epilepsy patients. The mechanisms underlying epilepsy development are not well understood. Here the authors show that loss of a key component of the so called blood-brain barrier drives seizures in mice and is also lost in humans with treatment resistant epilepsy

Background: Chronic kidney disease (CKD) impairs intestinal barrier function which leads to endotoxemia and systemic inflammation. We have found depletion of intestinal epithelial tight junction (TJ) proteins in animals with CKD. We further showed that addition of end-stage renal disease patients’ plasma to the culture medium provokes a marked drop in transepithelial electrical resistance (TER) and depletion of TJ proteins in cultured human enterocytes. These effects were less severe with post- than with prehemodialysis plasma, suggesting a role of dialyzable agent(s). This study tested the hypothesis that intestinal barrier dysfunction in uremia may be due to diffusion of urea into the gut and its conversion to ammonia by microbial urease. Methods: Human enterocytes (T84 cells) were seeded on Transwell plates and utilized when TER exceeded 1,000 mΩ·cm 2 to ensure full polarization and TJ formation. Confluent cells were then incubated for 24 h in media containing 0, 42 or 74 mg/dl urea or urea plus urease to simulate presence of microbial flora. Results: At clinically relevant concentrations, urea caused a concentration-dependent fall in TER and the key TJ proteins claudin-1, occludin and zonula occludens 1. The effects of urea were dramatically amplified by urease causing cell detachment, dissipation of TER, and massive loss of TJ proteins. Conclusions: Uremia-induced disruption of intestinal TJ and barrier function is, in part, mediated by urea which is generally considered to be a nontoxic retained metabolite. These findings reveal a novel mechanism for a salutary effect of urea-lowering strategies, e.g. low-protein diet and longer and more frequent dialysis regimens in advanced CKD.

The intestinal epithelial barrier, composed of epithelial cells, tight junction proteins and intestinal secretions, prevents passage of luminal substances and antigens through the paracellular space. Dysfunction of the intestinal barrier integrity induced by toxins and pathogens is associated with a variety of gastrointestinal disorders and diseases. Although butyrate is known to enhance intestinal health, its role in the protection of intestinal barrier function is poorly characterized. Therefore, we investigated the effect of butyrate on intestinal epithelial integrity and tight junction permeability in a model of LPS-induced inflammation in IPEC-J2 cells. Butyrate dose-dependently reduced LPS impairment of intestinal barrier integrity and tight junction permeability, measured by trans-epithelial electrical resistance (TEER) and paracellular uptake of fluorescein isothiocyanate-dextran (FITC-dextran). Additionally, butyrate increased both mRNA expression and protein abundance of claudins-3 and 4, and influenced intracellular ATP concentration in a dose-dependent manner. Furthermore, butyrate prevented the downregulation of Akt and 4E-BP1 phosphorylation by LPS, indicating that butyrate might enhance tight junction protein abundance through mechanisms that included activation of Akt/mTOR mediated protein synthesis. The regulation of AMPK activity and intracellular ATP level by butyrate indicates that butyrate might regulate energy status of the cell, perhaps by serving as a nutrient substrate for ATP synthesis, to support intestinal epithelial barrier tight junction protein abundance. Our findings suggest that butyrate might protect epithelial cells from LPS-induced impairment of barrier integrity through an increase in the synthesis of tight junction proteins, and perhaps regulation of energy homeostasis.

The blood–brain barrier (BBB) is a functional interface that provides selective permeability, protection from toxic substances, transport of nutrients, and clearance of brain metabolites. Additionally, BBB disruption has been shown to play a role in many neurodegenerative conditions and diseases. Therefore, the aim of this study was to establish a functional, convenient, and efficient in vitro co-cultured BBB model that can be used for several physiological conditions related to BBB disruption. Mouse brain-derived endothelial (bEnd.3) and astrocyte (C8-D1A) cells were co-cultured on transwell membranes to establish an intact and functional in vitro model. The co-cultured model and its effects on different neurological diseases and stress conditions, including Alzheimer’s disease (AD), neuroinflammation, and obesity, have been examined by transendothelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran, and tight junction protein analyses. Scanning electron microscope images showed evidence of astrocyte end-feet processes passing through the membrane of the transwell. Moreover, the co-cultured model showed effective barrier properties in the TEER, FITC, and solvent persistence and leakage tests when compared to the mono-cultured model. Additionally, the immunoblot results showed that the expression of tight junction proteins such as zonula occludens-1 (ZO-1), claudin-5, and occludin-1 was enhanced in the co-culture. Lastly, under disease conditions, the BBB structural and functional integrity was decreased. The present study demonstrated that the co-cultured in vitro model mimicked the BBB’s structural and functional integrity and, under disease conditions, the co-cultured model showed similar BBB damages. Therefore, the present in vitro BBB model can be used as a convenient and efficient experimental tool to investigate a wide range of BBB-related pathological and physiological studies.

This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.