Tight Junction Barrier Research Articles

Propionate regulates tight junction barrier by increasing endothelial-cell selective adhesion molecule in human intestinal Caco-2 cells

Regulation of the intestinal barrier is closely associated with intestinal microbial metabolism. This study investigated the role of propionate, a major short-chain fatty acid produced by intestinal microorganisms, in the regulation of the tight junction (TJ) barrier in human intestinal Caco-2 cells. Propionate strengthened TJ barrier integrity, as indicated by decreased permeability to macromolecules and increased transepithelial electrical resistance in Caco-2 cells. DNA microarray analysis revealed that propionate upregulated endothelial cell-selective adhesion molecule (ESAM), a TJ-associated protein, without any increase in other TJ proteins. The upregulation of ESAM was confirmed using quantitative reverse transcription-PCR, immunoblotting, and immunofluorescence analyses. Luciferase promoter analysis demonstrated that propionate induced the transcriptional activation of ESAM. The effects of propionate were sensitive to nilotinib inhibition of NR2C2. Overexpression of human ESAM (hESAM) in canine kidney epithelial MDCK-II cells lowered the permeability to macromolecules in a manner similar to that of propionate-treated Caco-2 cells. hESAM overexpression facilitated calcium-induced assembly of the TJ complex in MDCK-II cells. Taken together, propionate strengthened the intestinal TJ barrier by increasing ESAM levels in Caco-2 cells.

Valine Treatment Enhances Antimicrobial Component Production in Mammary Epithelial Cells and the Milk of Lactating Goats Without Influencing the Tight Junction Barrier

The production of antimicrobial components and the formation of less-permeable tight junctions (TJs) are important in the defense system of lactating mammary glands and for safe dairy production. Valine is a branched-chain amino acid that is actively consumed in the mammary glands and promotes the production of major milk components like β-casein; additionally, branched-chain amino acids stimulate antimicrobial component production in the intestines. Therefore, we hypothesized that valine strengthens the mammary gland defense system without influencing milk production. We investigated the effects of valine in vitro using cultured mammary epithelial cells (MECs) and in vivo using the mammary glands of lactating Tokara goats. Valine treatment at 4 mM increased the secretion of S100A7 and lactoferrin as well as the intracellular concentration of β-defensin 1 and cathelicidin 7 in cultured MECs. In addition, an intravenous injection of valine increased S100A7 levels in the milk of Tokara goats without influencing milk yield and milk components (i.e., fat, protein, lactose, and solids). In contrast, valine treatment did not affect TJ barrier function either in vitro or in vivo. These findings indicate that valine enhances antimicrobial component production without influencing milk production and TJ barrier function in lactating mammary glands; thus, valine contributes to safe dairy production.

Open hepatic artery flow with portal vein clamping protects against bile duct injury compared to pringles maneuver

Background Conventional hepatic artery and portal vein clamping strategies can prevent blood loss and ischemia-reperfusion liver injury, and such preventative measures are the key to successful liver surgery. However, ischemic-induced damage to cholangiocytes is rarely considered. Here, we aimed to investigate the effect of different hepatic inflow interruption methods on bile duct injury. Methods Forty rats were randomly grouped as sham, Pringle maneuver (PM) and hepatic arterial blood flow open (HAFO) groups. We evaluated liver histology and function in liver sections, and biliary histology, cholangiocyte apoptosis and proliferation, cytokine production, and bile composition. RNA sequencing is performed to explore possible molecular mechanisms. The Blood-biliary barrier permeability and tight junctions were analyzed by HRP injection, immunofluorescence staining and analysis of ZO-1 expression by immunoblotting. Results HAFO significantly attenuated ischemia-induced liver injury and decreased ALT, ALP, TBIL, and DBIL levels in serum. The histopathological observations showed that bile duct injury in the PM group was more serious than that in the HAFO group. The numbers of apoptotic biliary epithelial cells in HAFO-treated rat bile ducta were lower than those in the PM group. RNA-seq showed that tight junctions may be related to the mechanism underlying the protective effect of HAFO, as shown by the reduced HRP levels and increased ZO-1 and claudin-1/3 expression in the HAFO group compared to the PM group. Conclusion Compared with PM, HAFO alleviated the ischemic injury to the biliary system, which was characterized by decreased biliary epithelial cell apoptosis, reduced inflammatory responses and decreased blood-biliary-barrier permeability.

Advanced pathophysiology mimicking lung models for accelerated drug discovery

BackgroundRespiratory diseases are the 2nd leading cause of death globally. The current treatments for chronic lung diseases are only supportive. Very few new classes of therapeutics have been introduced for lung diseases in the last 40 years, due to the lack of reliable lung models that enable rapid, cost-effective, and high-throughput testing. To accelerate the development of new therapeutics for lung diseases, we established two classes of lung-mimicking models: (i) healthy, and (ii) diseased lungs – COPD.MethodsTo establish models that mimic the lung complexity to different extents, we used five design components: (i) cell type, (ii) membrane structure/constitution, (iii) environmental conditions, (iv) cellular arrangement, (v) substrate, matrix structure and composition. To determine whether the lung models are reproducible and reliable, we developed a quality control (QC) strategy, which integrated the real-time and end-point quantitative and qualitative measurements of cellular barrier function, permeability, tight junctions, tissue structure, tissue composition, and cytokine secretion.ResultsThe healthy model is characterised by (i) continuous tight junctions, (ii) physiological cellular barrier function, (iii) a full thickness epithelium composed of multiple cell layers, and (iv) the presence of ciliated cells and goblet cells. Meanwhile, the disease model emulates human COPD disease: (i) dysfunctional cellular barrier function, (ii) depletion of ciliated cells, and (ii) overproduction of goblet cells. The models developed here have multiple competitive advantages when compared with existing in vitro lung models: (i) the macroscale enables multimodal and correlative characterisation of the same model system, (ii) the use of cells derived from patients that enables the creation of individual models for each patient for personalised medicine, (iii) the use of an extracellular matrix proteins interface, which promotes physiological cell adhesion and differentiation, (iv) media microcirculation that mimics the dynamic conditions in human lungs.ConclusionOur model can be utilised to test safety, efficacy, and superiority of new therapeutics as well as to test toxicity and injury induced by inhaled pollution or pathogens. It is envisaged that these models can also be used to test the protective function of new therapeutics for high-risk patients or workers exposed to occupational hazards.Graphical

Lactobacillus acidophilus Induces Enhancement of the Intestinal Epithelial Tight Junction Barrier Function by Toll-like Receptor-2 Independent of MyD88

A defective intestinal epithelial tight junction (TJ) barrier has been postulated to be a key pathogenic factor in allergic disorders, including food allergy. Enhancement of the TJ barrier might be a target for prevention or treatment of such diseases. Our lab recently showed that Lactobacillus acidophilus (LA) caused an enhancement of the intestinal TJ barrier that was mediated by attachment of live LA to toll-like receptor-2 (TLR2) on the apical membrane surface of enterocytes. The aim of this study was to delineate TLR2 signaling mechanisms mediating the LA-induced enhancement of the intestinal TJ barrier.

DOP68 Autophagy induction in the intestinal epithelium protects against inflammation-induced barrier loss in vivo.

Abstract Background Loss of the paracellular tight junction (TJ) barrier and defective autophagy in the gut are pathogenic factors for inflammatory bowel disease (IBD). Earlier, we demonstrated the role of autophagy in the degradation of the pore-forming claudin-2 protein and the resultant TJ barrier enhancement. Here, we investigated the role of autophagy in the regulation of the TJ barrier in vivo and in Crohn’s disease (CD) patient-derived tissues and patient-derived organoids. Methods Wild type and autophagy-deficient Atg7-/- mice were given intraperitoneal rapamycin injection to induce autophagy along with LPS or TNF-α to induce inflammation. Surgically resected tissues from CD patients were incubated with rapamycin and the paracellular barrier was measured by Ussing chambers. Autophagy was induced in human colonic organoids by incubation with either rapamycin or by nutrient starvation. Results Intraperitoneal rapamycin injection in WT mice successfully induced autophagy as evidenced through the degradation of the autophagy substrate p62/SQSTM. Rapamycin injection increased the colonic occludin levels and enhanced the paracellular TJ barrier as evidenced by the decreasing paracellular flux of inulin. Rapamycin prevented LPS and TNF-a induced colonic paracellular barrier loss and reduction in occludin levels in WT mice. Rapamycin reduced the LPS and TNF-a-induced increase in occludin’s association with caveolin-1 thereby preventing its caveolae-mediated endocytosis and degradation. The protective effect of rapamycin was dependent on autophagy. Conditional knockout of Atg7 (cAtg7) in mouse colon decreased baseline occludin levels and increased inulin flux in mouse colon. Furthermore, rapamycin failed to prevent the LPS and TNF-a-induced colonic barrier loss in the cAtg7 mice. Additionally, these mice lacking the ability to undergo autophagy in the colon were more susceptible to Dextran Sodium Sulphate (DSS)-induced colitis. In normal and inflamed colonic tissues from CD patients, incubation with rapamycin induced autophagy, evidenced through the reduction in p62/SQSTM1 protein levels, increased occludin levels and enhanced the paracellular TJ barrier. Autophagy induction by rapamycin or nutrient starvation also increased occludin levels and its membrane localization in patient-derived organoids. Conclusion Our data show that rapamycin successfully induced autophagy in WT mice’s colon and protected these mice from the barrier disruptive effect of both LPS and TNF-α, indicating the importance of autophagy in regulating the intestinal TJ barrier. Our data also highlights the application of autophagy induction as a potential therapeutic approach in the treatment of IBD.

HC067047 as a potent TRPV4 inhibitor repairs endotoxemia colonic injury

Colonic injury causes severe inflammation during systemic infections in patients with endotoxemia. The prevention of colonic injury could effectively reduce the progression of endotoxemia. We investigated the protective effects and detailed mechanisms of the TRPV4 inhibitor HC067047 in the treatment of colonic injury caused by endotoxemia. An LPS-induced endotoxemia colonic injury model was used to assess the in vivo effects of HC067047. Colon slices were detected by hematoxylin and eosin (HE) staining and immunofluorescence assays. Spectrophotometry was used to determine the levels of MDA, calcium, GSH, and GSSG. Alterations in oxidative stress/mitophagy/inflammatory pyroptosis-related markers were evaluated by Q-PCR and western blot assays. HC067047 reduced the body weight loss and spleen weight index of endotoxemic mice and partly recovered the normal morphology of the colonic mucous layer. As an inhibitor of the calcium permeant cation channel, HC067047 suppressed the phosphorylation of the CAMKIIɑ protein and levels of MDA and calcium, upregulated the ratio of GSH/GSSG, shortened the expression of oxidative stress-related proteins, and enhanced the expression of the anti-oxidative protein CAT in damaged colon tissues. Additionally, HC067047 maintained normal mitochondrial functions in endotoxemia colons by promoting mitochondrial fusion and biosynthesis and suppressing mitochondrial fission and the PINK/Parkin/mitophagy pathway. HC067047 potently blocked inflammatory pyroptosis and protected the colonic tight junction barrier. HC067047 restores endotoxemia colons against oxidative stress, mitophagy, inflammatory pyroptosis, and colonic barrier dysfunction. Hence, HC067047 therapy may be potentially useful in the treatment of colonic injury in endotoxemia.

Green tea extract and Piper retrofractum attenuate deoxycholic acid-induced damage and enhance the tight junction barrier: An analysis in a Caco-2 cell culture model and a DSS coinduced mouse model

This study investigated the effect of green tea extract (GTE) and green tea–Piper retrofractum complex (GPX), on tight junctions (TJs) in a cellular model of deoxycholic acid (DCA)-induced intestinal barrier disruption and a mouse DSS + DCA-induced colitis model. Green tea and P. retrofractum were extracted and coadministered to examine the bioavailability and effects of combined catechins and piperine on colonic TJ damage. The ability of GPX to alleviate TJ damage was determined by comparing the transepithelial electrical resistance (TEER), FITC-dextran flux, and mRNA expression of TJ-related components and target expression pathway in the GTE and GPX groups. Compared with the DCA-treated group, the GTE- and GPX-treated groups showed significantly enhanced TEER, FITC-dextran flux, and TJ-related mRNA expression (p < 0.05). Cell treatment also downregulated the mRNA encoding bile acid receptor and decreased ERK1/2 phosphorylation. Compared with the GTE-treated group, the GPX-treated group showed higher mRNA expression of zonula occludens-1, occludin, claudin-3, and claudin-4; greater restoration of TJs, as determined by immunofluorescence; and better modulation of ERK1/2 activation. In the in vivo model, GPX-pretreated mice showed an improved intestinal damage index and colon length compared with mice in the other colitis-induced groups. Both extracts recovered the distal colon mRNA expression of zonula occludens-1 and claudin-1. These results suggest that GTE improves intestinal health by protecting TJs from secondary bile acid damage and that P. retrofractum may potentiate the bioactivity of green tea catechins.

New Data on the Features of Skin Barrier in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a Th1/17-driven inflammatory skin disease of the apocrine gland-rich (AGR) skin regions, where keratinocytes seem to be the crucial drivers of the initial pathogenic steps. However, the possible role of permeability barrier alteration in activating keratinocytes during HS development has not been clarified. We compared the major permeability barrier elements of non-lesional HS (HS-NL; n = 10) and lesional HS (HS-L; n = 10) skin with healthy AGR regions (n = 10) via RT-qPCR and immunohistochemistry. Stratum corneum components related to cornified envelope formation, corneocyte desquamation and (corneo)desmosome organization were analyzed along with tight junction molecules and barrier alarmins. The permeability barrier function was also investigated with transepidermal water loss (TEWL) measurements (n = 16). Junction structures were also visualized using confocal microscopy. At the gene level, none of the investigated molecules were significantly altered in HS-NL skin, while 11 molecules changed significantly in HS-L skin versus control. At the protein level, the investigated molecules were similarly expressed in HS-NL and AGR skin. In HS-L skin, only slight changes were detected; however, differences did not show a unidirectional alteration, as KRT1 and KLK5 were detected in decreased levels, and KLK7, KRT6 and DSG1 in increased levels. No significant differences in TEWL or the expression of junction structures were assessed. Our findings suggest that the permeability barrier is not significantly damaged in HS skin and permeability barrier alterations are not the driver factors of keratinocyte activation in this disease.

Shoseiryuto Promotes the Formation of a Tight-Junction Barrier in Cultured Human Bronchial Epithelial Cells.

Shoseiryuto (SST) (Xiao-Qing-Long-Tang in Chinese) is an effective treatment for respiratory diseases, such as bronchial asthma and allergic rhinitis, but its effects on the bronchial tight-junction (TJ) barrier have not been clarified. This study aimed to evaluate the effect of SST on TJ-barrier function in human bronchial epithelial (16HBE) cells. The 16HBE cells were cultured in a culture medium without (control) and with SST in the absence and presence of bacterial endotoxin lipopolysaccharide (LPS) in transwell chambers. Transepithelial electrical resistance (TEER) and sodium fluorescein (Na-F) permeability of the cultured-cell monolayer were measured as TJ integrity markers. In addition, immunofluorescence staining and quantitative real-time polymerase chain reaction analysis were used to measure the expression of the TJ protein, occludin. SST increased TEER and decreased Na-F permeability of the 16HBE cell monolayers. Furthermore, SST increased both occludin mRNA and immunostained protein expressions, suggesting that SST has the effect of directly promoting epithelial TJ-barrier function. LPS decreased TEER, increased Na-F permeability, and decreased both occludin mRNA and protein expression. LPS-induced barrier dysfunction was completely blocked by pre/co- and posttreatment with SST. These results suggest that SST has protective and therapeutic effects against LPS-induced TJ-barrier damage. To our knowledge, these are the first results to demonstrate the protective and therapeutic effects conferred by TJ-barrier promoting, which may be a novel mechanism contributing to the efficacy of SST for respiratory diseases.

Advanced glycation end-products change placental barrier function and tight junction in rats with gestational diabetes mellitus via the receptor for advanced glycation end products/nuclear factor-κB pathway

The placenta plays an important role in nutrient transport to maintain the growth and development of the embryo. Gestational diabetes mellitus (GDM), the most common complication during pregnancy, highly affects placental function in late gestation. Advanced glycation end-products (AGEs), a complex and heterogeneous group of compounds engaged by the receptor for AGEs (RAGE), are closely associated with diabetes-related complications. In this study, AGEs induced a decrease in the expression of tight junction (TJ) proteins in BeWo cells and increased the paracellular permeability of trophoblast cells by regulating RAGE/NF-κB. Sprague-Dawley (SD) rats injected with 100 mg/kg AGEs-rat serum albumin (RSA) via the tail vein from embryo day 2 were set as the placental barrier dysfunction model group (n = 10). The effect of AGEs on placental permeability was determined using the EvansBlue dye extravasation method. The ultrastructure of the placenta samples was observed by transmission electron microscopy. The effects of AGEs on the placenta were confirmed by treating rats with RAGE antagonist FPS-ZM1 and soluble forms of RAGE (sRAGE). AGEs treatment increased placental permeability and disrupted the tight junctions in pregnant rat placenta, but has no effect on blood glucose. The expression of TJ-related proteins, including ZO-1, Occludin, and Claudin 5, were downregulated after AGEs treatment. Further, AGEs treatment increased the expression of RAGE and nuclear factor-κB in the placenta of rats and upregulated the levels of vascular endothelial growth factor. The effects of AGEs on the placenta were blocked by RAGE antagonist FPS-ZM1 and sRAGE. This study demonstrates the mechanism underlying AGEs-induced disturbance in placental function in pregnant rats and highlights the potential of AGEs in the treatment of GDM.

In vitro and in vivo evaluation of effects of high-purity magnesium on tight junction of intestinal epithelial cell.

After anastomosis of sutures or pins, the restoration of intestinal barrier function can avoid several complications, such as tissue damage and inflammation. Our previous studies demonstrated the feasibility of biodegradable magnesium (Mg) pins as novel anastomosing implants to spontaneously absorb in the body, avoiding secondary removal surgery and long-term inflammation. However, the effect of Mg pins on the intestinal tight junction barrier is rarely investigated. In this study, we conducted high-purity Mg pins inserted in the intestine of rats and prepared Mg extracts cultured intestinal epithelial cell line to investigate the biological effect on the intestinal barrier associated with tight junction protein expression. We discovered that the concentration of released Mg ions over 1.7 mM was the critical threshold, above which mRNA expression of intestinal tight junction and cell apoptosis were affected considerably. Results of the immunohistochemical analysis revealed that Mg functions to stimulate ZO-1, caspase-3, occluding, and claudin-3 expressions. We offer new insight into the effectiveness of biodegradable Mg materials as the next generation of intestinal anastomosis pins, which effectively filters toxins as well as bacteria, and reduces inflammation.

Selective Pharmacological Inhibition of NOX2 by GSK2795039 Improves Bladder Dysfunction in Cyclophosphamide-Induced Cystitis in Mice.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. Among the many mediators implicated in cystitis, the overproduction of reactive oxygen species (ROS) seems to play a key role, although the main source of ROS remains unclear. This study aimed to investigate the contribution of NADPH oxidase (NOX) isoforms in ROS generation and the voiding dysfunction of cyclophosphamide (CYP, 300 mg/Kg, ip, 24 h)-induced cystitis in adult female mice, a well-recognized animal model to study IC/BPS, by using GKT137831 (5 mg/Kg, ip, three times in a 24 h period) or GSK2795039 (5 mg/Kg, ip, three times in a 24 h period) to inhibit NOX1/4 or NOX2, respectively. Our results showed that treatment with GSK2795039 improved the dysfunctional voiding behavior induced by CYP, reduced bladder edema and inflammation, and preserved the urothelial barrier integrity and tight junction occludin expression, besides inhibiting the characteristic vesical pain and bladder superoxide anion generation. In contrast, the NOX1/4 inhibitor GKT137831 had no significant protective effects. Taken together, our in vivo and ex vivo data demonstrate that NOX2 is possibly the main source of ROS observed in cystitis-induced CYP in mice. Therefore, selective inhibition of NOX2 by GSK2795039 may be a promising target for future therapies for IC/BPS.

Aflatoxin B1 Induces Intestinal Barrier Dysfunction by Regulating the FXR-Mediated MLCK Signaling Pathway in Mice and in IPEC-J2 Cells

Aflatoxin B1 (AFB1) is a widespread mycotoxin in food and feed. Although the liver is the main target organ of AFB1, the intestine is the first exposure organ to AFB1. However, the mechanism by which AFB1 induced intestinal barrier dysfunction via regulating the farnesoid X receptor (FXR)-mediated myosin light chain kinase (MLCK) signaling pathway has rarely been studied. In vivo, AFB1 exposure significantly decreased the small intestine length and increased the intestinal permeability. Meanwhile, AFB1 exposure markedly suppressed the protein expressions of FXR, ZO-1, occludin, and claudin-1 and enhanced the protein expression of MLCK. In vitro, AFB1 exposure induced intestinal barrier dysfunction by the elevation in the FITC-Dextran 4 kDa flux and inhibition in the transepithelial electrical resistance in a dose-dependent manner. In addition, AFB1 exposure downregulated the mRNA and protein expressions of FXR, ZO-1, occludin, and claudin-1, redistributed the ZO-1 protein, and enhanced the protein expressions of MLCK and p-MLC. However, fexaramine (Fex, FXR agonist) pretreatment markedly reversed the AFB1-induced FXR activity reduction, MLCK protein activation, and intestinal barrier impairment in vitro and in vivo. Moreover, pretreatment with the inhibition of MLCK with ML-7 significantly alleviated the AFB1-induced intestinal barrier dysfunction and tight junction disruption in vitro. In conclusion, AFB1 induced intestinal barrier impairment via regulating the FXR-mediated MLCK signaling pathway in vitro and in vivo and provided novel insights to prevent mycotoxin poisoning in the intestine.

Cathelicidin LL-37 Activates Human Keratinocyte Autophagy through the P2X₇, Mechanistic Target of Rapamycin, and MAPK Pathways

Human cathelicidin LL-37 is a multifunctional antimicrobial peptide that exhibits antimicrobial and immunomodulatory activities. LL-37 regulates skin barrier function and was recently reported to activate autophagy in macrophages. Because autophagy deficiency is associated with skin diseases characterized by a dysfunctional epidermal barrier, we hypothesized that LL-37 might regulate the skin barrier through autophagy modulation. We showed that LL-37 activated autophagy in human keratinocytes and three-dimensional skin equivalent models as indicated by increases in LC3 puncta formation, decreases in p62, and autophagosome and autolysosome formation. LL-37‒induced autophagy was suppressed by P2X7 receptor, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 inhibitors, suggesting that the P2X7, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 pathways are involved. Moreover, LL-37 enhanced the phosphorylation of adenosine monophosphate‒activated protein kinase and unc-51-like kinase 1. In addition, LL-37‒mediated autophagy involves the mechanistic target of rapamycin and MAPK pathways. Interestingly, the LL-37‒induced distribution of tight junction proteins and improvement in the tight junction barrier were inhibited in autophagy-deficient keratinocytes and keratinocytes and skin models treated with autophagy inhibitors, indicating that the LL-37‒mediated tight junction barrier is associated with autophagy activation. Collectively, these findings suggest that LL-37 is a potential therapeutic target for skin diseases characterized by dysfunctional autophagy and skin barriers.

192 Betacellulin alleviates Th2 cytokine-mediated impairment of skin tight junction barrier through epidermal growth factor receptor and protein kinase C pathway

Betacellulin (BTC) is a peptide ligand that belongs to the epidermal growth factor family, the members of which are implicated in skin morphogenesis, homeostasis, repair, and angiogenesis; however, the role of BTC in regulation of skin barrier remains unknown so far. To examine the role of BTC in skin barrier function, we analysed data from two independent Gene Expression Omnibus (GEO) datasets and evaluated the effects of BTC on primary human keratinocytes using real-time PCR, Western blot and transepidermal electrical resistance (TER), a functional parameter to monitor the tight junction barrier.

199 Ex vivo infection of human skin with herpes simplex virus 1: Lesional AD skin and IL-4/IL-13-stimulated skin provide facilitated viral invasion

Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that can target skin to establish infection in the epithelium. Since the human skin provides effective barriers against pathogens under normal conditions, HSV-1 must find opportunistic pathways in order to penetrate the skin and initiate infection. As mechanical wounds of the human skin surface do not provide ad hoc entry portals for HSV-1, we explored the role of pathological skin conditions. Ex vivo infection studies revealed successful invasion in lesional AD skin implying that the virus can overcome both the stratum corneum and the tight junction barriers. To dissect the parameters that contribute to HSV-1 invasion, we treated human skin without pre-existing barrier defects with interleukin (IL)-4 and IL-13 to investigate whether Th2 cytokine-driven inflammation already induces modifications that allow viral penetration. Strikingly, we detected infected cells in the epidermis indicating that the IL-induced, AD-like skin phenotype can indeed facilitate HSV-1 invasion. Potential modifications allowing for facilitated viral access include impaired tight junctions, an altered stratum corneum, or a redistribution of the HSV-1 receptor nectin-1. Studies using human epidermal equivalents highlighted the importance of functional tight junctions in addition to the stratum corneum in restricting HSV-1 entry and provide a tool to mimic the pathological alterations in AD skin that allow HSV-1 invasion.

Transepithelial Effect of Probiotics in a Novel Model of Gut Lumen to Nerve Signaling.

Recent studies have shown that the gut microbiome changes brain function, behavior, and psychiatric and neurological disorders. The Gut-Brain Axis (GBA) provides a neuronal pathway to explain this. But exactly how do commensal bacteria signal through the epithelial layer of the large intestine to activate GBA nerve afferents? An in vitro model is described. We differentiated two human cell lines: Caco2Bbe1 into mature epithelium on 0.4-micron filters and then SH-SY5Y into mature neurons in 24-well plates. These were co-cultured by placing the epithelium-laden filters 1 mm above the neurons. Twenty-four hours later they were tri-cultured by apical addition of 107Lactobacillus rhamnosus or Lactobacillus fermentum which settled on the epithelium. Alone, the Caco2bbe1 cells stimulated neurite outgrowth in underlying SH-SY5Y. Beyond this, the lactobacilli were well tolerated and stimulated further neurite outgrowth by 24 h post-treatment, though not passing through the filters. The results provide face validity for a first-of-kind model of transepithelial intestinal lumen-to nerve signaling. The model displays the tight junctional barrier characteristics found in the large intestine while at the same time translating stimulatory signals from the bacteria through epithelial cells to attracted neurons. The model is easy to set-up with components widely available.

The Epidermal Barrier Structure and Function of Re-Harvested Skin from Non-Scalp Donor Sites

Aim The aim of this study was to explore the epidermal barrier structure and function of re-harvested skin from non-scalp donor sites. Methods Six patients with large-area deep burns who met the inclusion and exclusion criteria were subjected to split-thickness skin excision three times on the same healthy non-scalp donor sites, with an interval of 14 days. The donor skin thus harvested was labeled as primary skin (S1), secondary skin (S2), and tertiary skin (S3). The transepidermal water loss (TEWL) and stratum corneum water content (SCH) of donor skin were detected before each surgery, and the donor skin was harvested during the surgery. The donor skin was stained with hematoxylin and eosin (HE) and involucrin, loricrin, filaggrin, small molecule proline-rich protein 3 (SPRR3), ZO-3, JAM-A, and JAM-C, or observed by transmission electron microscopy. Results The epidermal barrier function of the re-harvested skin from the non-scalp donor sites became impaired. The histopathological structure of the re-harvested skin from non-scalp donor sites became abnormal. The barrier of the epidermal stratum corneum of the re-harvested skin from non-scalp donor sites was damaged. The epidermal tight junction barrier in the re-harvested skin from non-scalp donor sites was damaged. Conclusions As the number of harvesting increases, the epidermal barrier function of the skin decreased, and the damage to the barrier structure increased. Hence, it is vitally important to restore the epidermal barrier function for re-harvesting in non-scalp donor sites.

EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier.

TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM-claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.