Epithelial Tight Junction Protein Expression Research Articles

CircMIRLET7BHG, upregulated in an m6A-dependent manner, induces the nasal epithelial barrier dysfunction in allergic rhinitis pathogenesis

ObjectiveAllergic rhinitis (AR) remains a frequent aspiratory allergic inflammatory disorder with a high incidence. Circular RNAs (circRNAs) have been revealed to participate in the pathogenesis of AR. This study investigated the biological function of circMIRLET7BHG (hsa_circ_0008668) in AR progression. MethodsOvalbumin (OVA)-exposed human nasal epithelial cell line (HNEpC) and mice were adopted as the in vitro and in vivo models of AR. Immunofluorescence staining was used to determine epithelial tight junction protein expression. Target molecule levels were assessed by RT-qPCR and Western blotting. Localization of circMIRLET7BHG and IGF2BP1 was observed by RNA-FISH and immunofluorescence. Epithelial barrier damage was determined by transepithelial electrical resistance and fluorescein isothiocyanate-dextran (FD4) permeability. Serum concentrations of IgE, sIgE, IFN-γ, IL-4, and IL-5 were detected by ELISA. Apoptosis, pathological changes, and eosinophil infiltration in nasal mucosa tissues were evaluated by TUNEL, H&E, and Sirius red staining, respectively. Molecular mechanism was analyzed by RNA pull-down, RIP, and MeRIP assays. ResultsAn increased expression of circMIRLET7BHG was found in AR patients and experimental models. Down-regulation of circMIRLET7BHG attenuated OVA-induced allergic symptoms via relieving epithelial thicknesses, eosinophil infiltration, apoptosis, and inflammatory response in mice. Subsequently, circMIRLET7BHG deficiency prevented OVA-induced epithelial barrier dysfunction by reducing epithelial permeability, and inhibiting tight junction proteins. Mechanistically, methyltransferase-like 3 (METTL3) enhanced circMIRLET7BHG expression via m6A methylation, which enhanced ADAM10 mRNA stability via interaction with IGF2BP1. ConclusionMETTL3-mediated m6A modification increased circMIRLET7BHG expression that consequently raised ADAM10 mRNA stability via interplay with IGF2BP1, thereby promoting AR by inducing epithelial barrier dysfunction.

Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis.

To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. NA Laryngoscope, 134:374-381, 2024.

Cis-Nerolidol Inhibits MAP Kinase and NF-κB Signaling Pathways and Prevents Epithelial Tight Junction Dysfunction in Colon Inflammation: In Vivo and In Vitro Studies

Inflammation of the GI tract leads to compromised epithelial barrier integrity, which increases intestine permeability. A compromised intestinal barrier is a critical event that leads to microbe entry and promotes inflammatory responses. Inflammatory bowel diseases that comprise Crohn's disease (CD) and ulcerative colitis (UC) show an increase in intestinal permeability. Nerolidol (NED), a naturally occurring sesquiterpene alcohol, has potent anti-inflammatory properties in preclinical models of colon inflammation. In this study, we investigated the effect of NED on MAPKs, NF-κB signaling pathways, and intestine epithelial tight junction physiology using in vivo and in vitro models. The effect of NED on proinflammatory cytokine release and MAPK and NF-κB signaling pathways were evaluated using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Subsequently, the role of NED on MAPKs, NF-κB signaling, and the intestine tight junction integrity were assessed using DSS-induced colitis and LPS-stimulated Caco-2 cell culture models. Our result indicates that NED pre-treatment significantly inhibited proinflammatory cytokine release, expression of proteins involved in MAP kinase, and NF-κB signaling pathways in LPS-stimulated RAW macrophages and DSS-induced colitis. Furthermore, NED treatment significantly decreased FITC-dextran permeability in DSS-induced colitis. NED treatment enhanced tight junction protein expression (claudin-1, 3, 7, and occludin). Time-dependent increases in transepithelial electrical resistance (TEER) measurements reflect the formation of healthy tight junctions in the Caco-2 monolayer. LPS-stimulated Caco-2 showed a significant decrease in TEER. However, NED pre-treatment significantly prevented the fall in TEER measurements, indicating its protective role. In conclusion, NED significantly decreased MAPK and NF-κB signaling pathways and decreased tight junction permeability by enhancing epithelial tight junction protein expression.

Microbiota, a key player in alcoholic liver disease.

Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. Only 20% of heavy alcohol consumers develop alcoholic liver cirrhosis. The intestinal microbiota (IM) has been recently identified as a key player in the severity of liver injury in ALD. Common features of ALD include a decrease of gut epithelial tight junction protein expression, mucin production, and antimicrobial peptide levels. This disruption of the gut barrier, which is a prerequisite for ALD, leads to the passage of bacterial products into the blood stream (endotoxemia). Moreover, metabolites produced by bacteria, such as short chain fatty acids, volatile organic compounds (VOS), and bile acids (BA), are involved in ALD pathology. Probiotic treatment, IM transplantation, or the consumption of dietary fiber, such as pectin, which all alter the ratio of bacterial species, have been shown to improve liver injury in animal models of ALD and to be associated with an improvement in gut barrier function. Although the connections between the microbiota and the host in ALD are well established, the underlying mechanisms are still an active area of research. Targeting the microbiome through the use of prebiotic, probiotic, and postbiotic modalities could be an attractive new approach to manage ALD.

Su1945 Histone Deacetylases Regulate Epithelial Tight Junction Protein Expression and Prevent Chronic Stress-induced Visceral Hyperalgesia

Su1945 Histone Deacetylases Regulate Epithelial Tight Junction Protein Expression and Prevent Chronic Stress-induced Visceral Hyperalgesia

Tu1876 Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Differential Effects of Lubiprostone on Tight Junction Protein Expression in the Colon

Introduction: In humans, chronic psychological stress is associated with increased intestinal permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. Previously, we reported that WAS rats demonstrated increased intestinal permeability for macromolecules specifically in the colon, which correlated with reduced epithelial tight junction protein expression in the colon epithelium. Lubiprostone (Lub), a prostaglandin receptor agonist used to treat chronic constipation and IBS-C, has been shown to restore barrier function and epithelial tight junction protein expression in the ischemic porcine intestine. Hypothesis: Lub treatment ameliorates increased colon permeability and visceral hyperalgesia in the WAS rat model. Methods: Male Sprague Dawley rats were subjected to one hour of WAS for 10 consecutive days with or without oral administration of Lub. A 20 μg/kg/day dose of Lub was chosen based on a dose response study. On day 11, visceral motor response (VMR) to colorectal distention (CRD) or colon permeability were measured. Permeability was assayed by measuring plasma levels of the non-ionic molecule FITC-Dextran (4KDa), which was given by gavage at 400 mg/Kg. Distal colon tissue was used for Western blot analysis. Results: WAS was associated with increased colon permeability compared to controls in 7 of 14 rats (p = 0.0036) and a decrease in the level of the epithelial tight junction protein occludin (p < 0.05). Lub prevented the stress-induced increase in colon permeability (p = 0.01), as well as the decrease in occludin. WAS rats had a significant increase in pellet output over controls (p < 0.0001), however there were no significant difference in pellet output between Lub treated and untreated WAS rats. All WAS rats showed visceral hyperalgesia in response to CRD (60 mmHg, p < 0.0001) in comparison to controls. Lub treatment of WAS rats prevented visceral hyperalgesia in response to CRD for 7 out of 14 rats (p < 0.0001), whereas the other 7 had VMR to CRD levels equivalent to untreated WAS rats. Conclusion: Lub prevents WASassociated: 1) decrease in epithelial tight junction protein expression in a subset of rats that showed an increase in colon permeability and 2) hyperalgesia in a subset of rats, despite genetic homogeneity. We hypothesize that increased gut permeability and hyperalgesia in response to WAS are linked, and the differential effects of WAS on colon permeability and Lub treatment on visceral hyperalgesia reflects differences in epigenetic regulation of relevant target genes.

Tu1875 BDNF Regulates NMDA Receptor Phosphorylation via the PLC/PKC Pathway in the Spinal Cord During Colitis

Introduction: In humans, chronic psychological stress is associated with increased intestinal permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. Previously, we reported that WAS rats demonstrated increased intestinal permeability for macromolecules specifically in the colon, which correlated with reduced epithelial tight junction protein expression in the colon epithelium. Lubiprostone (Lub), a prostaglandin receptor agonist used to treat chronic constipation and IBS-C, has been shown to restore barrier function and epithelial tight junction protein expression in the ischemic porcine intestine. Hypothesis: Lub treatment ameliorates increased colon permeability and visceral hyperalgesia in the WAS rat model. Methods: Male Sprague Dawley rats were subjected to one hour of WAS for 10 consecutive days with or without oral administration of Lub. A 20 μg/kg/day dose of Lub was chosen based on a dose response study. On day 11, visceral motor response (VMR) to colorectal distention (CRD) or colon permeability were measured. Permeability was assayed by measuring plasma levels of the non-ionic molecule FITC-Dextran (4KDa), which was given by gavage at 400 mg/Kg. Distal colon tissue was used for Western blot analysis. Results: WAS was associated with increased colon permeability compared to controls in 7 of 14 rats (p = 0.0036) and a decrease in the level of the epithelial tight junction protein occludin (p < 0.05). Lub prevented the stress-induced increase in colon permeability (p = 0.01), as well as the decrease in occludin. WAS rats had a significant increase in pellet output over controls (p < 0.0001), however there were no significant difference in pellet output between Lub treated and untreated WAS rats. All WAS rats showed visceral hyperalgesia in response to CRD (60 mmHg, p < 0.0001) in comparison to controls. Lub treatment of WAS rats prevented visceral hyperalgesia in response to CRD for 7 out of 14 rats (p < 0.0001), whereas the other 7 had VMR to CRD levels equivalent to untreated WAS rats. Conclusion: Lub prevents WASassociated: 1) decrease in epithelial tight junction protein expression in a subset of rats that showed an increase in colon permeability and 2) hyperalgesia in a subset of rats, despite genetic homogeneity. We hypothesize that increased gut permeability and hyperalgesia in response to WAS are linked, and the differential effects of WAS on colon permeability and Lub treatment on visceral hyperalgesia reflects differences in epigenetic regulation of relevant target genes.

Spontaneous, Immune-Mediated Gastric Inflammation in SAMP1/YitFc Mice, a Model of Crohn's-Like Gastritis

Crohn's disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn's gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp was tested in fecal pellets by polymerase chain reaction analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose, and epithelial tight junction protein expression was measured by quantitative reverse-transcription polymerase chain reaction analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured, and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4(+) T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn's gastritis.

Postinjury Vagal Nerve Stimulation Protects Against Intestinal Epithelial Barrier Breakdown

Vagal nerve stimulation (VNS) can have a marked anti-inflammatory effect. We have previously shown that preinjury VNS prevented intestinal barrier breakdown and preserved epithelial tight junction protein expression. However, a pretreatment model has little clinical relevance for the care of the trauma patient. Therefore, we postulated that VNS conducted postinjury would also have a similar protective effect on maintaining gut epithelial barrier integrity. Male balb/c mice were subjected to a 30% total body surface area, full-thickness steam burn followed by right cervical VNS at 15, 30, 60, 90, 120, and 150 minutes postinjury. Intestinal barrier dysfunction was quantified by permeability to 4 kDa fluorescein isothiocyanate-Dextran, histologic evaluation, gut tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay, and expression of tight junction proteins (myosin light chain kinase, occludin, and ZO-1) using immunoblot and immunoflourescence. Histologic examination documented intestinal villi appearance similar to sham if cervical VNS was performed within 90 minutes of burn insult. VNS done after injury decreased intestinal permeability to fluorescein isothiocyanate-Dextran when VNS was ≤90 minutes after injury. Burn injury caused a marked increase in intestinal TNF-α levels. VNS-treated animals had TNF-α levels similar to sham when VNS was performed within 90 minutes of injury. Tight junction protein expression was maintained at near sham values if VNS was performed within 90 minutes of burn, whereas expression was significantly altered in burn. Postinjury VNS prevents gut epithelial breakdown when performed within 90 minutes of thermal injury. This could represent a therapeutic window and clinically relevant strategy to prevent systemic inflammatory response distant organ injury after trauma.

Comparison of effects of alcaftadine and olopatadine on conjunctival epithelium and eosinophil recruitment in a murine model of allergic conjunctivitis

Background:Antihistamines constitute the first line of therapy for allergic conjunctivitis, and are safe and effective in relieving the signs and symptoms of ocular allergy. Despite this, they are less effective than some other drugs in relieving delayed symptoms of allergic conjunctivitis. Recent evidence suggests that changes in the conjunctival epithelium may underlie aspects of delayed reactions. In this study we compared two antihistamines, olopatadine and alcaftadine, for their ability to modify epithelial cell changes associated with allergic conjunctivitis at time points selected to reflect late-phase reactions.Methods:Studies employed a modified conjunctival allergen challenge model. Sensitized mice were challenged with topical allergen with or without drug treatments. Treatment groups were assayed for acute-phase (15 minutes) and delayed-phase (24 hours) responses. Groups were scored for allergy symptoms (redness, itch, tearing, and edema) and for conjunctival mast cell numbers. Delayed-phase groups were also examined for eosinophil numbers and for tight junctional protein expression.Results:Olopatadine-treated and alcaftadine-treated animals had similar efficacy profiles and mast cell numbers, suggesting both were effective at ameliorating symptoms of the acute phase. In contrast, alcaftadine-treated animals had significantly lower conjunctival eosinophil infiltration than either controls or olopatadine-treated animals. Allergen challenge caused a significant decrease in expression of the junctional protein, ZO-1, and this decrease was prevented by alcaftadine but not by olopatadine.Conclusion:Alcaftadine displays therapeutic properties beyond its antihistamine action. These include an ability to reduce conjunctival eosinophil recruitment, and a protective effect on epithelial tight junction protein expression.

Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.

Probiotic mixture VSL#3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis

Changes in epithelial tight junction protein expression and apoptosis increase epithelial permeability in inflammatory bowel diseases. The effect of the probiotic mixture VSL#3 on the epithelial barrier was studied in dextran sodium sulfate (DSS)-induced colitis in mice. Acute colitis was induced in BALB/c mice (3.5% DSS for 7 days). Mice were treated with either 15 mg VSL#3 or placebo via gastric tube once daily during induction of colitis. Inflammation was assessed by clinical and histological scores. Colonic permeability to Evans blue was measured in vivo. Tight junction protein expression and epithelial apoptotic ratio were studied by immunofluorescence and Western blot. VSL#3 treatment reduced inflammation (histological colitis scores: healthy control 0.94 +/- 0.28, DSS + placebo 14.64 +/- 2.55, DSS + VSL#3 8.43 +/- 1.82; P = 0.011). A pronounced increase in epithelial permeability in acute colitis was completely prevented by VSL#3 therapy [healthy control 0.4 +/- 0.07 (extinction/g), DSS + placebo 5.75 +/- 1.67, DSS + VSL#3 0.26 +/- 0.08; P = 0.003]. In acute colitis, decreased expression and redistribution of the tight junction proteins occludin, zonula occludens-1, and claudin-1, -3, -4, and -5 were observed, whereas VSL#3 therapy prevented these changes. VSL#3 completely prevented the increase of epithelial apoptotic ratio in acute colitis [healthy control 1.58 +/- 0.01 (apoptotic cells/1,000 epithelial cells), DSS + placebo 13.33 +/- 1.29, DSS + VSL#3 1.72 +/- 0.1; P = 0.012]. Probiotic therapy protects the epithelial barrier in acute colitis by preventing 1) decreased tight junction protein expression and 2) increased apoptotic ratio.

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis