Disruption Of Epithelial Tight Junctions Research Articles

Streptococcus salivarius ameliorates the destructive effect on the epithelial barrier by inhibiting the growth of Prevotella melaninogenica via metabolic acid production.

Oral lichen planus (OLP) is one of the most common oral mucosal diseases, exhibiting a higher prevalence in women than men, but its pathogenesis is still unclear. Current research suggests that microbial dysbiosis may play an important role in the pathogenesis of OLP. Our previous research has found that the increase of Prevotella melaninogenica and decrease of Streptococcus salivarius have been identified as a potential pathogenic factor in OLP. Consequently, the objective of this study is to examine whether S. salivarius can counteract the detrimental effects of P. melaninogenica on the integrity of the epithelial barrier function. Epithelial barrier disruption was induced by P. melaninogenica in human keratinocytes (HaCaT cells). HaCaT cells were pretreated with S. salivarius(MOI=20) or cell-free supernatant for 3h, followed by treatment with P. melaninogenica (MOI=5) for 3h. The epithelial barrier integrity of HaCaT cells was detected by FD4 permeability. The mRNA level of tight junction protein was detected by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and Western Blot were used to detect the protein expression of zonula occludin-1 (ZO-1). The serial dilution-spotting assay was applied to monitor the viability of P. melaninogenica at the end of 8 and 24h incubation. Challenge by P. melaninogenica decreased the levels of tight junction proteins, including occludin, ZO-1, and claudin in HaCaT cells. S. salivarius or its cell-free supernatant inhibited the down-regulation of ZO-1 mRNA and protein expression levels induced by P. melaninogenica and thus improved the epithelial barrier function. The inhibitory effect of the cell-free supernatant of S. salivarius on the growth of P. melaninogenica is associated with metabolic acid production rather than with bacteriocins and hydrogen peroxide. These results suggest that live S. salivarius or its cell-free supernatant significantly ameliorated the disruption of epithelial tight junctions induced by P. melaninogenica, likely through the inhibition of P. melaninogenica growth mediated by metabolic acid production.

Hydrogen alleviates impaired lung epithelial barrier in acute respiratory distress syndrome via inhibiting Drp1-mediated mitochondrial fission through the Trx1 pathway

Acute respiratory distress syndrome (ARDS) is an acute and severe clinical complication lacking effective therapeutic interventions. The disruption of the lung epithelial barrier plays a crucial role in ARDS pathogenesis. Recent studies have proposed the involvement of abnormal mitochondrial dynamics mediated by dynamin-related protein 1 (Drp1) in the mechanism of impaired epithelial barrier in ARDS. Hydrogen is an anti-oxidative stress molecule that regulates mitochondrial function via multiple signaling pathways. Our previous study confirmed that hydrogen modulated oxidative stress and attenuated acute pulmonary edema in ARDS by upregulating thioredoxin 1 (Trx1) expression, but the exact mechanism remains unclear. This study aimed to investigate the effects of hydrogen on mitochondrial dynamics both in vivo and in vitro. Our study revealed that hydrogen inhibited lipopolysaccharide (LPS)-induced phosphorylation of Drp1 (at Ser616), suppressed Drp1-mediated mitochondrial fission, alleviated epithelial tight junction damage and cell apoptosis, and improved the integrity of the epithelial barrier. This process was associated with the upregulation of Trx1 in lung epithelial tissues of ARDS mice by hydrogen. In addition, hydrogen treatment reduced the production of reactive oxygen species in LPS-induced airway epithelial cells (AECs) and increased the mitochondrial membrane potential, indicating that the mitochondrial dysfunction was restored. Then, the expression of tight junction proteins occludin and zonula occludens 1 was upregulated, and apoptosis in AECs was alleviated. Remarkably, the protective effects of hydrogen on the mitochondrial and epithelial barrier were eliminated after applying the Trx1 inhibitor PX-12. The results showed that hydrogen significantly inhibited the cell apoptosis and the disruption of epithelial tight junctions, maintaining the integrity of the epithelial barrier in mice of ARDS. This might be related to the inhibition of Drp1-mediated mitochondrial fission through the Trx1 pathway. The findings of this study provided a new theoretical basis for the application of hydrogen in the clinical treatment of ARDS.

Roxadustat Attenuates the Disruption of Epithelial Tight Junction in Caco2 Cells and a Rat Model of CKD Through MicroRNA-223.

IntroductionIncreasing evidence supports the idea that the disruption of epithelial tight junction proteins (TJPs) caused by accumulation of uremia toxins, such as homocysteine (Hcy), is one of the most important mechanisms underlying the damage of intestinal barrier function (IBF) in chronic kidney disease (CKD). Since the decrease of hypoxia inducible factor-1α (HIF-1α) is reported to be involved in Hcy-induced cell injury, and the upregulation of microRNA-223 (miR-223) plays a vital protective role in the impairment of IBF in the experimental colitis, we investigated the effect of HIF-1α stabilizer roxadustat on the disruption of TJPs induced by Hcy and CKD and the underlying mechanism.MethodsChronic kidney disease was induced in rats via 5/6 nephrectomy. In a series of experiments, the rats were treated orally with roxadustat of different doses. The expression of tight junction proteins, HIF-1α, and miR-223 was analyzed in different groups by western blotting analysis, RT-qPCR techniques and immunofluorescence. A series of experiments with cultured Caco2 cells was performed.ResultsThe results showed that the expression of TJPs (occludin, claudin-1, and ZO-1) decreased significantly, accompanied by the reduction of HIF-1α and miR-223 in Hcy-treated Caco2 cells and colonic mucosa of uremic rats. The reduction of HIF-1α and miR-223 was reversed by roxadustat and the decrease of TJPs expression was attenuated in both Caco2 cells induced by Hcy and colon tissue of CKD rats. Furthermore, transfection with miR-223 mimics increased the expression of TJPs, while transfection with miR-223 inhibitor decreased their expression in Caco2 cells. MiR-223 inhibitor applied before roxadustat treatment partly diminished the effect of roxadustat on TJPs expression in Caco2 cells.ConclusionThese results indicated that roxadustat attenuated the disruption of epithelial TJPs induced by Hcy in Caco2 cells and the damage of colonic epithelium in CKD rats through the upregulation of miR-223 induced by HIF-1α. A novel insight into the IBF dysfunction in CKD was provided, and it suggests a potential therapeutic use of roxadustat for the IBF dysfunction besides anemia in CKD.

Central role of intestinal epithelial glucocorticoid receptor in alcohol- and corticosterone-induced gut permeability and systemic response.

Corticosterone, the stress hormone, exacerbates alcohol‐associated tissue injury, but the mechanism involved is unknown. We examined the role of the glucocorticoid receptor (GR) in corticosterone‐mediated potentiation of alcohol‐induced gut barrier dysfunction and systemic response. Hepatocyte‐specific GR‐deficient (GRΔHC ) and intestinal epithelial‐specific GR‐deficient (GRΔIEC ) mice were fed ethanol, combined with corticosterone treatment. Intestinal epithelial tight junction integrity, mucosal barrier function, microbiota dysbiosis, endotoxemia, systemic inflammation, liver damage, and neuroinflammation were assessed. Corticosterone potentiated ethanol‐induced epithelial tight junction disruption, mucosal permeability, and inflammatory response in GRΔHC mouse colon; these effects of ethanol and corticosterone were absent in GRΔIEC mice. Gut microbiota compositions in ethanol‐fed GRΔHC and GRΔIEC mice were similar to each other. However, corticosterone treatment in ethanol‐fed mice shifted the microbiota composition to distinctly different directions in GRΔHC and GRΔIEC mice. Ethanol and corticosterone synergistically elevated the abundance of Enterobacteriaceae and Escherichia coli and reduced the abundance of Lactobacillus in GRΔHC mice but not in GRΔIEC mice. In GRΔHC mice, corticosterone potentiated ethanol‐induced endotoxemia and systemic inflammation, but these effects were absent in GRΔIEC mice. Interestingly, ethanol‐induced liver damage and its potentiation by corticosterone were observed in GRΔHC mice but not in GRΔIEC mice. GRΔIEC mice were also resistant to ethanol‐ and corticosterone‐induced inflammatory response in the hypothalamus. These data indicate that the intestinal epithelial GR plays a central role in alcohol‐ and corticosterone‐induced gut barrier dysfunction, microbiota dysbiosis, endotoxemia, systemic inflammation, liver damage, and neuroinflammation. This study identifies a novel target for potential therapeutic for alcohol‐associated tissue injury.

GUCY2C‐Directed CAR‐T Cell Therapy for Upper‐GI Cancers

Gastrointestinal (GI) cancers have dismal 5-year survival rates (19% in esophagus, 30% in stomach, 9% in pancreas, 65% in colorectal) and account for 20% of all cancer-related deaths. Development of safe and effective therapies is limited, and the impact of groundbreaking immunotherapies has been only minor for GI cancers. The intestinal epithelial protein, guanylyl cyclase C (GUCY2C) is found in nearly all primary and metastatic colorectal cancers and about 65% of pancreatic, gastric, and esophageal cancers. Disruption of epithelial tight junctions, cell polarity, and apical localization of GUCY2C makes it a desired target for systemic agents in tumor tissues while leaving normal tissues unaffected. Indeed, we have safely translated GUCY2C-targeted vaccines into clinical trials to prevent GI cancer recurrence in early-stage patients. CAR-T cell therapy employs bulk T cells of unknown specificity obtained from patients, engineered to express chimeric antigen-specific receptors (CARs), and administered to the patient to mediate remarkable antitumor effects for advanced hematologic cancers. We have generated GUCY2C-directed CAR-T cells which produce effector cytokines and lyse colorectal cancer cell lines. The joint safety and antitumor efficacy of GUCY2C-specific CAR-T cells were demonstrated in mice receiving CAR-T cells directed towards mouse GUCY2C. Here, we established patient-derived xenograft (PDX) models in mice by surgical implantation of GI cancer specimens and a clinical manufacturing process to explore the efficacy of GUCY2C-directed CAR-T cells that can be translated to GI cancer patients. While GUCY2C was present in nearly all colorectal cancer specimens collected by surgical resection, it was present in only 33% of gastroesophageal resection samples. Interestingly, those upper GI patients with detectable GUCY2C were treatment-naïve, while neoadjuvant-treated patients lacked viable tumor, revealing neoadjuvant therapy as a significant barrier to upper GI cancer model generation (neoadjuvant chemoradiation is uncommon in colon cancer patients). In contrast, 66% of gastroesophageal specimens collected by endoscopic biopsy in treatment-naïve patients express GUCY2C and >50% successfully formed PDX models. Gastroesophageal adenocarcinoma PDX models were histologically similar to their matched primary tumors and retained GUCY2C expression. Importantly, GUCY2C-directed CAR-T cells controlled GI cancer PDX growth, maintaining a >12-fold reduction in tumor volume compared to control and in some cases produced complete tumor elimination. In the context of previously established safety in mouse models, these studies support future translation of GUCY2C CAR-T cell therapy to patients with advanced upper-GI cancers.

Differential relieving effects of shikonin and its derivatives on inflammation and mucosal barrier damage caused by ulcerative colitis.

BackgroundUlcerative colitis (UC) is one of the most challenging human diseases. Natural shikonin (SK) and its derivatives (with have higher accumulation) isolated from the root of Lithospermum erythrorhizon have numerous beneficial effects, such as wound healing and anti-inflammatory activities. Some researchers have reported that hydroxynaphthoquinone mixture (HM) and SK attenuate the acute UC induced by dextran sulfate sodium (DSS). However, no existing study has systemically investigated the effectiveness of SK and other hydroxynaphthoquinone natural derivative monomers on UC.MethodsIn this study, mice were treated with SK and its derivatives (25 mg/kg) and mesalazine (200 mg/kg) after DSS administration daily for one week. Disease progression was monitored daily by observing the changes in clinical signs and body weight.ResultsIntragastric administration natural single naphthoquinone attenuated the malignant symptoms induced by DSS. SK or its derivatives remarkably suppressed the serum levels of pro-inflammatory cytokines while increasing the inflammatory cytokine interleukin (IL)-10 . Additionally, both SK and alkanin restrained the activities of cyclooxygenase-2 (COX-2), myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) in serum and colonic tissues. SK and its derivatives inhibited the activation of nucleotide binding oligomerization domain-like receptors (NLRP3) inflammasome and NF-κB signaling pathway, thereby relieving the DSS-induced disruption of epithelial tight junction (TJ) in colonic tissues.ConclusionsOur findings shed more lights on the pharmacological efficacy of SK and its derivatives in UC against inflammation and mucosal barrier damage.

Anisakis simplex products impair intestinal epithelial barrier function and occludin and zonula occludens-1 localisation in differentiated Caco-2 cells.

BackgroundAnisakis spp. are nematode parasites found in a wide range of marine organisms. Human beings may accidentally become infected, showing the symptoms of anisakiasis and allergic responses. There has been evidence of increased intestinal permeability in A. simplex–sensitized subjects and that specific IgE titres increase in some allergic patients when fishery products are re-introduced into their diet. The aims of this work were to study the effect of A. simplex crude extract on the intestinal integrity and permeability by using Caco-2 cell monolayer. To analyse the capacity of Ani s 4 allergen to cross the epithelial barrier.Methodology/Principal findingsCellular bioenergetics, transepithelial electrical resistance, viability, permeability, reactive oxygen species generation and immunofluorescent staining of tight junction proteins were analysed. A. simplex crude extract compromises the Caco-2 cell monolayer integrity in a dose-dependent manner. This effect is detected at 1 hour of culture and integrity is recovered after 24 hours of culture. The epithelial barrier disruption is accompanied by an increase in paracellular permeability and reactive oxygen species production and by a delocalization of occludin and zonula occludens-1. Finally, Ani s 4, a thermostable and resistant to digestion allergen with cystatin activity, is able to cross the epithelial barrier in Caco-2 monolayer and reach a cumulative mean percentage of 22.7% of total concentration in the basolateral side after 24 hours of culture.Conclusions/SignificanceOur results demonstrate that A. simplex induces an early and reversible alteration of integrity and permeability of Caco-2 cell monolayer and that an underlying mechanism of this effect would involve the oxidative stress and disruption of epithelial tight junctions. Additionally, it has been shown that Ani s 4 allergen is able to cross the epithelial barrier. These findings could explain the increased intestinal permeability observed in Anisakis-sensitized patients, the changes over time in IgE sensitization to A. simplex allergens, and the specific IgE persistence in Anisakis allergy.

Acute Respiratory Barrier Disruption by Ozone Exposure in Mice.

Ozone exposure causes irritation, airway hyperreactivity (AHR), inflammation of the airways, and destruction of alveoli (emphysema), the gas exchange area of the lung in human and mice. This review focuses on the acute disruption of the respiratory epithelial barrier in mice. A single high dose ozone exposure (1 ppm for 1 h) causes first a break of the bronchiolar epithelium within 2 h with leak of serum proteins in the broncho-alveolar space, disruption of epithelial tight junctions and cell death, which is followed at 6 h by ROS activation, AHR, myeloid cell recruitment, and remodeling. High ROS levels activate a novel PGAM5 phosphatase dependent cell-death pathway, called oxeiptosis. Bronchiolar cell wall damage and inflammation upon a single ozone exposure are reversible. However, chronic ozone exposure leads to progressive and irreversible loss of alveolar epithelial cells and alveoli with reduced gas exchange space known as emphysema. It is further associated with chronic inflammation and fibrosis of the lung, resembling other environmental pollutants and cigarette smoke in pathogenesis of asthma, and chronic obstructive pulmonary disease (COPD). Here, we review recent data on the mechanisms of ozone induced injury on the different cell types and pathways with a focus on the role of the IL-1 family cytokines and the related IL-33. The relation of chronic ozone exposure induced lung disease with asthma and COPD and the fact that ozone exacerbates asthma and COPD is emphasized.

Intestinal epithelial PKM2 serves as a safeguard against experimental colitis via activating β-catenin signaling.

The pyruvate kinase M2 (PKM2)-mediated aerobic glycolysis has been shown to play a critical role in promoting cell survival and proliferation. However, little is known about the function of intestinal epithelial PKM2 in intestine homeostasis. Here we investigate whether and how intestinal epithelial PKM2 modulates the morphology and function of the adult intestine in experimental colitis. Analyzing colonoscopic biopsies from Crohn's disease and ulcerative colitis patients, we found significantly decreased level of intestinal epithelial PKM2 in patients compared to that in non-inflamed tissues. Similar reduction of intestinal epithelial PKM2 was observed in mice with dextran sulfate sodium-induced colitis. Moreover, intestinal epithelial-specific PKM2-knockout (Pkm2-/-) mice displayed more severe intestinal inflammation, as evidenced by a shortened colon, disruption of epithelial tight junctions, an increase in inflammatory cytokine levels, and immune cell infiltration, when compared to wild-type mice. Gene profiling, western blot, and function analyses indicated that cell survival signals, particularly the Wnt/β-catenin pathways, were associated with PKM2 activity. Increasing mouse intestinal epithelial PKM2 expression via delivery of a PKM2-expressing plasmid attenuated experimental colitis. In conclusion, our studies demonstrate that intestinal epithelial PKM2 increases cell survival and wound healing under the colitic condition via activating the Wnt/β-catenin signaling.

Specifically Sized Hyaluronan (35kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.

Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4days) and treated with or without HA35 via daily gavage during the last 3days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.

Pathogenicity of In Vivo Generated Intestinal Th17 Lymphocytes is IFNγ Dependent.

T helper 17 [Th17] cells are crucially involved in the immunopathogenesis of inflammatory bowel diseases in humans. Nevertheless, pharmacological blockade of interleukin 17A [IL17A], the Th17 signature cytokine, yielded negative results in patients with Crohn's disease [CD], and attempts to elucidate the determinants of Th17 cells' pathogenicity in the gut have so far proved unsuccessful. Here, we aimed to identify and functionally validate the pathogenic determinants of intestinal IL-17-producing T cells. In vivo-generated murine intestinal IL-17-producing T cells were adoptively transferred into immunodeficient Rag1-/- recipients to test their pathogenicity. Human IL-17, IFNγ/IL-17, and IFNγ actively secreting T cell clones were generated from lamina propria lymphocytes of CD patients. The pathogenic activity of intestinal IL-17-producing T cells against the intestinal epithelium was evaluated. IL-17-producing cells with variable colitogenic activity can be generated in vivo using different experimental colitis models. The pathogenicity of IL-17-secreting cells was directly dependent on their IFNγ secretion capacity, as demonstrated by the reduced colitogenic activity of IL-17-secreting cells isolated from IFNγ-/- mice. Moreover, IFNγ production is a distinguished attribute of CD-derived lamina propria Th17 cells. IFNγ secretion by CD-derived IL-17-producing intestinal clones is directly implicated in the epithelial barrier disruption through the modulation of tight junction proteins. Intestinal Th17 cell pathogenicity is associated with IFNγ production, which directly affects intestinal permeability through the disruption of epithelial tight junctions.

Pneumolysin plays a key role at the initial step of establishing pneumococcal nasal colonization

Nasopharyngeal colonization by Streptococcus pneumoniae is an important initial step for the subsequent development of pneumococcal infections. Pneumococci have many virulence factors that play a role in colonization. Pneumolysin (PLY), a pivotal pneumococcal virulence factor for invasive disease, causes severe tissue damage and inflammation with disruption of epithelial tight junctions. In this study, we evaluated the role of PLY in nasal colonization of S. pneumoniae using a mouse colonization model. A reduction of numbers of PLY-deficient pneumococci recovered from nasal tissue, as well as nasal wash, was observed at days 1 and 2 post-intranasal challenges, but not later. The findings strongly support an important role for PLY in the initial establishment nasal colonization. PLY-dependent invasion of local nasal mucosa may be required to establish nasal colonization with S. pneumoniae. The data help provide a rationale to explain why an organism that exists as an asymptomatic colonizer has evolved virulence factors that enable it to occasionally invade and kill its hosts. Thus, the same pneumococcal virulence factor, PLY that can contribute to killing the host, may also play a role early in the establishment of nasopharynx carriage.

Epithelial barrier and oral bacterial infection.

The oral epithelial barrier separates the host from the environment and provides the first line of defense against pathogens, exogenous substances and mechanical stress. It consists of underlying connective tissue and a stratified keratinized epithelium with a basement membrane, whose cells undergo terminal differentiation resulting in the formation of a mechanically resistant surface. Gingival keratinocytes are connected by various transmembrane proteins, such as tight junctions, adherens junctions and gap junctions, each of which has a specialized structure and specific functions. Periodontal pathogens are able to induce inflammatory responses that lead to attachment loss and periodontal destruction. A number of studies have demonstrated that the characteristics of pathogenic oral bacteria influence the expression and structural integrity of different cell-cell junctions. Tissue destruction can be mediated by host cells following stimulation with cytokines and bacterial products. Keratinocytes, the main cell type in gingival epithelial tissues, express a variety of proinflammatory cytokines and chemokines, including interleukin-1alpha, interleukin-1beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha. Furthermore, the inflammatory mediators that may be secreted by oral keratinocytes are vascular endothelial growth factor, prostaglandin E2 , interleukin-1 receptor antagonist and chemokine (C-C motif) ligand 2. The protein family of matrix metalloproteinases is able to degrade all types of extracellular matrix protein, and can process a number of bioactive molecules. Matrix metalloproteinase activities under inflammatory conditions are mostly deregulated and often increased, and those mainly relevant in periodontal disease are matrix metalloproteinases 1, 2, 3, 8, 9, 13 and 24. Viral infection may also influence the epithelial barrier. Studies show that the expression of HIV proteins in the mucosal epithelium is correlated with the disruption of epithelial tight junctions, suggesting a possible enhancement of human papilloma virus infection by HIV-associated disruption of tight junctions. Altered expression of matrix metalloproteinases was demonstrated in keratinocytes transformed with human papilloma virus-16 or papilloma virus-18,. To summarize, the oral epithelium is able to react to a variety of exogenous, possibly noxious influences.

NOS-2 Inhibition in Phosgene-Induced Acute Lung Injury.

Phosgene exposure via an industrial or warfare release produces severe acute lung injury (ALI) with high mortality, characterized by massive pulmonary edema, disruption of epithelial tight junctions, surfactant dysfunction, and oxidative stress. There are no targeted treatments for phosgene-induced ALI. Previous studies demonstrated that nitric oxide synthase 2 (NOS-2) is upregulated in the lungs after phosgene exposure; however, the role of NOS-2 in the pathogenesis of phosgene-induced ALI remains unknown. We previously demonstrated that NOS-2 expression in lung epithelium exacerbates inhaled endotoxin-induced ALI in mice, mediated partially through downregulation of surfactant protein B (SP-B) expression. Therefore, we hypothesized that a selective NOS-2 inhibitor delivered to the lung epithelium by inhalation would mitigate phosgene-induced ALI. Inhaled phosgene produced increases in bronchoalveolar lavage fluid protein, histologic lung injury, and lung NOS-2 expression at 24 h. Administration of the selective NOS-2 inhibitor 1400 W via inhalation, but not via systemic delivery, significantly attenuated phosgene-induced ALI and preserved epithelial barrier integrity. Furthermore, aerosolized 1400 W augmented expression of SP-B and prevented downregulation of tight junction protein zonula occludens 1 (ZO-1), both critical for maintenance of normal lung physiology and barrier integrity. We also demonstrate for the first time that NOS-2-derived nitric oxide downregulates the ZO-1 expression at the transcriptional level in human lung epithelial cells, providing a novel target for ameliorating vascular leak in ALI. Our data demonstrate that lung NOS-2 plays a critical role in the development of phosgene-induced ALI and suggest that aerosolized NOS-2 inhibitors offer a novel therapeutic strategy for its treatment.

Mo1719 Epithelial NF-κB Activation via TNF Signaling May Be Involved in the Development of Colitis-Associated Carcinogenesis

Background & Aim: Prolonged inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease may promote carcinogenesis in the epithelia. It has been reported that activation of NF-κB pathway in both intestinal epithelial cells and myeloid cells are significant for the development of colitis-associated cancer (CAC). We previously reported that specific up-regulation of the type 2 receptor for tumor necrosis factor (TNFR2) rather than TNFR1 expression was observed in the inflamed colonic epithelia and further in the CAC. It is known that TNFR2 signaling may induce NF-κB activation, but the role of its expression in the setting of CAC has not been elucidated. Here we analyzed TNFR2 signaling in the colonic epithelial cells. Methods & Results: As previously observed in animal models of colitis and CAC, the expression of TNFR2 was up-regulated in an epithelial cell line, MOC1, which was derived from murine colonic ‘non-cancer' tissue, when stimulated with recombinant (r) IFN-γ. MOC1 cells incubated with both rIFN-γ and rTNF showed that NFκB pathway was activated rather than apoptosis pathway. Furthermore, the activated NFκB in MOC1 cells was associated with the expression of myosin light chain kinase (MLCK) as well as disrupted tight junction (TJ) in a rTNF dose-dependent manner. Such MLCK upregulation and TJ disruption in MOC1 cells were abrogated by either anti-TNF mAb (MP6XT22), TNFR2-specific siRNA or even MLCK inhibitor (ML-7). Using an animal model of CAC involving azoxymethane (AOM) and dextran sodium sulfate (DSS), the colonic lamina propria was found to have pro-tumorigenic cytokine production such as IL-1β, IL-6 and MIP-2 in association with epithelial NF-κB activation, TNFR2 and MLCK up-regulations and epithelial TJ disruption. AOM and DSS-administered mice with either MP6-XT22 or ML7 treatment failed to show significant abrogation of colitis severity, however such treatments revealed the restored epithelial TJ and decreased pro-tumorigenic cytokine production in the colonic tissues in association with the reduced CAC development. Conclusions: Our studies showed that epithelial NF-κB activation via TNFR2 signaling in the context of IBD may be involved in the epithelial permeabilization and pro-tumorigenic cytokine production that result in the induction of CAC development.

Role of Nrf2 dysfunction in uremia-associated intestinal inflammation and epithelial barrier disruption.

Gut inflammation is prevalent in chronic kidney disease (CKD) and likely contributes to systemic inflammation via disruption of the epithelial tight junction with subsequent endotoxin and bacterial translocation. To study the expression profile of inflammatory and tight junction proteins in the colon from CKD rats compared to healthy controls, and demonstrate the role of Nrf2 (transcription factor nuclear factor erythroid 2-related factor 2) using a potent Nrf2 activator. CKD was induced via 5/6 nephrectomy in Sprague-Dawley rats, and dh404 (2 mg/kg/day) was used to study the effects of systemic Nrf2 activation. The experimental groups included sham, CKD and CKD+ dh404 rats. Blood and colon tissues were analyzed after a 10-week study period. Colon from CKD rats showed histological evidence of colitis, depletion of epithelial tight junction proteins, significant reduction of Nrf2 and its measured target gene products (NQO1, catalase, and CuZn SOD), activation of NFkB, and upregulation of pro-inflammatory molecules (COX-2, MCP-1, iNOS, and gp91(phox)). Treatment with dh404 attenuated colonic inflammation, restored Nrf2 activity and levels of NQO1, catalase and CuZn SOD, decreased NFkB and lowered expression of COX-2, MCP-1, iNOS, and gp91(phox). This was associated with restoration of colonic epithelial tight junction proteins (occludin and claudin-1). CKD rats exhibited colitis, disruption of colonic epithelial tight junction, activation of inflammatory mediators, and impairment of Nrf2 pathway. Treatment with an Nrf2 activator restored Nrf2 activity, attenuated colonic inflammation, and restored epithelial tight junction proteins.

Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions.

The in vitro analysis of bacterial–epithelial interactions in the intestine has been hampered by a lack of suitable intestinal epithelium culture systems. Here, we report a new experimental model using an organoid culture system to study pathophysiology of bacterial–epithelial interactions post Salmonella infection. Using crypt‐derived mouse intestinal organoids, we were able to visualize the invasiveness of Salmonella and the morphologic changes of the organoids. Importantly, we reported bacteria‐induced disruption of epithelial tight junctions in the infected organoids. In addition, we showed the inflammatory responses through activation of the NF‐κB pathway in the organoids. Moreover, our western blot, PCR, and immunofluorescence data demonstrated that stem cell markers (Lgr5 and Bmi1) were significantly decreased by Salmonella infection (determined using GFP‐labeled Lgr5 organoids). For the first time, we created a model system that recapitulated a number of observations from in vivo studies of the Salmonella‐infected intestine, including bacterial invasion, altered tight junctions, inflammatory responses, and decreased stem cells. We have demonstrated that the Salmonella‐infected organoid culture system is a new experimental model suitable for studying host–bacterial interactions.

Hirsutenone reduces deterioration of tight junction proteins through EGFR/Akt and ERK1/2 pathway both converging to HO-1 induction

Oxidative stress-induced disruption of epithelial tight junctions (TJ) plays a critical role in the pathogenesis of intestinal disorders, including inflammatory bowel disease (IBD). The current study investigated the protective effect of hirsutenone against disruption of the intestinal barrier in vitro and in a mouse model of colitis. Caco-2 cells were stimulated with tert-butyl hydroperoxide (t-BH). Hirsutenone prevented the t-BH-induced increase in permeability by inhibiting the reduction in zonula occludens-1 (ZO-1) expression, and rapidly stimulated tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Hirsutenone-mediated protection against the loss of ZO-1 depends on the activation of both ERK1/2 and Akt signaling pathways. Interestingly, hirsutenone-mediated activation of Akt, but not ERK1/2, signaling was EGFR-dependent. Hirsutenone increased heme oxygenase-1 (HO-1) expression through both EGFR/Akt- and ERK1/2-dependent pathways, contributing to the protective effects against TJ dysfunction. Colitis was induced in mice by intrarectal administration of 2,4,6,-trinitrobenzene sulfonic acid (TNBS). Hirsutenone administration improved the clinical parameters and tissue histological appearance, increased HO-1 expression, attenuated reduction of ZO-1 and occludin mRNA, and promoted BrdU incorporation in the colonic epithelium of TNBS-treated mice. Taken together, our results demonstrate that hirsutenone reverse disordered intestinal permeability by activating EGFR/Akt and ERK1/2 pathways, which are involved in the regulation of HO-1 expression. These findings highlight the potential of hirsutenone for clinical applications in the treatment of IBD.

Mo1946 Interaction of EPEC EspF With 14-3-3 Drives the Redistribution of the Polarity Protein Par1b Leading to Disruption of Epithelial Tight Junctions

Mo1946 Interaction of EPEC EspF With 14-3-3 Drives the Redistribution of the Polarity Protein Par1b Leading to Disruption of Epithelial Tight Junctions

Regulation of epithelial cell tight junctions by protease-activated receptor 2.

ABSTRACTA layer of epithelial cells prevents the invasion of bacteria and the entry of foreign substances into the underlying tissue. The disruption of epithelial tight junctions initiates and exacerbates inflammation. However, the precise mechanism underlying the disruption of the epithelial tight junction remains unclear. The activation of protease-activated receptor 2 (PAR2) by serine proteases produced by some bacteria and mast cells contributes to inflammation in many tissues. In the present study, we tested the hypothesis that PAR2 activation affects the structure and function of tight junctions in Madin-Darby canine kidney (MDCK) cells. Although the application of a PAR2-activating peptide, PAR2-AP, from the apical side of MDCK cells failed to modify the transepithelial resistance (TER), its application from the basal side markedly suppressed the TER. In 3-dimensional cultures of MDCK cells expressing the mCherry-tagged PAR2, a lateral localization of PAR2 was observed. The application of PAR2-AP from the basal side changed the localization of the tight junctional protein, zonula occludin-1. Furthermore, PAR2-AP induced the phosphorylation of p38 MAP kinase. A p38 MAP kinase inhibitor, SB202190, inhibited PAR2-AP-induced changes in TER. Our results suggest that the activation of PAR2 leads to the disruption of tight junctions and increases the barrier permeability through the activation of p38 MAPK, which may cause the initiation and exacerbation of inflammation.