Junction Expression Research Articles

Role of intestinal epithelial AMPK in the protective effect of metformin on dextran sodium sulfate‐induced colitis in mice

Background Dysfunctions in the intestinal barrier, associated with an altered paracellular pathway, are commonly observed in gastrointestinal disorders, such as inflammatory bowel disease (IBD). The AMP-activated protein kinase (AMPK), principally known as a cellular energy sensor, has also been shown to play a key role in the stabilization and assembly of tight junctions following injury. Here, we aimed to investigate the effect of metformin in strengthening intestinal barrier function by activating intestinal epithelial AMPK. Methods A dextran sodium sulfate (DSS)-induced colitis model was used to assess disease progression in WT and intestinal epithelial cell-specific AMPK KO (IEC AMPK KO) mice in response to metformin treatment. Barrier integrity was analyzed by measuring paracellular permeability following dextran-4kDa gavage and, pro-inflammatory cytokines and tight junctions expression. Results The deletion of intestinal epithelial AMPK delayed intestinal injury repair after DSS-induced colitis and was associated with a slower re-epithelization of the intestinal mucosa coupled to a reduction in tight junctions expression and a higher paracellular permeability, inflammation and histological defects. Metformin administration attenuated the severity of DSS-induced colitis with improvement in intestinal permeability and inflammation in control mice but only partially in IEC AMPK KO mice. Conclusions Taken together these findings suggest that IEC AMPK contributes to intestinal healing and epithelial integrity after injury and may be effective as a therapeutic target to ameliorate dysfunctions of the intestinal barrier.

Human iPSC Colonoid Function is Improved by Exposure to Fecal Fermentates

The host-microbe interaction is critical for intestinal homeostasis. By-products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by-product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human induced pluripotent (iPSC) colon organoid model. Short-chain and branch-chain fatty acid concentrations of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin-A, SCFA transport monocarboxylate transporter-1, (6.2-fold, 2.1-fold, 1.8-fold, respectively; P<0.05). Mucin production (mucin-2, mucin-4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin-3) was noted by immunofluorescence in 25% supernatant treated cells. A dose-response increase in barrier function was observed over the 72-hour time course, with a 2-fold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (P<0.05). To further investigate host effects, clarified supernatants from a continuous multi-stage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL-1β, 6.15pg/mL; IL-6, 27.58pg/mL; TNFα, 4.49pg/mL; P < 0.05) in DC treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by-products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Pumpless, modular, microphysiological systems enabling tunable perfusion for long-term cultivation of endothelialized lumens.

Given the increased recognition of the importance of physiologically relevant microenvironments when designing in vitro assays, microphysiological systems (MPS) that mimic the critical function and structure of tissues and organs have gained considerable attention as alternatives to traditional experimental models. Accordingly, the field is growing rapidly, and some promising MPS are being tested for use in pharmaceutical development and toxicological testing. However, most MPS are complex and require additional infrastructure, which limits their successful translation. Here, we present a pumpless, modular MPS consisting of 1) a resistance module that controls flow rate and 2) a physiologically relevant, three-dimensional blood vessel module. Flow is provided by an attached reservoir tank that feeds fluid into the resistance channel via hydrostatic pressure. The flow rate is controlled by the height of the media in the tank and the resistance channel's dimensions. The flow from the resistance module is streamed into the blood vessel module using a liquid bridge. We utilize optical coherence tomography (OCT) to measure fluid velocity at regions of interest. The endothelial cells cultured in the MPS remain viable for up to 14days and demonstrate the functional characteristics of the human blood vessels verified by tight junction expression and diffusion assay. Our results show that a modular MPS can simulate a functional endothelium in vitro while simplifying the operation of the MPS. The simplicity of the system allows for modifications to incorporate other microenvironmental components and to build other organ-modeling systems easily.

Differential effects of intense exercise and pollution on the airways in a murine model

BackgroundExercise-induced bronchoconstriction (EIB) is a transient airway narrowing, occurring during or shortly after intensive exercise. It is highly prevalent in non-asthmatic outdoor endurance athletes suggesting an important contribution of air pollution in the development of EIB. Therefore, more research is necessary to investigate the combination of exercise and pollutants on the airways.MethodsBalbc/ByJ mice were intranasally challenged 5 days a week for 3 weeks with saline or 0.2 mg/ml diesel exhaust particles (DEP), prior to a daily incremental running session or non-exercise session. Once a week, the early ventilatory response was measured and lung function was determined at day 24. Airway inflammation and cytokine levels were evaluated in bronchoalveolar lavage fluid. Furthermore, innate lymphoid cells, dendritic cells and tight junction mRNA expression were determined in lung tissue.ResultsSubmaximal exercise resulted in acute alterations of the breathing pattern and significantly improved FEV0.1 at day 24. DEP exposure induced neutrophilic airway inflammation, accompanied with increased percentages of CD11b+ DC in lung tissue and pro-inflammatory cytokines, such as IL-13, MCP-1, GM-CSF and KC. Occludin and claudin-1(Cldn-1) expression were respectively increased and decreased by DEP exposure. Whereas, exercise increased Cldn-3 and Cldn-18 expression. Combining exercise and DEP exposure resulted in significantly increased SP-D levels in the airways.ConclusionDEP exposure induced typical airway neutrophilia, DC recruitment and pro-inflammatory cytokine production. Whereas, intensive exercise induced changes of the breathing pattern. The combination of both triggers resulted in a dysregulation of tight junction expression, suggesting that intensive exercise in polluted environments can induce important changes in the airway physiology and integrity.

Predicting Hyperoxia-Induced Lung Injury from Associated Intestinal and Lung Dysbiosis in Neonatal Mice

Background: Preclinical studies have demonstrated that hyperoxia disrupts the intestinal barrier, changes the intestinal bacterial composition, and injures the lungs of newborn animals. Objectives: The aim of the study was to investigate the effects of hyperoxia on the lung and intestinal microbiota and the communication between intestinal and lung microbiota and to develop a predictive model for the identification of hyperoxia-induced lung injury from intestinal and lung microbiota based on machine learning algorithms in neonatal mice. Methods: Neonatal C57BL/6N mice were reared in either room air or hyperoxia (85% O₂) from postnatal days 1–7. On postnatal day 7, lung and intestinal microbiota were sampled from the left lung and lower gastrointestinal tract for 16S ribosomal RNA gene sequencing. Tissue from the right lung and terminal ileum were harvested for Western blot and histology analysis. Results: Hyperoxia induced intestinal injury, decreased intestinal tight junction expression, and impaired lung alveolarization and angiogenesis in neonatal mice. Hyperoxia also altered intestinal and lung microbiota and promoted bacterial translocation from the intestine to the lung as evidenced by the presence of intestinal bacteria in the lungs of hyperoxia-exposed neonatal mice. The relative abundance of these bacterial taxa was significantly positively correlated with the increased lung cytokines. Conclusions: Neonatal hyperoxia induced intestinal and lung dysbiosis and promoted bacterial translocation from the intestine to the lung. Further studies are needed to clarify the pathophysiology of bacterial translocation to the lung.

Apolipoprotein E genotype-dependent nutrigenetic effects to prebiotic inulin for modulating systemic metabolism and neuroprotection in mice via gut-brain axis

ABSTRACT Objective The goal of the study was to identify the potential nutrigenetic effects to inulin, a prebiotic fiber, in mice with different human apolipoprotein E (APOE) genetic variants. Specifically, we compared responses to inulin for the potential modulation of the systemic metabolism and neuroprotection via gut-brain axis in mice with human APOE ϵ3 and ϵ4 alleles. Method We performed experiments with young mice expressing the human APOE3 (E3FAD mice and APOE4 gene (E4FAD mice). We fed mice with either inulin or control diet for 16 weeks starting from 3 months of age. We determined gut microbiome diversity and composition using16s rRNA sequencing, systemic metabolism using in vivo MRI and metabolomics, and blood–brain barrier (BBB) tight junction expression using Western blot. Results In both E3FAD and E4FAD mice, inulin altered the alpha and beta diversity of the gut microbiome, increased beneficial taxa of bacteria and elevated cecal short chain fatty acid and hippocampal scyllo-inositol. E3FAD mice had altered metabolism related to tryptophan and tyrosine, while E4FAD mice had changes in the tricarboxylic acid cycle, pentose phosphate pathway, and bile acids. Differences were found in levels of brain metabolites related to oxidative stress, and levels of Claudin-1 and Claudin-5 BBB tight junction expression. Discussion We found that inulin had many similar beneficial effects in the gut and brain for both E3FAD and E4FAD mice, which may be protective for brain functions and reduce risk for neurodegeneration. . E3FAD and E4FAD mice also had distinct responses in several metabolic pathways, suggesting an APOE-dependent nutrigenetic effects in modulating systemic metabolism and neuroprotection.

Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells.

Vinculin, a mechanotransducer associated with both adherens junctions (AJs) and focal adhesions (FAs), plays a central role in force transmission through cell-cell and cell-substratum contacts. We generated the conditional knockout (cKO) of vinculin in murine skin that results in the loss of bulge stem cell (BuSC) quiescence and promotes continual cycling of the hair follicles. Surprisingly, we find that the AJs in vinculin cKO cells are mechanically weak and impaired in force generation despite increased junctional expression of E-cadherin and α-catenin. Mechanistically, we demonstrate that vinculin functions by keeping α-catenin in a stretched/open conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, at the AJs. Altogether, our data provide mechanistic insights into the hitherto-unexplored regulatory link between the mechanical stability of cell junctions and contact-inhibition-mediated maintenance of BuSC quiescence.

Protective Effects of Baicalin on Peritoneal Tight Junctions in Piglets Challenged with Glaesserella parasuis

Glaesserella parasuis (G. parasuis) causes inflammation and damage to piglets. Whether polyserositis caused by G. parasuis is due to tight junctions damage and the protective effect of baicalin on it have not been examined. Therefore, this study aims to investigate the effects of baicalin on peritoneal tight junctions of piglets challenged with G. parasuis and its underlying molecular mechanisms. Piglets were challenged with G. parasuis and treated with or without baicalin. RT-PCR was performed to examine the expression of peritoneal tight junctions genes. Immunofluorescence was carried out to detect the distribution patterns of tight junctions proteins. Western blot assays were carried out to determine the involved signaling pathways. Our data showed that G. parasuis infection can down-regulate the tight junctions expression and disrupt the distribution of tight junctions proteins. Baicalin can alleviate the down-regulation of tight junctions mRNA in peritoneum, prevent the abnormalities and maintain the continuous organization of tight junctions. Our results provide novel evidence to support that baicalin has the capacity to protect peritoneal tight junctions from G. parasuis-induced inflammation. The protective mechanisms of baicalin could be associated with inhibition of the activation of PKC and MLCK/MLC signaling pathway. Taken together, these data demonstrated that baicalin is a promising natural agent for the prevention and treatment of G. parasuis infection.

Protective effect of ethyl pyruvate on gut barrier function through regulations of ROS-related NETs formation during sepsis

BackgroundSepsis impairs the function of the intestinal barrier through neutrophil extracellular traps (NETs). Reactive oxygen species (ROS)-induced activation of mitogen-activated protein kinase (MAPK) is involved in NET formation. Ethyl pyruvate (EP), a potent and effective ROS scavenger, ameliorates sepsis-associated intestinal barrier dysfunction, but the detailed mechanism is unknown. The current study aimed to explore the eff ;ects of EP on sepsis-induced intestinal barrier dysfunction and whether ROS and NETs were involved. MethodsA sepsis model was induced in mice by intraperitoneal injection of LPS (10 mg/kg). The mice were divided into 4 groups: (1) sham group; (2) LPS group; (3) DNase-1 + LPS group; and (4) EP + LPS group. EP or DNase-1 was intraperitoneally injected after the LPS model was established. After 24 h, the small intestine and blood were collected for analysis. Human neutrophils were harvested and incubated with phorbol-12-myristate-13-acetate (PMA) or PMA + EP, and ROS and NET generation was measured. ResultsEP significantly decreased proinflammatory cytokines and MPO-DNA in the LPS model. In addition, EP suppressed NET formation in the intestines of endotoxemic mice. The decrease in NETs induced by EP or DNase-1 alleviated histopathological damage, intestinal cell apoptosis and increased tight junction expression. In vitro, the treatment of EP abolished PMA-induced ROS production and NET formation which could be reversed by H2O2 treatment. Meanwhile, EP also abolished MAPK ERK1/2 and p38 activation during PMA-induced NET formation. ConclusionThis study was the first to demonstrate that EP alleviated NET formation and sepsis-induced intestinal damage through blockage of ROS mediated MAPK ERK1/2 and p38 activation.

Environmental contaminant BPA causes intestinal damage by disrupting cellular repair and injury homeostasis in vivo and in vitro

Our previous studies have shown that the environmental contaminant bisphenol A (BPA) exhibits strong intestinal toxicity and can readily cause intestinal barrier dysfunction. However, the causal relationship between adverse biological processes of BPA-induced intestinal tissue and the role of key signaling molecules in it requires further investigation. In this study, we established a mouse and intestinal epithelial cell model of BPA treatment to determine the underlying molecular mechanisms of BPA-induced intestinal injury. The results showed that the BPA treatment increased the intestinal permeability and disrupted the barrier function by increasing the chemical marker content and tight junction expression in intestinal tissues and blood circulation. BPA also altered the oxidative and antioxidant status of intestinal epithelial cells by increasing ROS and RNS contents and decreasing the activity levels of SOD, GPx, CAT, and T-AOC. BPA further induced inflammatory responses by upregulating the gene abundance of key factors of the innate immune system (TLR2, TLR4, MyD88, and NF-κB), the transcriptional activity of NF-kB, and the secretion of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α). Moreover, apoptosis was activated by BPA, whereas cell proliferation was inhibited by BPA. Mechanistically, co-treatment of intestinal epithelial cells with BPA using the oxidative stress scavenger NAC, the NF-κB-specific inhibitor JSH-23, and the apoptosis inhibitor Z-VAD-FMK, respectively, showed that BPA activates the innate immune response by inducing oxidative stress. Consequently, apoptosis is promoted, and cell proliferation is inhibited, ultimately disrupting the intestinal barrier function. Our findings provide insight into the pathogenesis of BPA-induced gut injury.

ODAM promotes junctional epithelium‐related gene expression via activation of WNT1 signaling pathway in an ameloblast‐like cell line ALC

In this study, we investigated the potential and mechanism of odontogenic ameloblast-associated protein (ODAM) in the promoting junctional epithelium-related gene expression in an ameloblast-like cell line ALC. ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM-ARHGEF5-RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear. A murine ameloblast-like cell line, ALC, was used to investigate the effects of ODAM on the JE-like changes of ALC cells in an epithelium-induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real-time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays. ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM-overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells. In conclusion, the research work showed that ODAM promotes junctional epithelium-related gene expression in ALC via the ODAM-WNT1 axis, which may provide new insight into the function of ODAM and JE formation.

HO-1/CO Maintains Intestinal Barrier Integrity through NF-κB/MLCK Pathway in Intestinal HO-1-/- Mice.

Background Intestinal barrier injury is an important contributor to many diseases. We previously found that heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal barrier. This study is aimed at elucidating the molecular mechanisms of HO-1/CO in barrier loss. Materials and Methods We induced gut leakiness by injecting carbon tetrachloride (CCl4) to wildtype or intestinal HO-1-deficient mice. In addition, we administrated tumor necrosis factor-α (TNF-α) to cells with gain- or loss-of-HO-1 function. The effects of HO-1/CO maintaining intestinal barrier integrity were investigated in vivo and in vitro. Results Cobalt protoporphyrin and CO-releasing molecule-2 alleviated colonic mucosal injury and TNF-α levels; upregulated tight junction (TJ) expression; and inhibited epithelial IκB-α degradation and phosphorylation, NF-κB p65 phosphorylation, long MLCK expression, and MLC-2 phosphorylation after administration of CCl4. Zinc protoporphyrin completely reversed these effects. These findings were further confirmed in vitro, using Caco-2 cells with gain- or loss-of-HO-1-function after TNF-α. Pretreated with JSH-23 (NF-κB inhibitor) or ML-7 (long MLCK inhibitor), HO-1 overexpression prevented TNF-α-induced TJ disruption, while HO-1 shRNA promoted TJ damage even in the presence of JSH-23 or ML-7, thus suggesting that HO-1 dependently protected intestinal barrier via the NF-κB p65/MLCK/p-MLC-2 pathway. Intestinal HO-1-deficient mice further demonstrated the effects of HO-1 in maintaining intestinal barrier integrity and its relative mechanisms. Alleviated hepatic fibrogenesis and serum ALT levels finally confirmed the clinical significance of HO-1/CO repairing barrier loss in liver injury. Conclusion HO-1/CO maintains intestinal barrier integrity through the NF-κB/MLCK pathway. Therefore, the intestinal HO-1/CO-NF-κB/MLCK system is a potential therapeutic target for diseases with a leaky gut.

Intestinal epithelial tight junctions and permeability can be rescued through the regulation of endoplasmic reticulum stress by amniotic fluid stem cells during necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases affecting premature infants. It has been shown that NEC is associated with disrupted intestinal barrier and dysregulated endoplasmic reticulum (ER)-stress response. It has also been shown that stem cells derived from amniotic fluid (AFSC) rescued intestinal injury in experimental NEC. Herein, we hypothesized that the beneficial effects of AFSC in the injured intestine are due to the restoration of intestinal barrier function. We evaluated intestinal barrier function using an ex vivo intestinal organoid model of NEC. We found that AFSC restored the expression and localization of tight junction proteins in intestinal organoids, and subsequently decreased epithelial permeability. AFSC rescued tight junction expression by inducing a protective ER stress response that prevents epithelial cell apoptosis in injured intestinal organoids. Finally, we validated these results in our experimental mouse model of NEC and confirmed that AFSC induced sustained ER stress and prevented intestinal apoptosis. This response led to the restoration of tight junction expression and localization, which subsequently reduced intestinal permeability in NEC pups. These findings confirm that intestinal barrier function is disrupted during NEC intestinal injury, and further demonstrate the disruption can be reversed by the administration of AFSC through the activation of the ER stress pathway. This study provides insight into the pathogenesis of NEC and highlights potential therapeutic targets for the treatment of NEC.

Mechanisms underlying age-associated manifestation of cardiac sodium channel gain-of-function

Cardiac action potentials are initiated by sodium ion (Na+) influx through voltage-gated Na+ channels. Na+ channel gain-of-function (GOF) can arise in inherited conditions due to mutations in the gene encoding the cardiac Na+ channel, such as Long QT syndrome type 3 (LQT3). LQT3 can be a “concealed” disease, as patients with LQT3-associated mutations can remain asymptomatic until later in life; however, arrhythmias can also arise early in life in LQT3 patients, demonstrating a complex age-associated manifestation. We and others recently demonstrated that cardiac Na+ channels preferentially localize at the intercalated disc (ID) in adult cardiac tissue, which facilitates ephaptic coupling and formation of intercellular Na+ nanodomains that regulate pro-arrhythmic early afterdepolarization (EAD) formation in tissue with Na+ channel GOF. Several properties related to ephaptic coupling vary with age, such as cell size and Na+ channel and gap junction (GJ) expression and distribution: neonatal cells have immature IDs, with Na+ channels and GJs primarily diffusively distributed, while adult myocytes have mature IDs with preferentially localized Na+ channels and GJs. Here, we perform an in silico study varying critical age-dependent parameters to investigate mechanisms underlying age-associated manifestation of Na+ channel GOF in a model of guinea pig cardiac tissue. Simulations predict that total Na+ current conductance is a critical factor in action potential duration (APD) prolongation. We find a complex cell size/ Na+ channel expression relationship: increases in cell size (without concurrent increases in Na+ channel expression) suppress EAD formation, while increases in Na+ channel expression (without concurrent increases in cell size) promotes EAD formation. Finally, simulations with neonatal and early age-associated parameters predict normal APD with minimal dependence on intercellular cleft width; however, variability in cellular properties can lead to EADs presenting in early developmental stages. In contrast, for adult-associated parameters, EAD formation is highly dependent on cleft width, consistent with a mechanism underlying the age-associated manifestation of the Na+ channel GOF.

Doxorubicin increases permeability of murine small intestinal epithelium and cultured T84 monolayers

Enteric bacteria and/or their products are necessary for doxorubicin (DXR)-induced small intestine mucosal damage. While DXR does not induce gross loss of epithelium, others have shown elevated serum endotoxin after DXR administration. However, the mechanism of movement is unknown. We hypothesized that DXR treatment resulted in increased paracellular translocation of bacteria or bacterial products through the small intestinal epithelium. We measured permeability after DXR administration using transepithelial resistance and macromolecular flux and assessed tight junctional gene expression and protein localization both in vitro using T84 cells and ex vivo using murine jejunum. DXR treatment increased flux of 4 kDa dextrans in mouse jejenum, but increased flux of 4, 10 and 20 kDa dextrans in T84 cells. Following DXR, we observed increased permeability, both in vitro and ex vivo, independent of bacteria. DXR induced increased expression of Cldn2 and Cldn4 in murine small intestine but increased only CLDN2 expression in T84 cells. DXR treatment induced disorganization of tight junctional proteins. We conclude that DXR increases paracellular transit of small macromolecules, including bacterial products, through the epithelium, by altering expression of tight junctional components and dynamic loosening of cellular tight junctions.

Prophylactic and therapeutic supplementation using fructo-oligosaccharide improves the intestinal homeostasis after mucositis induced by 5- fluorouracil

The beneficial effects of prebiotic, such as fructo-oligosaccharides (FOS), in intestinal inflammation have been demonstrated in several studies. Herein, we evaluate whether joint treatment with FOS, both before and during mucositis, had additional beneficial effects and investigated the mechanisms underlying in the action of FOS on the intestinal barrier. BALB/c mice were randomly divided into five groups: CTR (without mucositis + saline solution), FOS (without mucositis + 6 % FOS), MUC (mucositis + saline solution), PT (mucositis + 6 % FOS supplementation before disease induction), and TT (mucositis + 6 % FOS supplementation before and during disease induction). Mucositis was induced by intraperitoneal injection (300 mg/kg) of 5-fluorouracil (5-FU). After 72 h, the animals were euthanized and intestinal permeability (IP), tight junction, bacterial translocation (BT), histology and morphometry, and immunoglobulin A secretory (sIgA), inflammatory infiltrate, and production of short-chain fatty acids (acetate, butyrate and propionate) were evaluated. The MUC group showed an increase in the IP, BT, and inflammatory infiltrate but a decrease in the tight junction expression and butyrate and propionate levels (P < 0.05). In the PT and TT groups, FOS supplementation maintained the IP, tight junction expression, and propionate concentration within physiologic levels, increased butyrate levels, and reduced BT and inflammatory infiltrate (P < 0.05). Total treatment with FOS (TT group) was more effective in maintaining histological score, morphometric parameters, and sIgA production. Thus, total treatment (prophylactic and therapeutic supplementation) with FOS was more effective than pretreatment alone, in reducing 5-FU-induced damage to the intestinal barrier.

Butyrate as a bioactive human milk protective component against food allergy.

BackgroundFood allergy (FA) is a growing health problem worldwide. Effective strategies are advocated to limit the disease burden. Human milk (HM) could be considered as a protective factor against FA, but its mechanisms remain unclear. Butyrate is a gut microbiota‐derived metabolite able to exert several immunomodulatory functions. We aimed to define the butyrate concentration in HM, and to see whether the butyrate concentration detected in HM is able to modulate the mechanisms of immune tolerance.MethodsHM butyrate concentration from 109 healthy women was assessed by GS‐MS. The effect of HM butyrate on tolerogenic mechanisms was assessed in in vivo and in vitro models.ResultsThe median butyrate concentration in mature HM was 0.75 mM. This butyrate concentration was responsible for the maximum modulatory effects observed in all experimental models evaluated in this study. Data from mouse model show that in basal condition, butyrate up‐regulated the expression of several biomarkers of gut barrier integrity, and of tolerogenic cytokines. Pretreatment with butyrate significantly reduced allergic response in three animal models of FA, with a stimulation of tolerogenic cytokines, inhibition of Th2 cytokines production and a modulation of oxidative stress. Data from human cell models show that butyrate stimulated human beta defensin‐3, mucus components and tight junctions expression in human enterocytes, and IL‐10, IFN‐γ and FoxP3 expression through epigenetic mechanisms in PBMCs from FA children. Furthermore, it promoted the precursors of M2 macrophages, DCs and regulatory T cells.ConclusionThe study's findings suggest the importance of butyrate as a pivotal HM compound able to protect against FA.

Low junctional adhesion molecule-A expression is associated with an epithelial to mesenchymal transition and poorer outcomes in high-grade serous carcinoma of uterine adnexa

High-grade serous carcinoma of uterine adnexa (HGSC) is the most frequent histotype of epithelial ovarian cancer and has a poor 5-year survival rate due to late-stage diagnosis and the poor efficacy of standard treatments. Novel biomarkers of cancer outcome are needed to identify new targetable pathways and improve personalized treatments. Cell-surface screening of 26 HGSC cell lines by high-throughput flow cytometry identified junctional adhesion molecule 1 (JAM-A, also known as F11R) as a potential biomarker. Using a multi-labeled immunofluorescent staining coupled with digital image analysis, protein levels of JAM-A were quantified in tissue microarrays from three HGSC patient cohorts: a discovery cohort (n = 101), the Canadian Ovarian Experimental Unified Resource cohort (COEUR, n = 1158), and the Canadian Cancer Trials Group OV16 cohort (n = 267). Low JAM-A level was associated with poorer outcome in the three cohorts by Kaplan–Meier (p = 0.023, p < 0.001, and p = 0.036, respectively) and was an independent marker of shorter survival in the COEUR cohort (HR = 0.517 (0.381–703), p < 0.001). When analyses were restricted to patients treated by taxane–platinum-based chemotherapy, low JAM-A protein expression was associated with poorer responses in the COEUR (p < 0.001) and OV16 cohorts (p = 0.006) by Kaplan–Meier. Decreased JAM-A gene expression was an indicator of poor outcome in gene expression datasets including The Cancer Genome Atlas (n = 606, p = 0.002) and Kaplan–Meier plotter (n = 1816, p = 0.024). Finally, we observed that tumors with decreased JAM-A expression exhibited an enhanced epithelial to mesenchymal transition (EMT) signature. Our results demonstrate that JAM-A expression is a robust prognostic biomarker of HGSC and may be used to discriminate tumors responsive to therapies targeting EMT.

Staphylococcus aureus enterotoxin B disrupts nasal epithelial barrier integrity.

Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been associated with severe chronic rhinosinusitis with nasal polyps (CRSwNP). The pathogenic mechanism of SEB on epithelial barriers, however, is largely unexplored. We investigated the effect of SEB on nasal epithelial barrier function. SEB was apically administered to air-liquid interface (ALI) cultures of primary polyp and nasal epithelial cells of CRSwNP patients and healthy controls, respectively. Epithelial cell integrity and tight junction expression were evaluated. The involvement of Toll-like receptor 2 (TLR2) activation was studied in vitro with TLR2 monoclonal antibodies and in vivo in tlr2-/- knockout mice. SEB applied to ALI cultures of polyp epithelial cells decreased epithelial cell integrity by diminishing occludin and zonula occludens (ZO)-1 protein expression. Antagonizing TLR2 prevented SEB-induced barrier disruption. SEB applied in the nose of control mice increased mucosal permeability and decreased mRNA expression of occludin and ZO-1, whereas mucosal integrity and tight junction expression remained unaltered in tlr2-/- mice. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6 and IL-8, which was prevented by TLR2 antagonization. SEB damages nasal polyp epithelial cell integrity by triggering TLR2 in CRSwNP. Our results suggest that SEB might represent a driving factor of disease exacerbation, rather than a causal factor for epithelial defects in CRSwNP. Interfering with TLR2 triggering might provide a way to avoid the pathophysiological consequences of S.aureus on inflammation in CRSwNP.

Experimental Modeling of Necrotizing Enterocolitis in Human Infant Intestinal Enteroids

Background: Experimental model systems are of paramount importance in advancing our understanding of human disease. Methods There are several limitations when using a single cell culture to recapitulate the findings in a complex organism and results often vary between species, when proxy animal models are studied. Results Human enteroids have allowed for study of human disease in complex multicellular culture systems. Here we present the novel use of human infant enteroids generated from premature infant intestine to study necrotizing enterocolitis (NEC), which is a devastating intestinal disorder that affects our most vulnerable pediatric population. Conclusions We demonstrate that NEC can be induced in premature human enteroids as supported by corresponding alterations in inflammation, apoptosis, tight junction expression, and permeability by treatment with lipopolysaccharide.