Localization Of Tight Junction Proteins Research Articles

Euphorbium compositum SN improves the innate defenses of the airway mucosal barrier network during rhinovirus infection

BackgroundRhinoviruses (RV) are the major cause of common colds in healthy individuals and are associated with acute exacerbations in patients with chronic lung diseases. Yet, no vaccines or effective treatment against RV are available. This study investigated the effect of Euphorbium compositum SN (ECSN6), a multicomponent, multitarget medication made from natural ingredients, on the mucosal barrier network during RV infection.MethodsMucociliary-differentiated airway epithelial cell cultures were infected with RV or sham, and treated with 20% ECSN6 or placebo twice daily. Barrier integrity was assessed by measuring transepithelial resistance (TER), permeability to inulin, and expression and localization of intercellular junctions proteins (IJ). Ciliary beat frequency (CBF), expression of pro-inflammatory cytokines, antiviral interferons and mucins, and viral load were also measured. C57BL/6 mice were infected intranasally with RV or sham and treated with 40% ECSN6 or placebo twice daily. Inflammation of sinunasal mucosa, localization of E-cadherin, viral load and mucin gene expression were determined.ResultsECSN6-treated, uninfected cell cultures showed small, but significant increase in TER over placebo, which was associated with enhanced localization of E-cadherin and ZO-1 to IJ. In RV-infected cultures, treatment with ECSN6, but not placebo prevented RV-induced (1) reduction in TER, (2) dissociation of E-cadherin and ZO-1 from the IJ, (3) mucin expression, and (4) CBF attenuation. ECSN6 also decreased RV-stimulated expression of pro-inflammatory cytokines and permeability to inulin. Although ECSN6 significantly increased the expression of some antiviral type I and type III interferons, it did not alter viral load. In vivo, ECSN6 reduced RV-A1-induced moderate inflammation of nasal mucosa, beneficially affected RV-A1-induced cytokine responses and Muc5ac mRNA expression and prevented RV-caused dissociation of E-cadherin from the IJ of nasal mucosa without an effect on viral clearance.ConclusionsECSN6 prevents RV-induced airway mucosal barrier dysfunction and improves the immunological and mucociliary barrier function. ECSN6 may maintain integrity of barrier function by promoting localization of tight and adherence junction proteins to the IJ. This in turn may lead to the observed decrease in RV-induced pro-inflammatory responses in vitro. By improving the innate defenses of the airway mucosal barrier network, ECSN6 may alleviate respiratory symptoms caused by RV infections.

Impaired learning and memory in male mice induced by sodium arsenite was associated with MMP-2/MMP-9-mediated blood-brain barrier disruption and neuronal apoptosis

Arsenic is a widespread environmental contaminant known to accumulate in the brain, leading to cognitive impairment. However, the exact mechanisms by which arsenic causes cognitive deficits remain unclear. The present study aims to discover whether the destruction of the blood-brain barrier (BBB) mediated by matrix metalloproteinases 2 and matrix metalloproteinases 9 (MMP-2 and MMP-9) and subsequent neuronal apoptosis are involved in arsenic-induced cognitive impairment. Ninety male mice were given 0, 25, and 50 mg/L NaAsO2 in drinking water and 30 mg/kg doxycycline hyclate (DOX, an inhibitor of MMPs) gavage for 12 weeks to observe the alterations in learning and memory of mice, the morphology of hippocampal neurons, as well as the BBB permeability and ultrastructure, the localization and expression of tight junction proteins, MMP-2, and MMP-9. Our findings indicated that arsenic exposure induced learning and memory impairment in mice, accompanied by neuronal loss and apoptosis. Furthermore, arsenic exposure increased hematogenous IgG leakage into the brain, disrupted the tight junctions, reduced the expression of Claudin5, Occludin, and ZO1 in the endothelial cells, and increased the expression of MMP-2 and MMP-9 in the endothelial cells and astrocytes. Finally, DOX intervention preserved BBB integrity, alleviated hippocampal neuronal apoptosis, and improved cognitive impairment in mice caused by arsenic exposure. Our research demonstrates that cognitive disfunction in mice induced by arsenic exposure is associated with MMP-2 and MMP-9-mediated BBB destruction and neuronal apoptosis. The current investigation provides new insights into mechanisms of arsenic neurotoxicity and suggests that MMP-2 and MMP-9 may serve as potential therapeutic targets for treating arsenic-induced cognitive dysfunction in the future.

Hyperlipidemia negatively impacts implantation by dysregulating tight junction and Claudin-3 and Claudin-4 expression in the endometrium

Clinical observational studies have suggested hyperlipidemia may disturb embryo implantation through endometrium; however, the mechanism has been unclear. With its profound implications for reproductive health, the present study aims to investigate whether hyperlipidemia affects endometrial epithelial cell tight junctions for implantation failures. By constructing hyperlipidemia mice model, the number and distribution of embryo implantation status were investigated after both natural mating and in vitro fertilization and embryo transfer (IVF-ET). Transmission electron microscopy (TEM) was used to compare the ultrastructure of tight junctions in endometrial endothelial cells. Western blot and immunofluorescence were used to explore the expression and localization of tight junction proteins, such as Claudin (CDLN)3, CLDN4, occludin (OCLN), and zonula occludens-1 (ZO1). For women with reproductive failure, mid-luteal phase endometrial tissues were collected, and gene expression of tight junction proteins was investigated using RNA sequencing and qRT-PCR. In hyperlipidemic mice, the number of embryo implantation sites significantly decreased with uneven distribution after natural mating and IVF-ET. Disrupted tight junctions were found, characterized by a decreased number of tight junctions by TEM, downregulated expressions of CDLN4, OCLN, and ZO1, and an increased expression of CLDN3 by western blot. In hyperlipidemic women with reproductive failure, the dysregulated expression of CLDN3 and CLDN4 was also present in the luteal phase endometrium. In this study, evaluation of both animal models and infertile women in vivo demonstrated that hyperlipidemia reduced female fertility, accompanied by disruption of tight junction structures and dysregulation of CLDN3 and CLDN4 expression in the endothelial cells of luteal phase endometrium.

A human pluripotent stem cell-derived in vitro model of the blood–brain barrier in cerebral malaria

BackgroundBlood–brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but their physiological relevance remains uncertain. Using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs), we aimed to develop a novel in vitro model of the BBB in CM, exhibiting enhanced barrier properties.MethodshiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 h. Barrier integrity was measured using transendothelial electrical resistance (TEER) and sodium fluorescein permeability assays. Localization and expression of tight junction (TJ) proteins (occludin, zonula occludens-1, claudin-5), cellular adhesion molecules (ICAM-1, VCAM-1), and endothelial surface markers (EPCR) were determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were measured using multiplex proteome profiler arrays.ResultsAfter 6-h of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and increased sodium fluorescein permeability compared to co-culture with uninfected RBCs, indicative of a leaky barrier. We observed disruptions in localization of occludin, zonula occludens-1, and claudin-5 by IF, but no change in protein expression by WB in Pf-iRBC co-cultures. Expression of ICAM-1 and VCAM-1 but not EPCR was elevated in hiPSC-BMECs with Pf-iRBC co-culture compared to uninfected RBC co-culture. In addition, there was an increase in expression of angiogenin, platelet factor-4, and phospho-heat shock protein-27 in the Pf-iRBCs co-culture compared to uninfected RBC co-culture.ConclusionThese findings demonstrate the validity of our hiPSC-BMECs based model of the BBB, that displays enhanced barrier integrity and appropriate TJ protein localization. In the hiPSC-BMEC co-culture with Pf-iRBCs, reduced TEER, increased paracellular permeability, changes in TJ protein localization, increase in expression of adhesion molecules, and markers of angiogenesis and cellular stress all point towards a novel model with enhanced barrier properties, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.

The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function.

Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.

PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

Evaluation of Tight Junction Integrity in Brain Endothelial Cells Using Confocal Microscopy.

The blood-brain barrier (BBB) is a highly complex and dynamic microvascular barrier that protects the brain parenchyma from the entry of pathogens, toxins, and other macromolecules and is a critical structure that helps to maintain brain homeostasis. The BBB is formed mainly by brain capillary endothelial cells and perivascular astrocytes and pericytes. One of the primary properties of the BBB is a tight regulation of paracellular permeability due to the presence of tight junctional complexes (also, adherens and gap junctions) between the neighboring microvascular endothelial cells. Alterations in the assembly of the tight junctions impair BBB properties, particularly influenced barrier integrity and permeability. The tight junctions of the BBB are mainly composed of proteins including claudins, occludin, and zonula occludens-1 (ZO-1). Zonula occludens-1 binds to the actin cytoskeleton, and its localization provides valuable information on the status of BBB integrity and permeability. Immunofluorescence localization of ZO-1 and/or other tight junction proteins is a reliable indicator of barrier integrity and permeability in microvascular endothelial cells. In microvascular endothelial cells, f-actin stress fiber formation significantly influences the rate and size of the inter-endothelial cell gap that form as cells retract from their borders. Rhodamine phalloidin is a popular conjugate used as a fluorescent label for f-actin. Herein, we describe the procedures for ZO-1 immunofluorescence and f-actin labeling followed by confocal microscopic imaging to determine barrier integrity and tight junction organization in brain microvascular endothelial cells in vitro.

Sinapic acid alleviates inflammatory bowel disease (IBD) through localization of tight junction proteins by direct binding to TAK1 and improves intestinal microbiota.

Introduction: Although sinapic acid is found in various edible plants and has been shown to have anti-inflammatory properties including colitis, its underlying mechanism and effects on the composition of the gut microbiota are largely unknown. We aimed to identify an early response kinase that regulates the localization of tight junction proteins, act at the onset of the inflammatory response, and is regulated by sinapic acid. Additionally, we analyzed the effects of sinapic acid on the homeostasis of the intestinal microbiome. Methods: We examined the aberrant alterations of early response genes such as nuclear factor-kappa B (NF-κB) and activating transcription factor (ATF)-2 within 2h of sinapic acid treatment in fully differentiated Caco-2 cells with or without lipopolysaccharide and tumor necrosis factor (TNF)-α stimulation. To confirm the effect of sinapic acid on stimulus-induced delocalization of tight junction proteins, including zonula occludens (ZO)-1, occludin, and claudin-2, all tight junction proteins were investigated by analyzing a fraction of membrane and cytosol proteins extracted from Caco-2 cells and mice intestines. Colitis was induced in C57BL/6 mice using 2% dextran sulfate sodium and sinapic acid (2 or 10mg/kg/day) was administrated for 15days. Furthermore, the nutraceutical and pharmaceutical activities of sinapic acid for treating inflammatory bowel disease (IBD) evaluated. Results: We confirmed that sinapic acid significantly suppressed the stimulus-induced delocalization of tight junction proteins from the intestinal cell membrane and abnormal intestinal permeability as well as the expression of inflammatory cytokines such as interleukin (IL)-1β and TNF-α in vitro and in vivo. Sinapic acid was found to bind directly to transforming growth factor beta-activated kinase 1 (TAK1) and inhibit the stimulus-induced activation of NF-κB as well as MAPK/ATF-2 pathways, which in turn regulated the expression of mitogen-activated protein kinase (MLCK). Dietary sinapic acid also alleviated the imbalanced of gut microbiota and symptoms of IBD, evidenced by improvements in the length and morphology of the intestine in mice with colitis. Discussion: These findings indicate that sinapic acid may be an effective nutraceutical and pharmaceutical agent for IBD treatment as it targets TAK1 and inhibits subsequent NF-κB and ATF-2 signaling.

Consumption of honey ameliorates lipopolysaccharide-induced intestinal barrier dysfunction via upregulation of tight junction proteins.

The leaky gut barrier is an important factor leading to various inflammatory gastrointestinal disorders. The nutritional value of honey and variety of its health benefits have long been recognized. This study was undertaken to assess the role of Indian mustard honey in preventing lipopolysaccharide (LPS)-induced intestinal barrier dysfunction using a combination of in vitro and in vivo experimental model systems. LPS was used to induce intestinal barrier damage in a trans-well model of Caco-2 cells (1µg/ml) and in Swiss albino mice (5mg/kg body weight). Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to analyse sugar and phenolic components in honey samples. The Caco-2 cell monolayer integrity was evaluated by transepithelial electrical resistance (TEER) and paracellular permeability assays. The histopathology of intestinal tissue was analysed by haematoxylin and eosin dual staining. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the transcription of genes. The protein expression was analysed by immunofluorescence, western blot and ELISA-based techniques. The in vitro data showed that honey prevented LPS-induced intestinal barrier dysfunction dose dependently as was measured by TEER and paracellular flux of FITC-dextran dye. Further, the in vivo data showed a prophylactic effect of orally administered honey as it prevented the loss of intestinal barrier integrity and villus structure. The cellular localization and expression of tight junction (TJ) proteins were upregulated along with downregulation of pro-inflammatory cytokines in response to the administration of honey with LPS. The findings of this study suggest a propitious role of honey in the maintenance of TJ protein integrity, thereby preventing LPS-induced intestinal barrier disintegration.

Effects of Yogurt with Carao (Cassia grandis) on Intestinal Barrier Dysfunction, α-glycosidase Activity, Lipase Activity, Hypoglycemic Effect, and Antioxidant Activity

Cell inflammation disrupts intestinal barrier functions and may cause disorders related to a leaky gut, possibly leading to diabetes. The objective of this study was to determine if carao (Cassia grandis) incorporated into yogurt enhances in vitro intestinal barrier function. To achieve this goal, Caco-2 cells were used as a model of intestinal barrier permeability. Caco-2 cells were treated with cytokines (interleukin-1β, tumor necrosis factor-α, interferon-γ, and lipopolysaccharide (LPS)) and yogurt with carao yogurt (CY) at different doses (1.3 g/L, 2.65 g/L, and 5.3 g/L). Real-time quantitative polymerase chain and immunofluorescence microscopy were applied to evaluate the expression and localization of tight junction proteins. Functional effects of the formulation of yogurt supplemented with carao were also evaluated in terms of the antioxidant activity, the α-glycosidase activity, and lipase inhibitory properties. In addition, the hypoglycemic potential was validated in vivo in a rat model. Compared to the control yogurt, Caco-2 TEER (transepithelial electrical resistance evaluation) by yogurt with 5.3 g/L of carao was significantly lower (p < 0.05) after 48 h. Yogurt with 5.3 g/L of carao had a considerably lower permeability (p < 0.05) than control yogurt in FD and LY flux. Yogurt with 5.3 g/L of carao enhanced the localization of ZO-1. Carao addition into yogurt increased the flavonoid content, apparent viscosity, lipase inhibition activity, and α-glycosidase activity. The rats fed with the yogurt with 5.3 g/L of carao demonstrated a higher blood glucose modulation.

Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 μm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 μm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

Loss of NKCC1 function increases epithelial tight junction permeability by upregulating claudin-2 expression.

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2, the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

Dynamic Changes in Colonic Structure and Protein Expression Suggest Regulatory Mechanisms of Colonic Barrier Function in Torpor-Arousal Cycles of the Daurian Ground Squirrel.

Background: Both pathological conditions and hibernation can affect the barrier function of small intestine mucosa. However, the effect of hibernation on the barrier function of colonic mucosa remains unclear. Methods: We investigated morphological changes in colonic mucosa, the concentrations of specific proteins and molecules, and the enzymatic activity of diamine oxidase (DAO), in serum and colonic tissue; the expression of tight junction proteins and mucin, and the changes in inflammatory, farnesoid X receptor (FXR)–small heterodimer partner (SHP), and apoptosis-related molecules that could play a role in gut permeability changes in Daurian ground squirrels in summer active (SA), late torpor (LT), and interbout arousal (IBA) periods. Results: The results show that hibernation reduced the thickness of the colonic mucosa and the depth of the crypt, decreased the number of goblet cells (GCs), and damaged the structure of some microvilli. The concentrations of proteins and molecules, and the enzymatic activity of DAO, were all increased in the serum and colon, and the localization of tight junction proteins and mucin in the colonic mucosa were altered (compensatory response). Although the ground squirrels ate during the interbout arousal period, the changes remained similar to the response to torpor. Inflammation, apoptosis–anti-apoptosis, and FXR–SHP signaling may be involved in the possible changes in intestinal gut permeability during the torpor–arousal cycle in Daurian ground squirrels. In addition, periodic interbout arousal may play an inflammation-correcting role during the long hibernation season of Daurian ground squirrels.

Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

Ectopic tertiary lymphoid organs (TLOs) have been identified in many organs, such as the lungs, nasal cavities, and kidneys of both mice and humans. Although lymphocyte aggregates have been observed in the mammary glands of ruminants, the details remain unclear. In this study, we investigated the mammary glands of lactating goats for the presence of TLOs. The localization of CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules), CD40 (follicular dendritic cells), BCL6 (germinal center), and IgA was examined by immunohistochemistry. The concentrations of IgG, IgA, lactoferrin, β-defensin-1, cathelicidin-2, cathelicidin-7, S100A7, and S100A8 in milk were measured by ELISA. The localization and amount of tight junction (TJ) proteins (claudin-3 and claudin-4) were examined using immunofluorescence and western blotting. We found that 19 out of 30 udders contained lymphocyte aggregates, which showed positive reactions against CD20, CD3, CD40, and MECA79. In addition, large-sized aggregations showed separate localization of B cells and T cells and a positive reaction against BCL6, although BCL6 was sparsely localized in the aggregations. These results indicate that mammary glands of lactating goats contain TLOs. The IgG and IgA concentrations in the milk of TLO-positive goats and the number of IgA-positive cells were higher than those in negative goats. Furthermore, claudin-4 was localized in the TJ region and the amount was higher in TLO-positive mammary glands than that in the negative group, indicating the presence of leakages at TJs. In conclusion, a majority of lactating goat udders have TLOs, which contribute to local immunity by producing immunoglobulins.

Delay of endothelial cell senescence protects cerebral barrier against age-related dysfunction: role of senolytics and senomorphics

ABSTRACT Accumulation of senescent cells in cerebrovasculature is thought to play an important role in age-related disruption of blood–brain barrier (BBB). Using an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, this study explored the so-called correlative link between BMEC senescence and the BBB dysfunction in the absence or presence of functionally distinct senotherapeutics. Replicative senescence was deemed present at passage ≥19 where BMECs displayed shortened telomere length, reduced proliferative and tubulogenic potentials and increased NADPH oxidase activity, superoxide anion production (markers of oxidative stress), S-β-galactosidase activity and γ-H2AX staining. Significant impairments observed in integrity and function of a model of BBB established with senescent BMECs, ascertained successively by decreases in transendothelial electrical resistance and increases in paracellular flux, revealed a close correlation between endothelial cell senescence and BBB dysfunction. Disruptions in the localization or expression of tight junction proteins, zonula occludens-1, occludin, and claudin-5 in senescent BMECs somewhat explained this dysfunction. Indeed, treatment of relatively old BMEC (passage 16) with a cocktail of senolytics (dasatinib and quercetin) or senomorphics targeting transcription factor NF-κB (QNZ), p38MAPK signaling pathway (BIRB-796) or pro-oxidant enzyme NADPH oxidase (VAS2870) until passage 20 rendered these cells more resistant to senescence and totally preserved BBB characteristics by restoring subcellular localization and expression of tight junction proteins. In conclusion, attempts that effectively mitigate accumulation of senescent endothelial cells in cerebrovasculature may prevent age-related BBB dysfunction and may be of prophylactic or therapeutic value to extend lifelong health and wellbeing.

Effects of SPA4 peptide on lipopolysaccharide-disrupted lung epithelial barrier, injury, and function in a human cell system and mouse model of lung injury.

Disrupted epithelial barrier, fluid accumulation, inflammation, and compromised physiology are hallmarks of lung injury. Here we investigated the structural stability of the Toll‐like receptor‐4 (TLR4)‐interacting SPA4 peptide, its effect on Pseudomonas aeruginosa lipopolysaccharide (LPS)‐disrupted epithelial barrier in a human cell system, and lung injury markers in a mouse model of LPS‐induced lung inflammation. The structural properties of SPA4 peptide were investigated using circular dichroism and UV–VIS spectroscopy. The transepithelial electrical resistance (TEER), an indicator of barrier function, was measured after the cells were challenged with 1 μg/ml LPS and treated with 10 or 100 μM SPA4 peptide. The expression and localization of tight junction proteins were studied by immunoblotting and immunocytochemistry, respectively. Mice were intratracheally challenged with 5 μg LPS per g body weight and treated with 50 μg SPA4 peptide. The lung wet/dry weight ratios or edema, surfactant protein‐D (SP‐D) levels in serum, lung function, tissue injury, body weights, and temperature, and survival were determined as study parameters. The spectroscopy results demonstrated that the structure was maintained among different batches of SPA4 peptide throughout the study. Treatment with 100 μM SPA4 peptide restored the LPS‐disrupted epithelial barrier, which correlated with the localization pattern of Zonula Occludens (ZO)‐1 and occludin proteins. Correspondingly, SPA4 peptide treatment helped suppress the lung edema and levels of serum SP‐D, improved some of the lung function parameters, and reduced the mortality risk against LPS challenge. Our results suggest that the anti‐inflammatory activity of the SPA4 peptide facilitates the resolution of lung pathology.

BATF3 Protects Against Metabolic Syndrome and Maintains Intestinal Epithelial Homeostasis.

The intestinal immune system and microbiota are emerging as important contributors to the development of metabolic syndrome, but the role of intestinal dendritic cells (DCs) in this context is incompletely understood. BATF3 is a transcription factor essential in the development of mucosal conventional DCs type 1 (cDC1). We show that Batf3-/- mice developed metabolic syndrome and have altered localization of tight junction proteins in intestinal epithelial cells leading to increased intestinal permeability. Treatment with the glycolysis inhibitor 2-deoxy-D-glucose reduced intestinal inflammation and restored barrier function in obese Batf3-/- mice. High-fat diet further enhanced the metabolic phenotype and susceptibility to dextran sulfate sodium colitis in Batf3-/- mice. Antibiotic treatment of Batf3-/- mice prevented metabolic syndrome and impaired intestinal barrier function. Batf3-/- mice have altered IgA-coating of fecal bacteria and displayed microbial dysbiosis marked by decreased obesity protective Akkermansia muciniphila, and Bifidobacterium. Thus, BATF3 protects against metabolic syndrome and preserves intestinal epithelial barrier by maintaining beneficial microbiota.

Differential expression and localization of tight junction proteins in the goat epididymis.

The blood-epididymis barrier (BEB) forms a unique microenvironment that is crucial for the maturation, protection, transport, and storage of spermatozoa in the epididymis. To characterize the function of tight junctions (TJs), which are constitutive components of the BEB, we determined the expression and localization of TJ proteins such as zonula occludens (ZO)-1, 2, and 3, occludin, and claudin3 (Cldn3) during postnatal development in the goat epididymis. To assess the expression patterns of TJ proteins in immature (3 months of age) and mature (14 months of age) goat epididymides, two different experimental methods were used including immunofluorescence labeling and western blotting. We show that, ZO-1, 2, and 3, and occludin, were strictly expressed and localized to the TJs of the goat epididymis, whereas Cldn3 was present in basolateral membranes as well as TJs. All TJ proteins examined were more highly expressed in the immature epididymis compared to levels in mature tissue. In conclusion, our study indicates that at least five TJ proteins, namely ZO-1, ZO-2, ZO-3, occludin, and Cldn3, are present in TJs, and the expression strength and pattern of TJ proteins tend to be age dependent in the goat epididymis. Together, these data suggest that the distinct expression patterns of TJ proteins are essential for regulating components of the luminal contents in the epididymal epithelium and for forming adequate luminal conditions that are necessary for the maturation, protection, transport, and storage of spermatozoa in the goat epididymis.

Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis

Intestinal epithelial barrier function is compromised in inflammatory bowel disease and barrier dysfunction contributes to disease progression. Extracellular nucleotides/nucleosides generated in gut inflammation may regulate barrier function through actions on diverse cell types. Enteric glia modulate extracellular purinergic signaling and exert pathophysiological effects on mucosal permeability. These glia may regulate inflammation with paracrine responses, theoretically mediated via adenosine 2B receptor (A2BR) signaling. As the cell-specific roles of A2BRs in models of colitis and barrier dysfunction are unclear, we studied glial A2BRs in acute dextran sodium sulfate (DSS) colitis. We performed and validated conditional ablation of glial A2BRs in Sox10CreERT2+/−;Adora2bf/f mice. Overt intestinal disease activity indices in DSS-colitis were comparable between Sox10CreERT2+/–;Adora2bf/f mice and littermate controls. However, ablating glial A2BRs protected against barrier dysfunction following acute DSS-colitis. These benefits were associated with the normalization of tight junction protein expression and localization including claudin-1, claudin-8, and occludin. Glial A2BR signaling increased levels of proinflammatory mediators in the colon and cell-intrinsic regulation of genes including Csf3, Cxcl1, Cxcl10, and Il6. Our studies show that glial A2BR signaling exacerbates immune responses during DSS-colitis and that this adenosinergic cell-specific mechanism contributes to persistent gut epithelial barrier dysfunction.