Permeability Of Epithelial Monolayers Research Articles

P111 Dietary emulsifier κ-carrageenan does not directly affect intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease

Abstract Background The dietary emulsifier carrageenan (CGN) is widely used to optimise the texture of processed foods such as ice cream, plant-based milk and processed meat products. However, CGN is suggested to play a role in IBD development by exerting pro-inflammatory effects and increasing intestinal permeability, as shown in immortalized cell lines and murine models. Specifically kappa-carrageenan (κ-CGN) has been shown to disrupt barrier function of intestinal epithelial cells. In this study, we investigated if κ-CGN can directly increase intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease (CD). Methods Organoid cultures were derived from colonic biopsies of 5 patients with CD (Table 1) and seeded on Transwell® inserts to obtain epithelial monolayers. Two independent experiments were performed per patient (n = 10). Once a confluent monolayer was formed, non-inflamed and inflamed conditions were established by adding regular culture medium or inflammatory stimuli (100 ng/mL TNF-α, 20 ng/mL IL-1β, 1 µg/mL flagellin) to the basolateral side, respectively. The next day, both inflamed and non-inflamed monolayers were stimulated with 100 µg/mL κ-CGN on the apical side, or with regular culture medium as control. The basolateral side was also renewed with either the inflammatory stimuli or regular culture medium. Transepithelial electrical resistance (TEER) was measured after 24 and 48 hours of κ-CGN exposure to study permeability of the epithelium. Relative TEER (% change compared to baseline) was compared between controls and κ-CGN stimulated conditions (repeated measures ANOVA, Šídák's multiple comparisons correction). Results As expected, exposure to inflammatory stimuli on the basolateral side resulted in a significant reduction of TEER of the monolayers after 48 hours (+ 24 hours pre-stimulation), compared to the non-inflamed conditions (-22.54 ± 4.557 %, p<0.0001, paired t-test) (Figure 1A). After 24 hours of stimulation with κ-CGN, TEER did not significantly change in both the non-inflamed (p=0.90) and inflamed conditions (p=0.80), compared to the control conditions. Also after 48 hours, exposure to κ-CGN did not significantly affect the TEER in both non-inflamed (p=0.94) and inflamed (p=0.74) conditions (Figure 1B). RNA analysis of the organoid-derived monolayers is ongoing to explore the inflammatory potential of κ-CGN and possible effects on mucus production. Conclusion The dietary emulsifier κ-CGN does not directly alter the permeability of the intestinal epithelium of CD patients. Further analysis of gene expression of inflammatory markers and mucus production is ongoing. Possible indirect effects, through alterations of the microbiota or immune cells, should still be explored.

Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study

Background and aimsWe have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions.MethodsWe identified, isolated, cultivated, and sequenced a unique novel strain (20220303-A2) of B. vulgatus found only in control subjects. Using a human gut organoid system developed from pre-celiac patients, we monitored epithelial phenotype and innate immune cytokines at baseline, after exposure to gliadin, or gliadin plus B. vulgatus cell free supernatant (CFS).ResultsFollowing gliadin exposure, we observed increases in epithelial cell death, epithelial monolayer permeability, and secretion of pro-inflammatory cytokines. These effects were mitigated upon exposure to B. vulgatus 20220303-A2 CFS, which had matched phenotype gene product mutations. These protective effects were mediated by epigenetic reprogramming of the organoids treated with B. vulgatus CFS.ConclusionsWe identified a unique strain of B. vulgatus that may exert a beneficial role by protecting CeD epithelium against a gluten-induced break of epithelial tolerance through miRNA reprogramming.ImpactGut dysbiosis precedes the onset of celiac disease in genetically at-risk infants.This dysbiosis is characterized by the loss of protective bacterial strains in those children who will go on to develop celiac disease.The paper reports the mechanism by which one of these protective strains, B. vulgatus, ameliorates the gluten-induced break of gut epithelial homeostasis by epigenetically re-programming the target intestinal epithelium involving pathways controlling permeability, immune response, and cell turnover.

Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: Focus on tight junction modulation.

Oxidative stress and inflammation lead by dietary oxidised lipids, as oxysterols, have been linked to the loss of intestinal barrier integrity, a crucial event in the initiation and progression of intestinal disorders. In the last decade, probiotic lactobacilli have emerged as an interesting tool to improve intestinal health, thanks to their antioxidant and anti-inflammatory properties. The aim of the present study was to evaluate the ability of two commercial probiotic strains of lactobacilli (Lactiplantibacillus plantarum 299v® (DMS 9843) and Lacticaseibacillus casei DG® (CNCMI-1572)), both as live bacteria and intracellular content, to attenuate the oxysterols-induced alteration of intestinal epithelial Caco-2cell monolayer permeability. Our investigation was focused on the modulation of tight junctions (TJs) proteins, occludin, ZO-1 and JAM-A, in relation to redox-sensitive MAPK p38 activation. Obtained results provided evidence on the ability of the two probiotics to counteract the alteration of monolayer permeability and loss of TJs proteins, at least in part, through the modulation of p38 pathway. The protective action was exerted by live bacteria, whose adhesion to Caco-2cells was not altered by oxysterols, and bacterial intracellular components equally able to interact with the signaling pathway.

Intestinal Epithelial Monolayer Permeability of Sweet Potato-Derived Anthocyanin and Carotenoid Extracts in Caco-2 Cells

Woo Sung Park, Kyung Ah Koo, Hye-Jin Kim, Ji-Min Kwon, Dong-Min Kang, Hye Jin Chung, Shin Woo Lee, Ho-Su Kim, Sang-Soo Kwak, and Mi-Jeong Ahn. Journal of the Korean Society of Food Science and Nutrition 2021;50:765-73. https://doi.org/10.3746/jkfn.2021.50.8.765

Tight junction protein claudin-1 is downregulated by TGF-β1 via MEK signaling in benign prostatic epithelial cells.

Benign prostatic hyperplasia (BPH) is arguably the most common disease in aging men. Although the etiology is not well understood, chronic prostatic inflammation is thought to play an important role in BPH initiation and progression. Our recent studies suggest that the prostatic epithelial barrier is compromised in glandular BPH tissues. The proinflammatory cytokine transforming growth factor beta 1 (TGF-β1) impacts tight junction formation, enhances epithelial barrier permeability, and suppresses claudin-1 messenger RNA expression in prostatic epithelial cells. However, the role of claudin-1 in the prostatic epithelial barrier and its regulation by TGF-β1 in prostatic epithelial cells are not clear. The expression of claudin-1 was analyzed in 22 clinical BPH specimens by immunohistochemistry. Human benign prostate epithelial cell lines BPH-1 and BHPrE1 were treated with TGF-β1 and transfected with small interfering RNAs specific to claudin-1. Epithelial monolayer permeability changes in the treated cells were measured using trans-epithelial electrical resistance (TEER). The expression of claudin-1, E-cadherin, N-cadherin, snail, slug, and activation of mitogen-activated proteins kinases (MAPKs) and AKT was assessed following TGF-β1 treatment using Western blot analysis. Claudin-1 expression was decreased in glandular BPH tissue compared with adjacent normal prostatic tissue in patient specimens. TGF-β1 treatment or claudin-1 knockdown in prostatic epithelial cell lines increased monolayer permeability. TGF-β1 decreased levels of claudin-1 and increased levels of snail and slug as well as increased phosphorylation of the MAPK extracellular signal-regulated kinase-1/2 (ERK-1/2) in both BPH-1 and BHPrE1 cells. Overexpression of snail or slug had no effect on claudin-1 expression. In contrast, PD98059 and U0126, inhibitors of the upstream activator of ERK-1/2 (ie, MEK-1/2) restored claudin-1 expression level as well as the epithelial barrier. Our findings suggest that downregulation of claudin-1 by TGF-β1 acting through the noncanonical MEK-1/2/ERK-1/2 pathway triggers increased prostatic epithelial monolayer permeability in vitro. These findings also suggest that elevated TGF-β1 may contribute to claudin-1 downregulation and compromised epithelial barrier in clinical BPH specimens.

Structure-function Analysis of E-cadherin Dimerization at the Plasma Membrane

This study identified a critical region of E-cadherin that both drives the formation of lateral dimers at the plasma membrane and affects intercellular junction assembly and function. Cadherin oligomers are fundamental building blocks of intercellular junctions. Prior findings indicate that cadherin oligomerization regulates tissue cohesion and paracellular permeability. Current models of cadherin dimerization/oligomerization focus on the cis-interactions between the extracellular domains. These studies used novel FullSpectral Imaging Förster Resonance Energy Transfer (FSI-FRET) technique to directly quantify binding interactions or E-cadherin and its mutants, to identify protein regions required for homodimerization at the plasma membrane. Although the extracellular region contributes modestly to the dimerization propensity, FSI-FRET results demonstrated that the intracellular domain is the major contributer to E-cadherin dimerization. The intracellular region is crucial for both lateral dimerization and the formation of large cadherin clusters at cell-cell adhesions. In studies of cadherin mutants that lack this domain, actin anchoring did not rescue clustering. Destabilizing E-cadherin dimers through domain deletions sharply impaired the ability of E-cadherins to cluster and increased epithelial monolayer permeability. Further structure-function analysis explored the mechanism by which the intracellular domain modulates E-cadherin dimerization.

Gastrointestinal microbiota alteration induced by Mucor circinelloides in a murine model.

Mucor circinelloides is a pathogenic fungus and etiologic agent of mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.

Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. RSV is known to infect epithelial cells and increase the permeability of model airway epithelial monolayers in vitro. We hypothesized that RSV infection also induces airway barrier dysfunction in vivo. C57BL/6 mice were intranasally inoculated with RSV, and on day 4 post-inoculation were examined for viral replication, lung inflammation, and barrier integrity as well as the structure and molecular composition of epithelial junctions. In parallel, primary mouse tracheal epithelial cells (mTEC) were cultured for in vitro studies. RSV-infected mice lost weight and showed significant peribronchial inflammation compared with noninfected controls and UV-inactivated RSV-inoculated animals. RSV infection increased the permeability of the airway epithelial barrier and altered the molecular composition of epithelial tight junctions. The observed RSV-induced barrier disruption was accompanied by decreased expression of several tight-junction proteins and accumulation of cleaved extracellular fragments of E-cadherin in bronchoalveolar lavage and mTEC supernatants. Similarly, in vitro RSV infection of mTEC monolayers resulted in enhanced permeability and disruption of tight-junction structure. Furthermore, incubation of mTEC monolayers with a recombinant fragment of E-cadherin caused tight-junction disassembly. Taken together, these data indicate that RSV infection leads to airway barrier dysfunction in vivo, mediated by either decreased expression or cleavage of junctional proteins. Our observations provide further insights into the pathophysiology of RSV infection and provide a rationale for development of barrier-protecting agents to alleviate the pathogenic effects of RSV infection.

Targeting Intestinal Vitamin D Receptor Signaling to Mitigate Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Impaired mucosal epithelial barrier contributes significantly to amplifying systemic inflammatory responses characteristic of GVHD. Emerging evidence demonstrates that protecting or restoring intestinal barrier function represents a feasible approach to mitigate GVHD.There is growing interest in how vitamin D and the vitamin D receptor (VDR) signaling regulate immune responses in intestinal inflammatory diseases. The role of VDR signaling in regulating alloimmunity is unknown. We therefore tested the novel hypothesis that VDR signaling plays an important role in maintaining intestinal epithelial barrier function during GVHD. In initial studies, we examined whether intestinal VDR signaling is altered in recipient mice undergoing either syngeneic or allogeneic bone marrow transplantation (BMT). We found a progressive decline in VDR gene expression over time in colon tissues of allogeneic, but not syngeneic, BMT recipients compared with that of naïve animals. Notably, the expression of CYP27B1, the key enzyme participating in the biosynthesis of the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25VitD3), was also significantly decreased in the intestines of allogeneic BMT recipients compared to that of naïve animals. Caco-2 is an epithelial cell line that is widely used as a model for the intestinal barrier. We showed that proinflammatory cytokines TNF-α and IL-6 reduced Caco-2 VDR protein levels in a dose and time dependent manner by western blot analysis. To assess monolayer barrier function, an in vitro permeability assay was performed using FITC-dextran. We found 1,25VitD3 significantly attenuated the increase in epithelial monolayer permeability induced by TNF-α and IL-6. Tight junctions are important components of the intestinal barrier. We also found proinflammatory cytokines reduced gene expression of tight junctions such as occludin in Caco-2 cells. Remarkably, 1,25VitD3 treatment significantly restored occludin expression.We then examined how vitamin D/VDR pathway regulates the development of GVHD. 1,25VitD3 supplementation after BMT significantly increased the survival of recipient mice in a well-established C57Bl/6→Balb/c GVHD model (p=0.0045). 1,25VitD3 treatment did not appear to modify donor T cell alloreactivity. Specifically, donor T cell activation, proliferation, and polarization were not affected by 1,25VitD3 treatment. Notably, infusion of donor T cells from VDR-WT (VDR+/+) or VDR-deficient (VDR-/-) mice caused similar GVHD-associated mortality in Balb/c recipient mice (p=0.98), further suggesting that the beneficial effects of 1,25VitD3 treatment is not due to its effects on donor T cells. 1,25VitD3 also did not appear to affect host and donor antigen presenting cells to modulate GVHD severity. Specifically, dendritic cells from VDR+/+ or VDR-/- mice showed equal ability to stimulate the proliferation of alloreactive T cells in mixed lymphocyte reactions. There was also no difference in post-transplant survival when recipient mice were reconstituted with donor T-cell-depleted bone marrow cells from either VDR+/+ or VDR-/- mice (recipients were transplanted with the same donor T cells from VDR-WT animals; p=0.58). Instead, 1,25VitD3 treatment significantly increased tight junction protein expression in the gut, suggesting its effect on intestinal epithelial cells. Finally, transgenic mice selectively overexpress VDR in the intestinal epithelium (villin promoter-driven FLAG-tagged human VDR gene expression) were used as BMT recipients in gain-of-function studies. We found a significantly decreased GVHD-associated mortality in these mice compared to that of control littermates (p=0.0002), providing direct evidence that intestinal epithelial VDR signaling protects against GVHD. Our results provide new evidence supporting the notion that impaired mucosal barrier function is a major pathogenic factor in the development of GVHD. Intestinal VDR signaling plays an important role in maintaining intestinal barrier integrity and enhancing this pathway protects against GVHD. Thus, our study provides pre-clinical data supporting the use of vitamin D or its analogs as a simple and inexpensive therapeutic modality with minimal side effects for mitigating GVHD. DisclosuresNo relevant conflicts of interest to declare.

VCAM-1-mediated neutrophil infiltration exacerbates ambient fine particle-induced lung injury

BackgroundFine ambient particle matter (PM2.5) induces inflammatory lung injury; however, whether intratracheal administration of PM2.5 increases pulmonary polymorphonuclear leukocyte (PMN) infiltration, the mechanism of infiltration, and if these cells exacerbate PM2.5-induced lung injury are unknown. MethodsUsing 32,704 subjects, the association between blood PMNs and ambient PM2.5 levels on the previous day was retrospectively analyzed. Neutropenia was achieved by injecting mice with PMN-specific antibodies. Inhibition of PMN infiltration was achieved by pretreating PMNs with soluble vascular cell adhesion molecule-1 (sVCAM-1). The effects of PMNs on PM2.5-induced lung injury and endothelial dysfunction were observed. ResultShort-term PM2.5 (> 75 μg/m3 air) exposure increased the PMN/white blood cell ratio and the PMN count in human peripheral blood observed during routine examination. A significant number of PM2.5-treated PMNs was able to bind sVCAM-1. In mice, intratracheally-instilled PM2.5 deposited in the alveolar space and endothelial cells, which caused significant lung edema, morphological disorder, increased permeability of the endothelial-alveolar epithelial barrier, and PMN infiltration with increased VCAM-1 expression. Depletion of circulatory PMNs inhibited these adverse effects. Replenishment of untreated PMNs, but not those pretreated with soluble VCAM-1, restored lung injury. In vitro, PM2.5 increased VCAM-1 expression and endothelial and epithelial monolayer permeability, and promoted PMN adhesion to, chemotaxis toward, and migration across these monolayers. PMNs, but not those pretreated with soluble VCAM-1, exacerbated these effects. ConclusionVCAM-1-mediated PMN infiltration was essential for a detrimental cycle of PM2.5-induced inflammation and lung injury. Results suggest that drugs that inhibit PMN function might prevent acute deterioration of chronic pulmonary and cardiovascular diseases triggered by PM2.5.

AB012. E-cadherin is down-regulated in benign prostate hyperplasia and required for tight junction formation and permeability barrier in prostatic epithelial cell monolayer

BackgroundProstate specific antigen (PSA), which is expressed by luminal epithelial cells in prostate was recently shown in the stromal compartment of benign prostatic hyperplasia (BPH). Since the stromal compartment does not express PSA, epithelial barrier integrity in BPH nodules might be compromised through loss of cell junctions, resulting in the leakage of PSA and other secreted proteins into the stromal compartment and subsequently promoting BPH pathogenesis. E-cadherin, an important cell junction regulator, is found to be down-regulated in epithelial cells in clinical BPH specimens. Whether E-cadherin downregulation affects epithelial barrier permeability is unknown. This research is aimed at examining epithelial barrier permeability change in BPH and exploring the potential role of E-cadherin in prostatic luminal epithelial permeability.MethodsExplants derived from BPH patients were used to study epithelial barrier permeability in BPH nodules and its normal adjacent tissues by FITC-dextran assay. These specimens were also used to study tight junctions in BPH nodules and normal prostate tissues using transmission electron microscopy (TEM), and used to study the expression of junctional proteins ZO-1, ZO-2, ZO-3 and E-cadherin using immunofluorescence staining. The expression of E-cadherin in these specimens was also determined by immunohistochemistry staining. Normal prostatic luminal epithelial cell lines BHPrE-1 and BPH-1 were utilized to perform in vitro studies. Two independent siRNAs were used to knockdown E-cadherin expression. Permeability and tight junctions of cell monolayers in trans-well inserts with/without E-cadherin knockdown were evaluated by trans-epithelium electrical resistant (TER) assay and FITC-dextran trans-well assay, and TEM respectively. Expression of E-cadherin following siRNAs treatment was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western-blot (WB).ResultsFITC-dextran assay detected an increased epithelial barrier permeability in BPH tissues but not in the adjacent normal prostate in explants derived from BPH patients. Tight junctions as well as junctional proteins were decreased in BPH tissues as compared to the normal prostate, suggesting the compromise of luminal epithelial barriers in BPH. E-cadherin was down-regulated and displayed a discontinuous pattern in BPH. E-cadherin expression was negatively correlated with prostatic epithelial monolayer permeability, and E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation.ConclusionsEpithelial barrier permeability is increased in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting blocking E-cadherin loss could be a potential approach to prevent or treat BPH.

Intestinal mucosal barrier dysfunction in SAP patients with MODS ameliorated by continuous blood purification.

Dysfunction of the intestinal mucosal barrier plays an important role in the pathophysiology of severe acute pancreatitis (SAP). Continuous blood purification (CBP) has been shown to improve the prognosis of SAP patients. In order to investigate the effect of CBP on intestinal mucosal barrier dysfunction in SAP patients with MODS, we conducted in vivo and in vitro experiments to explore the underlying mechanisms. The markers for the assessment of intestinal mucosal barrier function including serum diamine oxidase (DAO), endotoxin and intestinal epithelial monolayer permeability were detected during CBP therapy. The distribution and expression of cytoskeleton protein F-actin and tight junction proteins claudin-1 were observed. In addition, Rho kinase (ROCK) mRNA expression and serum tumor necrosis factor-alpha (TNF-α) levels during CBP were determined. SAP patients with MODS had increased levels of serum DAO, endotoxin and intestinal epithelial monolayer permeability when compared with normal controls. While the distribution of F-actin and claudin-1 was rearranged, and the expression of claudin-1 significantly decreased, but F-actin had no change. Meanwhile, ROCK mRNA expression and serum TNF-α level were increased. However, after CBP treatment, levels of serum DAO, endotoxin and intestinal epithelial monolayer permeability decreased. The F-actin and claudin-1 reorganization attenuated and the expression of claudin-1 increased. At the same time, ROCK mRNA expression and serum TNF-α level were decreased. CBP can effectively improve intestinal mucosal barrier dysfunction. The beneficial effect is associated with the improvement of cytoskeleton and tight junction proteins in stability by downregulation of ROCK mRNA expression through the removal of excess proinflammatory factors.

Microfluidic platform for rapid measurement of transepithelial water transport.

Water transport across epithelial monolayers is of central importance in mammalian fluid homeostasis, and epithelial aquaporin (AQP) water channels are potential drug targets. Current methods to measure transepithelial water permeability based on indicator dilution have limited accuracy and can require hours for a single measurement. We report here a microfluidics platform for rapid and accurate measurement of water transport across a conventionally cultured epithelial monolayer on a porous filter requiring only a single image obtained using a standard laboratory fluorescence microscope. The undersurface of a porous polyester filter containing cultured epithelial cells on top is contacted with a perfused microfluidic channel of 100 μm width, 20 μm height and 10 cm length with folded geometry, with in-plane size of 3.2 × 3.2 mm2 for visualization with a 2× objective lens. Osmotic water permeability is measured from the steady-state concentration profile along the length of the channel of a membrane-impermeant fluorescent dye in the perfusate, in which an osmotic gradient is imposed by an anisosmolar solution overlying the epithelial monolayer; diffusional water permeability is measured using a D2O/H2O-sensing fluorescent dye in the perfusate with a D2O-containing isosmolar solution overlying the cell layer. Permeability values are deduced from single fluorescence images. The method, named fluid transport on a chip (FT-on-Chip), was applied to measure transepithelial osmotic and diffusional water permeability in control and AQP4-expressing epithelial cell monolayers. FT-on-Chip allows for rapid, accurate and repeated measurements of transepithelial water permeability, and is generalizable to transport measurements of ions and solutes using suitable indicator dyes.

Reactive Oxygen Species/Hypoxia-Inducible Factor-1α/Platelet-Derived Growth Factor-BB Autocrine Loop Contributes to Cocaine-Mediated Alveolar Epithelial Barrier Damage.

Abuse of psychostimulants, such as cocaine, has been shown to be closely associated with complications of the lung, such as pulmonary hypertension, edema, increased inflammation, and infection. However, the mechanism by which cocaine mediates impairment of alveolar epithelial barrier integrity that underlies various pulmonary complications has not been well determined. Herein, we investigate the role of cocaine in disrupting the alveolar epithelial barrier function and the associated signaling cascade. Using the combinatorial electric cell-substrate impedance sensing and FITC-dextran permeability assays, we demonstrated cocaine-mediated disruption of the alveolar epithelial barrier, as evidenced by increased epithelial monolayer permeability with a concomitant loss of the tight junction protein zonula occludens-1 (Zo-1) in both mouse primary alveolar epithelial cells and the alveolar epithelial cell line, L2 cells. To dissect the signaling pathways involved in this process, we demonstrated that cocaine-mediated induction of permeability factors, platelet-derived growth factor (PDGF-BB) and vascular endothelial growth factor, involved reactive oxygen species (ROS)-dependent induction of hypoxia-inducible factor (HIF)-1α. Interestingly, we demonstrated that ROS-dependent induction of another transcription factor, nuclear factor erythroid-2-related factor-2, that did not play a role in cocaine-mediated barrier dysfunction. Importantly, this study identifies, for the first time, that ROS/HIF-1α/PDGF-BB autocrine loop contributes to cocaine-mediated barrier disruption via amplification of oxidative stress and downstream signaling. Corroboration of these cell culture findings in vivo demonstrated increased permeability of the alveolar epithelial barrier, loss of expression of Zo-1, and a concomitantly increased expression of both HIF-1α and PDGF-BB. Pharmacological blocking of HIF-1α significantly abrogated cocaine-mediated loss of Zo-1. Understanding the mechanism(s) by which cocaine mediates barrier dysfunction could provide insights into the development of potential therapeutic targets for cocaine-mediated pulmonary hypertension.

Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury.

Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes (P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury (P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.

Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability.

Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients.

Flavaglines Ameliorate Experimental Colitis and Protect Against Intestinal Epithelial Cell Apoptosis and Mitochondrial Dysfunction.

Flavaglines are a family of natural compounds shown to have anti-inflammatory and cytoprotective effects in neurons and cardiomyocytes. Flavaglines target prohibitins as ligands, which are scaffold proteins that regulate mitochondrial function, cell survival, and transcription. This study tested the therapeutic potential of flavaglines to promote intestinal epithelial cell homeostasis and to protect against a model of experimental colitis in which inflammation is driven by epithelial ulceration. Survival and homeostasis of Caco2-BBE and IEC-6 intestinal epithelial cell lines were measured during treatment with the flavaglines FL3 or FL37 alone and in combination with the proinflammatory cytokines tumor necrosis factor (TNF) α and interferon γ. Wild-type mice were intraperitoneally injected with 0.1 mg/kg FL3 or vehicle once daily for 4 days during dextran sodium sulfate-induced colitis to test the in vivo anti-inflammatory effect of FL3. FL3 and FL37 increased basal Caco2-BBE and IEC-6 cell viability, decreased apoptosis, and decreased epithelial monolayer permeability. FL3 and FL37 inhibited TNFα- and interferon γ-induced nuclear factor kappa B and Cox2 expression, apoptosis, and increased permeability in Caco2-BBE cells. FL3 and FL37 protected against TNFα-induced mitochondrial superoxide generation by preserving respiratory chain complex I activity and prohibitin expression. p38-MAPK activation was essential for the protective effect of FL3 and FL37 on barrier permeability and mitochondrial-derived reactive oxygen species production during TNFα treatment. Mice administered FL3 during dextran sodium sulfate colitis exhibited increased colonic prohibitin expression and p38-MAPK activation, preserved barrier function, and less inflammation. These results suggest that flavaglines exhibit therapeutic potential against colitis and preserve intestinal epithelial cell survival, mitochondrial function, and barrier integrity.

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2.

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2(-/-)) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2(-/-) mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2(-/-) mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2(-/-) mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.

Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

HbAHP-25, a peptide designed in silico, exhibits potent anti-HIV activity in vitro

Methods HbAHP-25 was designed in silico against CD4 binding domain of gp120 of HIV-1 by molecular docking using Z dock and PROSA softwares. ELISA and SPR were used to determine the binding ability of HbAHP-25 to gp120. Anti-HIV activity of this peptide was checked by two different assays, viz: a) On TZM bl cells, using luciferase assay; b) On CEM-GFP cells and PBMCs using p24 antigen assay. MTT assay, TER/microsphere assay and Immunofluorescence were used to determine the effect of HbAHP-25 on cell viability, epithelial monolayer integrity and permeability.