The epithelial barrier serves as a critical defense mechanism separating the human body from the external environment, fulfilling both physical and immune functions. This barrier plays a pivotal role in shielding the body from environmental risk factors such as allergens, pathogens, and pollutants. However, since the 19th century, the escalating threats posed by environmental pollution, global warming, heightened usage of industrial chemical products, and alterations in biodiversity have contributed to a noteworthy surge in allergic disease incidences. Notably, allergic diseases frequently exhibit dysfunction in the epithelial barrier. The proposed epithelial barrier hypothesis introduces a novel avenue for the prevention and treatment of allergic diseases. Despite increased attention to the role of barrier dysfunction in allergic disease development, numerous questions persist regarding the mechanisms underlying the disruption of normal barrier function. Consequently, this review aims to provide a comprehensive overview of the epithelial barrier\'s role in allergic diseases, encompassing influencing factors, assessment techniques, and repair methodologies. By doing so, it seeks to present innovative strategies for the prevention and treatment of allergic diseases.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn\'s disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

OBJECTIVE: Inflammatory bowel disease (IBD) is currently gaining an increasing global interest. Intestinal epithelial barrier dysfunction is crucial toward developing IBD; however, the underlying mechanisms are not yet elucidated. This study is aimed at elucidating the function of CRL4DCAF2, an E3 ligase, toward mediating intestinal homeostasis.
METHODS: Colon samples were collected from patients with IBD and healthy individuals to examine the expression of CRL4DCAF2. CRL4DCAF2 conditional knockdown in mouse intestinal epithelial cells (IECs) (DCAF2EKD) were constructed. DCAF2EKD and their littermate control (DCAF2EWT) were treated with dextran sodium sulfate (DSS) to induce acute colitis. Transcriptome analysis was performed on inflamed colon samples obtained from the mice. Cell cycle regulators were evaluated using real-time polymerase chain reaction (PCR), while tight junction and apoptosis proteins were examined via immunofluorescence and western blot.
RESULTS: CRL4DCAF2 expression was significantly decreased in the inflamed IBD epithelium, and low expression of CRL4DCAF2 associated with high recurrence risk. Mice with DCAF2 specific knockout in IECs suffer from embryonic death. Multiple genes involved in cell proliferation, immune response, and gap junction were differentially expressed in inflamed colon from DCAF2EKD compared with DCAF2EWT. Furthermore, conditional downregulation of CRL4DCAF2 in the intestinal epithelium induced primarily epithelial damage, increased intestinal permeability, and diminished tight junction protein expression. In vivo and in vitro cell transfection experiments revealed that CRL4DCAF2 enhanced cell proliferation by promoting p21 ubiquitination and degradation, thereby inhibiting G2/M cell cycle. In addition, CRL4DCAF2 can also inhibit IEC apoptosis and promote cell autophagy.
CONCLUSIONS: CRL4DCAF2 downregulation in IECs promotes intestinal barrier dysfunction and inhibits IEC proliferation, thus making it more susceptible to inflammation.

Quercetin (Que) is a flavonol compound found in plants, which has a variety of biological activities. Necroptosis, a special form of programmed cell death, plays a vital role in the development of many gastrointestinal diseases. This study aimed to explore whether Que could attenuate the intestinal injury and barrier dysfunction of piglets after deoxynivalenol (DON) exposure through modulating the necroptosis signaling pathway. Firstly, twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors, including Que (basal diet or diet supplemented with 100 mg/kg Que) and DON exposure (control feed or feed contaminated with 4 mg/kg DON). After feeding for 21 d, piglets were killed for samples. Next, the intestinal porcine epithelial cell line (IPEC-1) was pretreated with or without Que (10 μmol/mL) in the presence or absence of a DON challenge (0.5 μg/mL). Dietary Que increased the body weight, average daily gain, and average daily feed intake (p < 0.05) through the trial. Que supplementation improved the villus height, and enhanced the intestinal barrier function (p < 0.05) indicated by the higher protein expression of occludin and claudin-1 (p < 0.05) in the jejunum of the weaned piglets after DON exposure. Dietary Que also down-regulated the protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated RIP1 (p-RIP1), p-RIP3, total mixed lineage kinase domain-like protein (t-MLKL), and p-MLKL (p < 0.05) in piglets after DON exposure. Moreover, Que pretreatment increased the cell viability and decreased the lactate dehydrogenase (LDH) activity (p < 0.05) in the supernatant of IPEC-1 cells after DON challenge. Que treatment also improved the epithelial barrier function indicated by a higher transepithelial electrical resistance (TEER) (p < 0.001), lower fluorescein isothiocyanate-labeled dextran (FD4) flux (p < 0.001), and better distribution of occludin and claudin-1 (p < 0.05) after DON challenge. Additionally, pretreatment with Que also inhibited the protein abundance of t-RIP1, p-RIP1, t-RIP3, p-RIP3, t-MLKL, and p-MLKL (p < 0.05) in IPEC-1 cells after DON challenge. In general, our data suggest that Que can ameliorate DON-induced intestinal injury and barrier dysfunction associated with suppressing the necroptosis signaling pathway.

Since the 1960 s, our health has been compromised by exposure to over 350,000 newly introduced toxic substances, contributing to the current pandemic in allergic, autoimmune and metabolic diseases. The \"Epithelial Barrier Theory\" postulates that these diseases are exacerbated by persistent periepithelial inflammation (epithelitis) triggered by exposure to a wide range of epithelial barrier-damaging substances as well as genetic susceptibility. The epithelial barrier serves as the body\'s primary physical, chemical, and immunological barrier against external stimuli. A leaky epithelial barrier facilitates the translocation of the microbiome from the surface of the afflicted tissues to interepithelial and even deeper subepithelial locations. In turn, opportunistic bacterial colonization, microbiota dysbiosis, local inflammation and impaired tissue regeneration and remodelling follow. Migration of inflammatory cells to susceptible tissues contributes to damage and inflammation, initiating and aggravating many chronic inflammatory diseases. The objective of this review is to highlight and evaluate recent studies on epithelial physiology and its role in the pathogenesis of chronic diseases in light of the epithelial barrier theory.

Large-scale use of antimicrobials in agriculture and medicine contributes to antibiotic residues in raw foods, the spread of antimicrobial resistance (AMR) and drug pollution, which seriously threatens human health and imposes significant economic burdens on society, suggesting the need for novel therapeutic options that prevent or control zoonoses. In this study, four probiotics were selected to assess their capability to alleviate pathogen-induced damage. Results showed that a simulated gastrointestinal juice and bile tolerated L. plantarum Lac16 with high lactic acid secretion can significantly inhibit the growth of multiple zoonotic pathogens. Lac16 also significantly inhibited the biofilm formation and mRNA expression of virulence traits (genes related to virulence, toxins, flagella biogenesis and motility, antibiotic resistance, biofilm formation and AI-2 quorum sensing) of enterohemorrhagic E. coli O157:H7 (EHEC). Furthermore, Lac16 and Lac26 significantly protected C. elegans against zoonotic pathogen-induced (EHEC, S. typhimurium, C. perfringens) deaths. Moreover, Lac16 significantly promoted epithelial repair and ameliorated lipopolysaccharide (LPS)-induced intestinal epithelial apoptosis and barrier dysfunction by activating the Wnt/β-catenin signaling pathway, and markedly reduced LPS-induced inflammatory responses by inhibiting the TLR4/MyD88 signaling pathway. The present results indicate that Lac16 attenuates enterohemorrhagic E. coli infection-induced damage by inhibiting key virulence traits of E. coli, promoting epithelial repair and improving intestinal epithelial barrier function, which may be mediated by the activated Wnt/β-catenin signaling pathway and the inhibited TLR4/MyD88 signaling pathway of the intestinal epithelium.

The blood brain barrier (BBB) is a protective border that prevents noxious substances from gaining access to the central nervous system (CNS). CXCL13 is a chemokine from the CXC chemokine family, which has been shown to destroy the barrier function of umbilical vein endothelial cells with its receptor CXCR5. Here, we aimed to investigate the role of CXCL13/CXCR5 signaling axis in BBB. The invasive ability of bEnd.3 cells was determined by the Transwell invasion assay. The barrier integrity of bEnd.3 cells was assessed by detecting trans-endothelial electrical resistance, the permeability to fluorescein isothiocyanate-dextran, and the expression levels of the tight junction protein E-cadherin. Lipopolysaccharide (LPS)-activated microglia promoted invasion and barrier dysfunction, and upregulated CXCR5 and p-p38 expression levels in cocultured bEnd.3 cells. However, the effects of activated microglia were alleviated by knocking down CXCR5 in cocultured bEnd.3 cells. Furthermore, recombinant CXCL13 promoted invasion and barrier dysfunction, and upregulated the expression levels of p-p38 in bEnd.3 cells; however, its effects were abolished by treating bEnd.3 cells with the p38 inhibitor SB203580. Our data tentatively demonstrated that LPS-activated microglial cells may promote invasion and barrier dysfunction in bEnd.3 cells by regulating the CXCL13/CXCR5 axis and p38 signaling.

Paired related homeobox 1 (PRRX1) is a newly identified transcription factor that regulates the expression of various genes. We aimed to investigate the roles of PRRX1 and Matrix metalloproteinases (MMP)13 in dextran sulfate sodium (DSS)-induced inflammation and barrier dysfunction of NCM460 cells. PRRX1 expression in the mucosal tissues of patients with ulcerative colitis was analyzed using the GSE87466 microarray. PRRX1 and MMP13 expression was examined using Western blotting and RT-qPCR following the exposure of the NCM460 cells to DSS. The JASPAR database was used to predict the binding sites of PRRX1 to the MMP13 promoter, which was verified by luciferase reporter and chromatin immunoprecipitation assays. MMP13 expression was then detected following PRRX1 silencing or overexpression. The levels of inflammatory factors were determined using ELISA. Finally, the expression of intestinal barrier function-related proteins was evaluated using Western blotting and cellular permeability was detected by Transepithelial electrical resistance. PRRX1 was upregulated in the mucosal tissue samples of patients with UC. DSS induction upregulated PRRX1 and MMP13 expression. PRRX1 bound to the promoter of MMP13, which was further supported by the decreased expression of MMP13 observed following PRRX1 knockdown and its increased expression following PRRX1 overexpression. Furthermore, PRRX1 deletion decreased TNF-α, IL-1β and IL-6 levels in the DSS-challenged NCM460 cells, which were subjected to MMP13 overexpression. Moreover, PRRX1 silencing upregulated ZO-1, occludin and claudin-1 expression and elevated the TEER value, whereas MMP13 overexpression attenuated these effects. Collectively, PRRX1 activates MMP13, which in turn promotes the DSS-induced inflammation and barrier dysfunction of NCM460 cells.

Necrotizing enterocolitis, a devastating gastrointestinal disease with high mortality, poses great threats to global health. Therefore, we conducted this study to explore the role of ANGPT2, as well as the potential mechanism, in necrotizing enterocolitis. IEC-6 cells were stimulated with lipopolysaccharide (LPS) to induce necrotizing enterocolitis model in vitro. The expression of ANGPT2 was measured by RT-qPCR. The cell viability was detected using CCK-8. Besides, the expressions of endoplasmic reticulum (ER) stress-related proteins, Notch signaling pathway-related proteins and tight junction proteins were checked by western blot. The apoptosis and inflammatory response were detected by TUNEL and ELISA, respectively. Moreover, with the adoption of TEER, the cell monolayer permeability was detected. The results showed that ANGPT2 expression was greatly increased after LPS induction. In addition, ANGPT2 knockdown significantly decreased the apoptosis, inflammatory response, barrier dysfunction and endoplasmic reticulum stress of LPS-induced IEC-6 cells. What is more, ANGPT2 knockdown could block Notch signaling pathway. Additionally, with the treatment of Jagged-1, the protective effect of ANGPT2 knockdown on LPS-induced intestinal injury was partly abolished. To sum up, silencing ANGPT2 could improve LPS-induced inflammation, barrier dysfunction and ER stress of intestinal epithelial cells via blocking Notch signaling pathway.

Endothelial cells form the innermost layer of blood vessels with a fundamental role as the physical barrier. While regulation of endothelial cell function by reactive oxygen species (ROS) is critical in physiological processes such as angiogenesis, endothelial function is a major target for interruption by oxidative stress resulting from generation of high levels of ROS in endothelial cells by various pathological factors and also release of ROS by neutrophils. TRPM2 is a ROS-sensitive Ca2+-permeable channel expressed in endothelial cells of various vascular beds. In this review, we provide an overview of the TRPM2 channel and its role in mediating ROS-induced Ca2+ signaling in endothelial cells. We discuss the TRPM2-mediated Ca2+ signaling in vascular endothelial growth factor-induced angiogenesis and in post-ischemic neovascularization. In particular, we examine the accumulative evidence that supports the role of TRPM2-mediated Ca2+ signaling in endothelial cell dysfunction caused by various oxidative stress-inducing factors that are associated with tissue inflammation, obesity and diabetes, as well as air pollution. These findings provide new, mechanistic insights into ROS-mediated regulation of endothelial cells in physiology and diseases.