Upregulation of Basonuclin1 Is Associated with p63-Involved Epithelial Barrier Impairment and Type-2 Helper T-cell Inflammation in Chronic Rhinosinusitis with Nasal Polyps.

BACKGROUND: Tumor protein p63 has been shown to be important for epithelial dysfunction, including epithelial barrier defects and mucosal inflammation, in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). Basonuclin1 (BNC1), an epithelial-specific transcriptional factor, is a direct downstream target of p63 and thus might be involved in the pathogenesis of CRSwNP. OBJECTIVE: We sought to investigate whether BNC1 was associated with p63-mediated epithelial barrier defects and nasal mucosal inflammation in CRSwNP. METHODS: Nasal tissue biopsies were obtained from 91 patients to CRSwNP, 49 chronic rhinosinusitis without nasal polyps (CRSsNP) patients, and 28 control subjects. Immunohistochemistry and immunofluorescence staining were used to determine the distribution of BNC1 in tissues and localization in cells, respectively. Quantitative PCR was performed to detect the expression levels of BNC1, TP63, epithelial barrier proteins, and type-2 helper T-cell inflammation-related genes. RESULTS: BNC1 mRNA expression was significantly elevated in the tissues in CRSwNP patients compared with CRSsNP (1.96-fold, p = 0.0003) and control groups (2.40-fold, p < 0.0001). BNC1 staining was strongly positive in the nasal epithelium and co-localized with p63-positive epithelial cells. The expression of BNC1 mRNA was strongly correlated with TP63 mRNA level both in tissue biopsies (r = 0.78, p < 0.0001) and epithelial scrapings (r = 0.97, p < 0.0001). BNC1 expression was also positively correlated with epithelial barrier protein genes (CDH1, CLDN1, CLDN4, TJP1, and TJP2) and epithelial genes involved in TH2 inflammation (IL33, CCL26, CLC, and ALOX15). CONCLUSIONS: Overexpression of BNC1 may be associated with increased expression of TP63, and possibly contribute to the epithelial barrier defects and TH2 inflammation in CRSwNP.

PAD4 selective inhibitor TDFA protects lipopolysaccharide-induced acute lung injury by modulating nuclear p65 localization in epithelial cells.

Protein arginine deiminase 4 (PAD4) serves a critical role in differentiation, development and apoptosis through gene regulation and has emerged as a potential therapeutic target for the treatment of various diseases. However, the roles of PAD4 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remain largely unknown. To investigate the roles of PAD4 during LPS-induced ALI, the present study detected the trend of PAD4 expression in the lung tissues of ALI mice. Subsequently, the efficiency of TDFA on PAD4 and citrullinated H3 histone were detected. And then, histology, the wet/dry weight ratio, survival rate, activated cells infiltration, oxidative stress levels, tight junction proteins and proinflammatory cytokine expression were detected. In addition, the level of transepithelial electrical resistance (TEER) was assessed. Finally, the level of nuclear P65, total phosphorylated P65 and P65 were measured in vivo and in vitro. The results showed that PAD4 expression was upregulated in the lung tissues of LPS-induced ALI. TDFA efficiently decreased the severity of the lung edema, attenuated the severity of pulmonary injury and improved the survival rate following lethal LPS administration. Besides, TDFA reduced activated cells infiltration and suppressed inflammation related parameters, including proinflammatory cytokines production (TNF-α, IL-6 and IL-1ß) and oxidative stress (MDA, GSH and SOD). Furthermore, TDFA reversed the TEER downregulation tendency and tight junction proteins (ZO-1, Occludin, Claudin-4) levels that represent the integrity of alveolar epithelium. Eventually, TDFA exerts its protective roles through modulating nuclear localization of transcription factor NF-κB P65 in epithelial cells. Taken together, these results indicate that PAD4 inhibition may serve as a promising therapeutic approach for LPS-induced ALI.

Ginsenoside Rk3 alleviated DSS-induced ulcerative colitis by protecting colon barrier and inhibiting NLRP3 inflammasome pathway.

Ginsenosides have a variety of pharmacological activities, including immunomodulatory, antitumor and anti-inflammatory activities. However, the effect of Rk3 on ulcerative colitis has rarely been reported. This study evaluated the effect of Rk3 on DSS-induced ulcerative colitis and preliminarily explored the anti-inflammatory mechanisms. Rk3 administration significantly attenuated the weight loss, increased DAI scores, colonic shortening, and increased MPO and iNOS activities caused by DSS in mice. Histological improvement was apparent, tight junctions in the colon were restored, and the levels of short-chain fatty acids (acetic acid, butyric acid and isovaleric acid) were increased. In addition, Rk3 reduced the expression of proinflammatory factors (TNF-α, IL-1ß and IL-6), NLRP3, ASC, and Caspase-1, indicating blockade of the NLRP3 inflammasome pathway. These results show that Rk3 can improve DSS-induced ulcerative colitis by protecting intestinal barrier function and inhibiting NLRP3 inflammasome expression, indicating that Rk3 could be used as a potential drug for treating ulcerative colitis.

Thymol Improves Barrier Function and Attenuates Inflammatory Responses in Porcine Intestinal Epithelial Cells during Lipopolysaccharide (LPS)-Induced Inflammation.

It is well-known that essential oil thymol exhibits antibacterial activity. The protective effects of thymol on pig intestine during inflammation is yet to be investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cells was established. Cells were pretreated with thymol for 1 h and then exposed to LPS for various assays. Interleukin 8 (IL-8) secretion, the mRNA abundance of cytokines, reactive oxygen species (ROS), nutrient transporters, and tight junction proteins was measured. The results showed that LPS stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) mRNA abundance ( P < 0.05), but the mRNA abundance of sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1), and H+/peptide cotransporter 1 (PepT1) were decreased ( P < 0.05). Thymol blocked ROS production ( P < 0.05) and tended to decrease the production of LPS-induced IL-8 secretion ( P = 0.0766). The mRNA abundance of IL-8 and TNF-α was reduced by thymol pretreatment ( P < 0.05), but thymol did not improve the gene expression of nutrient transporters ( P > 0.05). The transepithelial electrical resistance (TEER) was reduced and cell permeability increased by LPS treatment ( P < 0.05), but these effects were attenuated by thymol ( P < 0.05). Moreover, thymol increased zonula occludens-1 (ZO-1) and actin staining in the cells. However, the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS or thymol treatments. These results indicated that thymol enhances barrier function and reduce ROS production and pro-inflammatory cytokine gene expression in the epithelial cells during inflammation. The regulation of barrier function by thymol and LPS may be at post-transcriptional or post-translational levels.

Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors.

The expression pattern of tight junction proteins (TJPs) varies among organs and tumor types. In this study, we examined the immunoreactivity of claudin (CLDN)-1, -4, and -7, and JAM-A in salivary gland tumors (SGTs) by histological types and cell types to estimate their usefulness as differential diagnostic markers. Immunoreactivity of CLDN1 was higher in ductal epithelium cells of SGTs than in non-tumor tissues. Conversely, immunoreactivity of CLDN1 was significantly decreased in basal/myoepithelium cells of SGTs compared with that in non-tumor tissues. There was no significant difference between the immunoreactivity of CLDN1 in benign tumors and that in malignant tumors. Immunoreactivity of CLDN4, CLDN7, and JAM-A in ductal epithelium cells was higher in many SGTs than in non-tumor tissues. There was a difference depending on the histological type of SGT in immunoreactivity of CLDN4, CLDN7, and JAM-A in basaloid/myoepithelial cells. It was possible to classify SGTs by a hierarchical clustering using immunoreactivity of TJPs. The results suggest that an immunohistochemical marker panel including these TJPs may be useful for differential diagnosis of SGTs and that CLDN1 is associated with tumorigenesis of SGTs.

Granny Smith apple procyanidin extract upregulates tight junction protein expression and modulates oxidative stress and inflammation in lipopolysaccharide-induced Caco-2 cells.

The present work is undertaken to characterize a Granny Smith apple procyanidin extract (AE) and investigate the beneficial effect of the AE in the intestine in vitro. Each AE was characterized via LC-ESI-MS. Caco-2 cells were used to study the preventive actions of the AE against the downregulation of tight junction protein expression, oxidative stress and inflammation induced by lipopolysaccharides (LPS). Phenolic compounds present in the AE, including chlorogenic acid, catechin, epicatechin, proanthocyanidin dimers, and proanthocyanidin trimers, were characterized. The expression of the tight junction protein, including occludin and zona occludens (ZO)-1, increased significantly in LPS + AE treated Caco-2 cells, compared to LPS induced Caco-2 cells. Proanthocyanidin dimers had the most potent effect on increasing tight junction protein expression. The addition of LPS to Caco-2 cells induced oxidative stress and inflammation. However, incubation with proanthocyanidin dimers prevented LPS-mediated oxidative stress, including the increase of SOD, HO-1, CAT, and GSH-Px mRNA expression, and counteracted LPS-mediated inflammation as evidenced by the down-regulation of inflammatory markers (NF-κß, IL-6, and TNF-α mRNA expression). Our findings provide evidence that AE could upregulate tight junction protein expression, probably acting via the reduction of oxidative stress and inflammation.

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP-9 expression but not direct TJ proteins expression regulation.

Blood-spinal cord barrier (BSCB) disruption is a major process for the secondary injury of spinal cord injury (SCI) and is considered to be a therapeutic target for SCI. Previously, we demonstrated that metformin could improve functional recovery after SCI; however, the effect of metformin on BSCB is still unknown. In this study, we found that metformin could prevent the loss of tight junction (TJ) proteins at day 3 after SCI in vivo, but in vitro there was no significant difference of these proteins between control and metformin treatment in endothelial cells. This indicated that metformin-induced BSCB protection might not be mediated by up-regulating TJ proteins directly, but by inhibiting TJ proteins degradation. Thus, we investigated the role of metformin on MMP-9 and neutrophils infiltration. Neutrophils infiltration is the major source of the enhanced MMP-9 in SCI. Our results showed that metformin decreased MMP-9 production and blocked neutrophils infiltration at day 1 after injury, which might be related to ICAM-1 down-regulation. Also, our in vitro study showed that metformin inhibited TNF-α-induced MMP-9 up-regulation in neutrophils, which might be mediated via an AMPK-dependent pathway. Together, it illustrated that metformin prevented the breakdown of BSCB by inhibiting neutrophils infiltration and MMP-9 production, but not by up-regulating TJ proteins expression. Our study may help to better understand the working mechanism of metformin on SCI.

Manganese deficiency or excess caused the depression of intestinal immunity, induction of inflammation and dysfunction of the intestinal physical barrier, as regulated by NF-κB, TOR and Nrf2 signalling, in grass carp (Ctenopharyngodon idella).

Intestinal mucosal immune components and mRNA levels of inflammatory cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules in young grass carp (Ctenopharyngodon idellus) under dietary manganese (Mn) deficiency or excess were investigated. Fish were fed the diets containing graded levels of Mn [3.65-27.86 mg Mn kg(-1) diet] for 8 weeks. The results demonstrated that Mn deficiency significantly decreased the lysozyme and acid phosphatase (ACP) activities, up-regulated tumour necrosis factor α (TNF-α), interleukin 8 and the signalling factor nuclear factor-κB p65, and down-regulated interleukin 10 (IL-10), transforming growth factor ß1, inhibitor of signalling factors κB-α and target of rapamycin mRNA levels in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI). However, Mn deficiency did not change the C3 content in the PI, whereas it decreased the C3 contents in the MI and DI. Additionally, Mn depletion also resulted in significantly low mRNA levels for tight junction proteins (claudin-b, claudin-c, claudin-15, occludin and zonula occludens-1), antioxidant enzymes (MnSOD, GPx and CAT) and NF-E2-related factor-2 in the intestines of fish. Excessive Mn exhibited toxic effects similar to Mn deficiency, where optimal Mn contents reversed those indicators. In conclusion, Mn deficiency or excess causes the depression of intestinal immunity, induction of inflammation and dysfunction of the intestinal physical barrier relating to NF-κB, TOR and Nrf2 signalling in grass carp. Furthermore, quadratic regression analysis at 95% maximum response of lysozyme and acid phosphatase activities in the distal intestine of young grass carp revealed the optimum dietary Mn levels to be 8.90 and 8.99 mg kg(-1) diet, respectively.

Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation.

Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.

Interferon-γ-induced intestinal epithelial barrier dysfunction by NF-κB/HIF-1α pathway.

Interferon-γ (IFN-γ) plays an important role in intestinal barrier dysfunction. However, the mechanisms are not fully understood. As hypoxia-inducible factor-1 (HIF-1) is a critical determinant response to hypoxia and inflammation, which has been shown to be deleterious to intestinal barrier function, we hypothesized that IFN-γ induces loss of barrier function through the regulation of HIF-1α activation and function. In this study, we detected the expressions of HIF-1α and tight junction proteins in IFN-γ-treated T84 intestinal epithelial cell line. IFN-γ led to an increase of HIF-1α expression in time- and dose-dependent manners but did not change the expression of HIF-1ß. The IFN-γ-induced increase in HIF-1α was associated with an activation of NF-κB. Treatment with the NF-κB inhibitor, pyrolidinedithiocarbamate (PDTC), significantly suppressed the activation of NF-κB and the expression of HIF-1α. In addition, IFN-γ also increased intestinal epithelial permeability and depletion of tight junction proteins; inhibition of NF-κB or HIF-1α prevented the increase in intestinal permeability and alteration in tight junction protein expressions. Interestingly, we demonstrated that a significant portion of IFN-γ activation NF-kB and modulation tight junction expression is mediated through HIF-1α. Taken together, this study suggested that IFN-γ induced the loss of epithelial barrier function and disruption of tight junction proteins, by upregulation of HIF-1α expression through NF-κB pathway.

Dysregulation of claudin-5 in HIV-induced interstitial pneumonitis and lung vascular injury. Protective role of peroxisome proliferator-activated receptor-γ.

RATIONALE: HIV-1-induced interstitial pneumonitis (IP) is a serious complication of HIV-1 infection, characterized by inflammation and cellular infiltration in lungs, often leading to respiratory failure and death. The barrier function of the pulmonary endothelium is caused in part by tight junction (TJ) proteins, such as claudin-5. Peroxisome proliferator-activated receptor (PPAR)-γ is expressed in lung tissues and regulates inflammation. We hypothesize that HIV-1 induces vascular lung injury, and HIV-1-mediated damage of the pulmonary endothelium and IP is associated with dysregulation of PPAR-γ. OBJECTIVES: Investigate the effects of HIV-1 infection on the pulmonary microvasculature and the modulatory effects of the PPAR-γ ligands. METHODS: Using human lung tissues, we demonstrated down-regulation of claudin-5 (marker of pulmonary barrier integrity), down-regulation of PPAR-γ transcription, and expression in lung tissues of HIV-1-infected humans with IP. MEASUREMENTS AND MAIN RESULTS: Human lung microvascular endothelial cells expressed the TJ proteins claudin-5, ZO-1, and ZO-2; HIV-1 decreased TJ proteins expression and induced nuclear factor-κB promoter activity, which was reversed by PPAR-γ agonist. Using two murine HIV/AIDS models, we demonstrated decreased claudin-5 expression and increased macrophage infiltration in the lungs of HIV-1-infected animals. Activation of PPAR-γ prevented HIV-1-induced claudin-5 down-regulation and significantly reduced viremia and pulmonary macrophage infiltration. CONCLUSIONS: HIV-induced IP is associated with injury to the lung vascular endothelium, with decreased TJ and PPAR-γ expression, and increased pulmonary macrophage infiltration. PPAR-γ ligands abrogated these effects. Thus, regulation of PPAR-γ can be a therapeutic approach against HIV-1-induced vascular damage and IP in infected humans. Removal of Expression of Concern: Issues leading to the previous expression of concern for this article have been resolved after further revisions and editorial review. No further concerns exist.

Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

BACKGROUND AND OBJECTIVE: Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. MATERIAL AND METHODS: An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. RESULTS: The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. CONCLUSION: There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin-like receptors of commensal oral streptococci could mediate the phenotype of health, whereas pathogenic organisms associated with periodontal disease might not signal effectively through CD24.

Immunologic and chemical targeting of the tight-junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells.

The tumorigenicity of human pluripotent stem cells is a major safety concern for their application in regenerative medicine. Here we identify the tight-junction protein Claudin-6 as a cell-surface-specific marker of human pluripotent stem cells that can be used to selectively remove Claudin-6-positive cells from mixed cultures. We show that Claudin-6 is absent in adult tissues but highly expressed in undifferentiated cells, where it is dispensable for human pluripotent stem cell survival and self-renewal. We use three different strategies to remove Claudin-6-positive cells from mixed cell populations: an antibody against Claudin-6; a cytotoxin-conjugated antibody that selectively targets undifferentiated cells; and Clostridium perfringens enterotoxin, a toxin that binds several Claudins, including Claudin-6, and efficiently kills undifferentiated cells, thus eliminating the tumorigenic potential of human pluripotent stem cell-containing cultures. This work provides a proof of concept for the use of Claudin-6 to eliminate residual undifferentiated human pluripotent stem cells from culture, highlighting a strategy that may increase the safety of human pluripotent stem cell-based cell therapies.