Tight Junction Barrier Function Research Articles

Effects of topical application of resveratrol on tight junction barrier and antimicrobial compound production in lactating goat mammary glands

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased β-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

Calcitriol modulates epidermal tight junction barrier function in human keratinocytes

BackgroundThe aberrant expression of tight junction (TJ) proteins play an important role in several diseases with impaired skin barriers, including atopic dermatitis, psoriasis, and chronic wounds. The evidence provided thus far suggests an important role of calcitriol in skin homeostasis. However, it is not known whether calcitriol improves the impaired skin barrier. ObjectiveTo investigate the effect of calcitriol on TJ barrier function in human primary keratinocytes. MethodsNormal human primary keratinocytes were stimulated with calcitriol, and the expression of TJ-related proteins was measured by real-time PCR and Western blotting. Immunofluorescence was used to examine the intercellular distribution of TJ-related proteins. TJ barrier function was assessed by the transepithelial electrical resistance (TER) assay. ResultsWe demonstrated that calcitriol increased the expression levels of TJ-related proteins, including claudin-4, claudin-7, occludin, and zonula occludens (ZO)− 1. Calcitriol enhanced the distribution of TJ-related proteins at cellcell borders and induced the phosphorylation of pathways involved in the regulation of TJ barrier function, such as atypical protein kinase C (aPKC), Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt), as evidenced by the effects of specific inhibitors on the above pathways. Indeed, we confirmed that calcitriol enhanced TER in keratinocyte monolayers. ConclusionThese findings showed that calcitriol could modify the expression of keratinocyte TJ proteins, contributing to the maintenance of homeostatic barrier function.

O-GlcNAcylation Inhibition Upregulates Connexin43 Expression in the Endothelium to Protect the Tight Junction Barrier in Diabetic Retinopathy.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on connexin43 (Cx43) expression and its subsequent effects on tight junction properties in diabetic retinopathy (DR). O-GlcNAcylation levels in primary human retinal vascular endothelial cells (HRVECs) and retinas from rats with diabetes were regulated by treatment with Thiamet G or alloxan. Immunoprecipitation was used to examine the relationship between O-GlcNAcylation and Cx43 expression. Stable overexpression and knockdown of Cx43 in HRVECs were achieved using lentivirus constructs; further, their effects on occludin and zonula occluden-1 (ZO-1) expression and tight junction barrier function were determined. O-GlcNAcylation level increased significantly, whereas Cx43 expression decreased in retinas from rats with diabetes and HRVECs cultured under high-glucose conditions. Immunoprecipitation revealed that Cx43 was modified by O-GlcNAcylation and phosphorylation simultaneously. O-GlcNAcylation inhibition negatively regulated both total Cx43 and phosphorylated Cx43 expression, subsequently disrupting tight junction properties. Conversely, Cx43 overexpression reversed the disruption of tight junction properties and downregulated vascular endothelial growth factor expression. Consistently, Cx43 overexpression increased transendothelial electrical resistance values in HRVEC layers. O-GlcNAcylation negatively regulated Cx43 expression, contributing to the disruption of the blood retinal barrier. However, O-GlcNAcylation inhibition and Cx43 overexpression could reverse the tight junction disruption. Therefore, O-GlcNAcylation inhibition is a potential target for avoiding tight junction disruption through the Cx43 pathway in DR.

Important denominator between autoimmune comorbidities: a review of class II HLA, autoimmune disease, and the gut.

Human leukocyte antigen (HLA) genes are associated with more diseases than any other region of the genome. Highly polymorphic HLA genes produce variable haplotypes that are specifically correlated with pathogenically different autoimmunities. Despite differing etiologies, however, many autoimmune disorders share the same risk-associated HLA haplotypes often resulting in comorbidity. This shared risk remains an unanswered question in the field. Yet, several groups have revealed links between gut microbial community composition and autoimmune diseases. Autoimmunity is frequently associated with dysbiosis, resulting in loss of barrier function and permeability of tight junctions, which increases HLA class II expression levels and thus further influences the composition of the gut microbiome. However, autoimmune-risk-associated HLA haplotypes are connected to gut dysbiosis long before autoimmunity even begins. This review evaluates current research on the HLA-microbiome-autoimmunity triplex and proposes that pre-autoimmune bacterial dysbiosis in the gut is an important determinant between autoimmune comorbidities with systemic inflammation as a common denominator.

Effects of functional nutrients on chicken intestinal epithelial cells induced with oxidative stress.

The objective of this study was to investigate the protective effects of functional nutrients including various functional amino acids, vitamins, and minerals on chicken intestinal epithelial cells (cIECs) treated with oxidative stress. The cIECs were isolated from specific pathogen free eggs. Cells were exposed to 0 mM supplement (control), 20 mM threonine (Thr), 0.4 mM tryptophan (Trp), 1 mM glycine (Gly), 10 μM vitamin C (VC), 40 μM vitamin E (VE), 5 μM vitamin A (VA), 34 μM chromium (Cr), 0.42 μM selenium (Se), and 50 μM zinc (Zn) for 24 h with 6 replicates for each treatment. After 24 h, cells were further incubated with fresh culture medium (positive control, PC) or 1 mM H2O2 with different supplements (negative control, NC and each treatment). Oxidative stress was measured by cell proliferation, whereas tight junction barrier function was analyzed by fluorescein isothiocyanate (FITC)-dextran permeability and transepithelial electrical resistance (TEER). Results indicated that cell viability and TEER values were less (p < 0.05) in NC treatments with oxidative stress than in PC treatments. In addition, FITC-dextran values were greater (p < 0.05) in NC treatments with oxidative stress than in PC treatments. The supplementations of Thr, Trp, Gly, VC, and VE in cells treated with H2O2 showed greater (p < 0.05) cell viability than the supplementation of VA, Cr, Se, and Zn. The supplementations of Trp, Gly, VC, and Se in cells treated with H2O2 showed the least (p < 0.05) cellular permeability. In addition, the supplementation of Thr, VE, VA, Cr, and Zn in cells treated with H2O2 decreased (p < 0.05) cellular permeability. At 48 h, the supplementations of Thr, Trp, and Gly in cells treated with H2O2 showed the greatest (p < 0.05) TEER values among all treatments, and the supplementations of VC and VE in cells treated with H2O2 showed greater (p < 0.05) TEER values than the supplementations of VA, Cr, Se, and Zn in cells treated with H2O2. In conclusion, Thr, Trp, Gly, and VC supplements were effective in improving cell viability and intestinal barrier function of cIECs exposed to oxidative stress.

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

Potential effects of gingerol topical application on components of the innate immunity in lactating goat mammary glands.

In the mammary glands, production of antimicrobial components and formation of less-permeable tight junctions (TJs) are important for safe milk production. Previously, we reported that local heat treatment of udders using disposable heating pad enhances the components of innate immunity in lactating goat mammary glands. Gingerol is a polyphenol present in ginger that can induce heat-like effects. However, oral administration of polyphenols causes a decrease in biological activity through conjugation and metabolic conversion. Here, we investigated the effects of gingerol on antimicrobial components and TJs by topically applying it to lactating goat udders. Gingerol application increased the somatic cell count, cathelicidin-2 concentration, and proportion of polymorphonuclear cells in the milk and interleukin-8 production. Moreover, gingerol treatment enhanced β-defensin-1 production in milk, cultured mammary epithelial cells, and cultured somatic cells. Contrastingly, gingerol treatment did not affect the concentrations of blood-derived components (Na+, albumin, and IgG) in the milk or the TJ barrier function of cultured mammary epithelial cells. These findings suggest that the topical application of gingerol, similar to local heat treatment, to udders enhances the components of innate immunity in mammary glands. These findings may be useful for the prevention of mastitis in milk-producing animals and, hence, safe and stable dairy production.

Knock Out of CGN and CGNL1 in MDCK Cells Affects Claudin-2 but Has a Minor Impact on Tight Junction Barrier Function.

Cingulin (CGN) and paracingulin (CGNL1) are cytoplasmic proteins of tight junctions (TJs), where they play a role in tethering ZO-1 to the actomyosin and microtubule cytoskeletons. The role of CGN and CGNL1 in the barrier function of epithelia is not completely understood. Here, we analyzed the effect of the knock out (KO) of either CGN or CGNL1 or both on the paracellular permeability of monolayers of kidney epithelial (MDCK) cells. KO cells displayed a modest but significant increase in the transepithelial resistance (TER) of monolayers both in the steady state and during junction assembly by the calcium switch, whereas the permeability of the monolayers to 3 kDa dextran was not affected. The permeability to sodium was slightly but significantly decreased in KO cells. This phenotype correlated with slightly increased mRNA levels of claudin-2, slightly decreased protein levels of claudin-2, and reduced junctional accumulation of claudin-2, which was rescued by CGN or CGNL1 but not by ZO-1 overexpression. These results confirm previous observations indicating that CGN and CGNL1 are dispensable for the barrier function of epithelia and suggest that the increase in the TER in clonal lines of MDCK cells KO for CGN, CGNL1, or both is due to reduced protein expression and junctional accumulation of the sodium pore-forming claudin, claudin-2.

Huanglian Ganjiang decoction alleviates ulcerative colitis by restoring gut barrier via APOC1-JNK/P38 MAPK signal pathway based on proteomic analysis

Ethnopharmacological relevanceUlcerative colitis (UC) is a kind of chronic intestinal inflammation accompanied with abdominal pain, diarrhea and hematochezia. Huanglian Ganjiang decoction (HGD) derived from “Beiji Qianjin Yao Fang” was used for UC patients clinically. However, the specific mechanism of HGD in treating UC remain unclear. Aim of studyOur study devoted to demonstrating the therapeutic effect of HGD for colitis and clarifying the underlying mechanism. Materials and methodsUPLC-MS was carried out to identify the ingredients of HGD. UC mice were induced by giving 3% dextran sulfate sodium (DSS) solution for one week and treated by HGD for another week. Body weight fluctuation, disease activity index (DAI), colon length and pathological change of colon tissues were observed to evaluate therapeutical effect of HGD. ELISA and qPCR were carried out to estimate the inflammatory state. Western blot, qPCR and immunofluorescence were used to access the expression of tight junction proteins. Tandem mass tag (TMT)-Based proteomics and network pharmacology was launched to screen and predict the potential targets and pathway regulated by HGD. ResultsBased on the UPLC-MS/MS analysis, 100 components were identified in HGD. After 7-day treatment, HGD significantly alleviated colitis-associated symptoms including body weight loss, shorted colon, increase of DAI score, histopathologic lesions. HGD also reduced inflammatory cytokines IL-6 and IL-1β levels, increased the number of goblet cells and restored tight junction proteins Occludin, Claudin-1 in colon. Network pharmacology study predicted that tight junction and MAPK pathway might be affected by HGD in colitis mice. APOC1 was screened out as key target in HGD-treated mice using TMT-based proteomics study. Further Western blot results showed that HGD reduced expressions of APOC1, p-P38 and p-JNK. ConclusionHGD improves general symptoms of colitis mice at medium and high doses, which may be associated with restoring tight junction and intestinal barrier integrity and function through suppression of APOC1-JNK/P38 MAPK signal pathway.

Calcitriol, an Active Form of Vitamin D3, Mitigates Skin Barrier Dysfunction in Atopic Dermatitis NC/Nga Mice.

Atopic dermatitis and psoriasis are prevalent chronic inflammatory skin diseases that are characterized by dysfunctional skin barriers and substantially impact patients' quality of life. Vitamin D3 regulates immune responses and keratinocyte differentiation and improves psoriasis symptoms; however, its effects on atopic dermatitis remain unclear. Here, we investigated the effects of calcitriol, an active form of vitamin D3, on an NC/Nga mouse model of atopic dermatitis. We observed that the topical application of calcitriol decreased the dermatitis scores and epidermal thickness of NC/Nga mice with atopic dermatitis compared to untreated mice. In addition, both stratum corneum barrier function as assessed by the measurement of transepidermal water loss and tight junction barrier function as evaluated by biotin tracer permeability assay were improved following calcitriol treatment. Moreover, calcitriol treatment reversed the decrease in the expression of skin barrier-related proteins and decreased the expression of inflammatory cytokines such as interleukin (IL)-13 and IL-33 in mice with atopic dermatitis. These findings suggest that the topical application of calcitriol might improve the symptoms of atopic dermatitis by repairing the dysfunctional epidermal and tight junction barriers. Our results suggest that calcitriol might be a viable therapeutic agent for the treatment of atopic dermatitis in addition to psoriasis.

Hazara orthonairovirus nucleoprotein facilitates viral cell-to-cell spread by modulating tight junction protein, claudin-1.

Tight junctions act as a barrier that prevents invasion of pathogens through epithelial cells. This study aims to elucidate the correlation between tight junctions and nairoviruses using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. mRNA, total protein, and cell surface protein levels of tight junction proteins were examined by quantitative real-time reverse transcription polymerase chain reaction, immunoblot and flow cytometry, respectively. HAZV growth was measured by plaque assay. Immunofluorescence assay was used to examine viral cell-to-cell spread. The interaction between HAZV nucleoprotein and claudin-1 was analyzed by immunoprecipitation. HAZV infection induced mRNA of several tight junction proteins, especially claudin-1. HAZV infection also induced cell surface expression of claudin-1 protein. Claudin-1 overexpression inhibited the growth of HAZV by blocking its cell-to-cell spread. In contrast, HAZV nucleoprotein completely inhibited HAZV-induced cell surface expression of claudin-1, and this inhibition required interaction between HAZV nucleoprotein and claudin-1. HAZV nucleoprotein was shown to bind to claudin-1 to negatively regulate its cell surface expression, and so can promote cell-to-cell spread of HAZV. This is the first presentation of a possible mechanism behind how nairoviruses counteract tight junction barrier function.

TLR/MyD88-Mediated Inflammation Induced in Porcine Intestinal Epithelial Cells by Ochratoxin A Affects Intestinal Barrier Function.

The intestinal epithelium performs vital functions such as nutrient absorption and acting as an intestinal barrier to maintain the host's homeostasis. Mycotoxin, which affects the processing and storage of animal feedstuff, is a problematic pollutant in farming products. Ochratoxin A generated by Aspergillus and Penicillium fungi causes inflammation, intestinal dysfunction, decline in growth, and reduced intake in porcine and other livestock. Despite these ongoing problems, OTA-related studies in intestinal epithelium are lacking. This study aimed to demonstrate that OTA regulates TLR/MyD88 signaling in IPEC-J2 cells and induces barrier function impairment through tight junction reduction. We measured expression of TLR/MyD88 signaling-related mRNAs and proteins. The indicator of intestinal barrier integrity was confirmed through immunofluorescence and transepithelial electrical resistance. Additionally, we confirmed whether inflammatory cytokines and barrier function were affected by MyD88 inhibition. MyD88 inhibition alleviated inflammatory cytokine levels, tight junction reduction, and damage to barrier function due to OTA. These results indicate that OTA induces TLR/MyD88 signaling-related genes and impairs tight junctions and intestinal barrier function in IPEC-J2 cells. MyD88 regulation in OTA-treated IPEC-J2 cells mitigates the tight junction and intestinal barrier function impairments. Our findings provide a molecular understanding of OTA toxicity in porcine intestinal epithelial cells.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Galectin-9 mediates gut-associated inflammation and disruption of tight junctions

Abstract Background: High levels of Galectin-9 (Gal-9), a β-galactoside-binding mammalian lectin, are associated with systemic inflammation in chronic viral infections like HIV, despite viral suppression by ART. Gut-systemic microbial translocation is a major contributor of persistent inflammation. Whether Gal-9 influences the integrity of the gut during viral infections remains unknown. Objective: Here we investigated the impact of Gal-9 in the interplay between gut-systemic microbial translocation in the setting of well-treated chronic HIV infection. Method: We assessed the levels of Gal-9 and sCD14 in the plasma of people with HIV on ART (PWH; n=25) and people without HIV (PWOH; n=15) by ELISA. The integrity of the gut epithelium in the presence and absence of rGal-9 was assessed in the HT-29 cell line by immunofluorescence. TNFa was used as a positive control to disrupt gut integrity. The levels of TNFa were measured by qPCR. Non parametric tests were used in the analysis. Results: Plasma Gal-9 and sCD14 were elevated in PWH compared to PWOH (all p&amp;lt;0.0001). Further, high Gal-9 levels correlated with high sCD14 in both the PWH (r=0.43, p=0.0317) and PWOH (r=0.79, p=0.0006) groups. Immunofluorescence revealed disruption of the Claudin tight junctions in vitroafter 24 hrs of rGal-9 treatment. rGal-9 also induced a 6-fold increase in the expression of TNFα in HT-29 cells. Conclusion: Our results show that Gal-9 is associated with markers of gut-systemic microbial translocation irrespective of HIV status and given the direct effects of rGal-9 on tight junction barrier function, targeting Gal-9 either by exploiting its secretory pathways or competitive blockade may serve as a novel therapeutic approach to control gut-associated inflammation.

Cinnamon (Cinnamomum cassia) hot water extract improves inflammation and tight junctions in the intestine in vitro and in vivo

The natural byproduct Cinnamomum cassia was widely used in ancient Asia to cure disease because of its various pharmacological effects. Despite its ethnomedicinal benefits, few studies on the intestinal anti-inflammatory effect of C. cassia have been reported. Therefore, this study aimed to evaluate the potential beneficial effects of C. cassia on the intestine in vitro and in vivo. Herein, the effects of cinnamon hot water extract (CWE) on tight junction (TJ) barrier function, transepithelial electrical resistance, and mRNA expression were confirmed in Caco-2 cells. The CWE treatment groups showed significantly enhanced cell permeability, proinflammatory cytokine mRNA expression, and TJ expression. CWE-treated mice showed an improved histological index and decreased cytokine concentrations compared with those of colitis model mice. These results suggest that CWE alleviated inflammatory damage and improved the TJ barrier, indicating that CWE may be used as a functional food to improve intestinal health.

The Effects of Mucopolysaccharide Polysulfate on Steroid-Induced Tight Junction Barrier Dysfunction in Human Epidermal Keratinocytes and a 3D Skin Model

Introduction: The long-term use of topical corticosteroids (TCS) is associated with side effects such as skin atrophy and barrier deterioration. Moisturizers, such as mucopolysaccharide polysulfate (MPS), have been reported to prevent relapses in atopic dermatitis (AD) when used in combination with TCS. However, the mechanisms underlying the positive effects of MPS in combination with TCS in AD are poorly understood. In the present study, we investigated the effects of MPS in combination with clobetasol 17-propionate (CP) on tight junction (TJ) barrier function in human epidermal keratinocytes (HEKa) and 3D skin models. Methods: The expression of claudin-1, which is crucial for TJ barrier function in keratinocytes, and transepithelial electrical resistance (TEER) was measured in CP-treated human keratinocytes incubated with and without MPS. A TJ permeability assay, using Sulfo-NHS-Biotin as a tracer, was also conducted in a 3D skin model. Results: CP reduced claudin-1 expression and TEER in human keratinocytes, whereas MPS inhibited these CP-induced effects. Moreover, MPS inhibited the increase in CP-induced TJ permeability in a 3D skin model. Conclusion: The present study demonstrated that MPS improved TJ barrier impairment induced by CP. The improvement of TJ barrier function may partially be responsible for the delayed relapse of AD induced by the combination of MPS and TCS.

The Antimicrobial Peptide AMP-IBP5 Suppresses Dermatitis-like Lesions in a Mouse Model of Atopic Dermatitis through the Low-Density Lipoprotein Receptor-Related Protein-1 Receptor

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) exhibits antimicrobial activities and immunomodulatory functions in keratinocytes and fibroblasts. However, its role in regulating skin barrier function remains unclear. Here, we investigated the effects of AMP-IBP5 on the skin barrier and its role in the pathogenesis of atopic dermatitis (AD). 2,4-Dinitrochlorobenzene was used to induce AD-like skin inflammation. Transepithelial electrical resistance and permeability assays were used to investigate tight junction (TJ) barrier function in normal human epidermal keratinocytes and mice. AMP-IBP5 increased the expression of TJ-related proteins and their distribution along the intercellular borders. AMP-IBP5 also improved TJ barrier function through activation of the atypical protein kinase C and Rac1 pathways. In AD mice, AMP-IBP5 ameliorated dermatitis-like symptoms restored the expression of TJ-related proteins, suppressed the expression of inflammatory and pruritic cytokines, and improved skin barrier function. Interestingly, the ability of AMP-IBP5 to alleviate inflammation and improve skin barrier function in AD mice was abolished in mice treated with an antagonist of the low-density lipoprotein receptor-related protein-1 (LRP1) receptor. Collectively, these findings indicate that AMP-IBP5 may ameliorate AD-like inflammation and enhance skin barrier function through LRP1, suggesting a possible role for AMP-IBP5 in the treatment of AD.

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.

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.

Mucin-degrading gut commensals isolated from healthy faecal donor suppress intestinal epithelial inflammation and regulate tight junction barrier function.

The intestinal epithelium surface is covered by a layer of mucus that harbors a complex and dynamic population of bacteria termed gut microbiota. In particular, some gut bacteria have the ability to degrade the mucin glycan for nutritional sources. However, the bacterial diversity of mucin-degrading bacteria in human gut microbiota and their role in the gut remains unclear. In this study, we characterized the diversity of mucin-degrading bacteria in the human gut microbiota by an established cultivation-based molecular profiling method. The results showed the gut commensals having the mucin degrading ability were widely distributed in the gut microbiota and were more abundant than previously thought. In addition, many previously uncharacterized mucin degraders were isolated from faecals samples, suggesting the mucin-degrading gut commensals were underappreciated. To gain a better understanding of the interaction between these mucin-degrading gut commensals and the host, the effect of the commensals on intestinal epithelial cells were examined, and the results revealed that the commensals (8 Bacteroides spp., 2 Parabacteroides spp, Akkermanisa muciniphila and Bifidobacterial dentium) incited low level of inflammatory response (IL-8 and TNF-α) but suppressed the inflammatory response induced by E. coli through downregulating the NF-κB pathway. The presence of gut commensals also showed potential in enhancing the epithelial tight junction (TJ) barrier function through regulating the mRNA expression of TJ protein genes such as Zo-1, Occludin, Claudin-1 and E-cadherin. Furthermore, the presence of commensal bacteria P. distasonis, B. thetaiotaomicron and A. muciniphila completely or partly restored the pro-inflammatory cytokine IL-1β induced TJ barrier disruption. In conclusion, these findings indicate that mucin-degrading gut commensals were widely distributed in the gut microbiota and showed anti-inflammatory effect against pathogen infection and potential in modulating the epithelial barrier function.