Decreased Barrier Function Research Articles

Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.

Filaggrin deficiency in Stat6 transgenic mice exacerbates allergic skin inflammation (HYP4P.310)

Abstract Atopic dermatitis (AD) is a chronic inflammatory skin disease induced by a complex interaction between susceptibility genes encoding skin barrier components and environmental antigen exposure. Although genome-wide association studies in AD have identified several susceptibility loci, loss of function mutations in the human filaggrin gene (FLG) is the major predisposing factor for AD. We have previously shown that 40% transgenic mice expressing constitutively active Stat6 (Stat6VT) in T cells spontaneously develop AD due to increased Th2 responses leading to decreased expression of barrier function genes including Flg. Flaky tail mice (FTM) have been widely used as a model of filaggrin deficiency-associated AD pathogenesis in patients. FTM have decreased barrier function, increased Th2 and Th17 responses following sensitization, but do not develop spontaneous allergic inflammation. In order to determine the effects of having both barrier dysfunction and increased Th2 responses, Stat6VT mice were mated to FTM. Our results demonstrate that over 90% of the STAT6VT mice carrying mutant Flg develop severe AD lesions by 3 months of age in comparison to 40% Stat6VT mice that develop disease between 4 to 5 months of age. Histopathological analysis of skin tissues revealed extensive thickening of the dermis with more severe infiltrates as compared to Stat6VT mice. Our studies suggest that skin barrier defects and altered Th2 responses separately contribute to the pathogenesis of AD.

117 Correlation of Histology to Endoscopic Mucosal Impedance in Patients With Eosinophilic Esophagitis

Background and Aim: An early event in the pathogenesis of eosinophilic esophagitis (EoE) is food antigen penetration of esophageal epithelium, but the precise relationship of eosinophilia, dilated intercellular spaces (DIS) and decreased barrier function is unclear. The aim of this study was to determine the correlation and accuracy of site-specific mucosal impedance (MI) measurement of ion flux with esophageal histology in distinguishing active from inactive EoE. Methods: In this study, 10 patients each with active and inactive EoE underwent MI measurement and mucosal biopsies at four esophageal sites (2, 5,10,15cm above the Zline). MI was compared to 10 control patients without esophageal symptoms. Data were analyzed by comparing MI values, Eos/HPF and DIS grade in the three groups at each level examined. Results: The esophageal MI values (ohms) were significantly lower in active when compared to inactive and control patients (1909 vs. 4349 and 5530 ohms, resp., p<.001). (Table) Four active patients had biopsies with eos< 15/HPF with lower DIS grade. There were significant and inverse correlations between MI and eos/HPF as well as DIS (rs=.584 and -.531, respectively, p<.001). Furthermore, MI cut off of 2300 ohms demonstrated 90% sensitivity and 91% specificity for diagnosis of active EoE and 89% and 82% for high grade DIS. Conclusions: In EoE, eosinophil eosinophilia and DIS correlate to MI measured ionic conductance and by inference barrier function. Endoscopic MI measurement in the esophagus is a promising, safe and easy to perform means to assess disease activity in diseases such as EoE. Impedance measurements in EoE patients and controls

118 Identification of a Novel Germ-Line Variant in an Uncharacterized Gene, FBE-1, and Its Putative Role in Familial Barrett's Esophagus

Background and Aim: An early event in the pathogenesis of eosinophilic esophagitis (EoE) is food antigen penetration of esophageal epithelium, but the precise relationship of eosinophilia, dilated intercellular spaces (DIS) and decreased barrier function is unclear. The aim of this study was to determine the correlation and accuracy of site-specific mucosal impedance (MI) measurement of ion flux with esophageal histology in distinguishing active from inactive EoE. Methods: In this study, 10 patients each with active and inactive EoE underwent MI measurement and mucosal biopsies at four esophageal sites (2, 5,10,15cm above the Zline). MI was compared to 10 control patients without esophageal symptoms. Data were analyzed by comparing MI values, Eos/HPF and DIS grade in the three groups at each level examined. Results: The esophageal MI values (ohms) were significantly lower in active when compared to inactive and control patients (1909 vs. 4349 and 5530 ohms, resp., p<.001). (Table) Four active patients had biopsies with eos< 15/HPF with lower DIS grade. There were significant and inverse correlations between MI and eos/HPF as well as DIS (rs=.584 and -.531, respectively, p<.001). Furthermore, MI cut off of 2300 ohms demonstrated 90% sensitivity and 91% specificity for diagnosis of active EoE and 89% and 82% for high grade DIS. Conclusions: In EoE, eosinophil eosinophilia and DIS correlate to MI measured ionic conductance and by inference barrier function. Endoscopic MI measurement in the esophagus is a promising, safe and easy to perform means to assess disease activity in diseases such as EoE. Impedance measurements in EoE patients and controls

Vitamin D prevents hypoxia/reoxygenation-induced blood-brain barrier disruption via vitamin D receptor-mediated NF-kB signaling pathways.

Maintaining blood-brain barrier integrity and minimizing neuronal injury are critical components of any therapeutic intervention following ischemic stroke. However, a low level of vitamin D hormone is a risk factor for many vascular diseases including stroke. The neuroprotective effects of 1,25(OH)2D3 (vitamin D) after ischemic stroke have been studied, but it is not known whether it prevents ischemic injury to brain endothelial cells, a key component of the neurovascular unit. We analyzed the effect of 1,25(OH)2D3 on brain endothelial cell barrier integrity and tight junction proteins after hypoxia/reoxygenation in a mouse brain endothelial cell culture model that closely mimics many of the features of the blood-brain barrier in vitro. Following hypoxic injury in bEnd.3 cells, 1,25(OH)2D3 treatment prevented the decrease in barrier function as measured by transendothelial electrical resistance and permeability of FITC-dextran (40 kDa), the decrease in the expression of the tight junction proteins zonula occludin-1, claudin-5, and occludin, the activation of NF—kB, and the increase in matrix metalloproteinase-9 expression. These responses were blocked when the interaction of 1,25(OH) )2D3 with the vitamin D receptor (VDR) was inhibited by pyridoxal 5’-phosphate treatment. Our findings show a direct, VDR-mediated, protective effect of 1,25(OH) )2D3 against ischemic injury-induced blood-brain barrier dysfunction in cerebral endothelial cells.

Endoscopic Mucosal Impedance Measurements Correlate With Eosinophilia and Dilation of Intercellular Spaces in Patients With Eosinophilic Esophagitis

Penetration of the esophageal epithelium by food antigens is an early event in the pathogenesis of eosinophilic esophagitis (EoE), but the precise relationship among eosinophilia, dilated intercellular spaces (DIS), and decreased barrier function is unclear. We investigated the correlation between site-specific mucosal impedance (MI) measurements of ion flux and esophageal histology, and whether MI measurements can be used to distinguish between patients with active and inactive EoE. MI was measured (in Ω) in 10 patients with active EoE (>15 eosinophils [eos]/high-power field [HPF]) and in 10 with inactive EoE (<15 eos/HPF, as a result of treatment), and mucosal biopsy specimens were collected from 4 esophageal sites (2, 5, 10, and 15 cm above the Z-line). MI also was measured in 10 individuals without esophageal symptoms (controls). MI measurements, eos/HPF, and DIS grade were compared among patients with EoE and controls. The esophageal MI values were significantly lower in patients with active EoE (1909 Ω) compared with inactive EoE (4349 Ω) or controls (5530 Ω) (P < .001). Biopsy specimens from 4 patients with active EoE contained fewer than 15 eos/HPF and lower-grade DIS than in patients with active disease. There were significant inverse correlations between MI and eos/HPF (rs = -.584), as well as between MI and DIS (rs = -.531; P < .001). The MI cut-off value of 2300 Ω identified patients with active EoE with 90% sensitivity and 91% specificity, and high-grade DIS with 89% sensitivity and 82% specificity. In patients with EoE, eosinophilia and DIS correlate with MI measurements of ion flux. Endoscopic MI measurement in the esophagus is safe and easy to perform, and can be used to assess activity of diseases such as EoE.

Astrocyte-derived retinoic acid: a novel regulator of blood–brain barrier function during neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by immune cell entry into the brain. In normal conditions, this infiltration of immune cells is prevented by the blood–brain barrier (BBB) which is characterized by highly specialized endothelial cells. Therefore, the BBB is of high importance for brain protection and homeostasis. However, during inflammatory processes, like the ones observed in the course of MS, the BBB is dysfunctional and leaky, allowing the entry of serum proteins and immune cells into the brain, aggravating lesion formation. Notch signaling is a pathway essential for proper vascular development; however its role during adult vascular homeostasis and during inflammation is not clearly understood. Several research groups have shown that during inflammatory conditions Notch signaling is altered in endothelial cells, hinting for a function of this pathway during inflammation. Therefore, we hypothesize that Notch signaling is involved in BBB function during homeostasis and during inflammatory processes. Here we show that inflammation alters Notch receptors and ligand expression in brain endothelial cells. Furthermore, when Notch signaling is inhibited, brain endothelial cells show a decrease in barrier function as demonstrated by decreased resistance and increased permeability of endothelial monolayers to fluorescent molecules. During inflammatory conditions we observe an even more pronounced effect, demonstrating the importance of Notch signaling in coping with inflammation. These results were also confirmed by the Notch receptors knockdown in brain endothelial cells. In addition, our results show that Notch ligands are important during migration processes of T cells through the brain vasculature. Taken together our results demonstrate the importance of Notch signaling in BBB homeostasis and function. We believe that by understanding how BBB function is regulated, we can develop targeted therapies to the brain vasculature and restore its function during disease.

Notch receptors and ligands mediate barrier function in brain endothelial cells

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by immune cell entry into the brain. In normal conditions, this infiltration of immune cells is prevented by the blood–brain barrier (BBB) which is characterized by highly specialized endothelial cells. Therefore, the BBB is of high importance for brain protection and homeostasis. However, during inflammatory processes, like the ones observed in the course of MS, the BBB is dysfunctional and leaky, allowing the entry of serum proteins and immune cells into the brain, aggravating lesion formation. Notch signaling is a pathway essential for proper vascular development; however its role during adult vascular homeostasis and during inflammation is not clearly understood. Several research groups have shown that during inflammatory conditions Notch signaling is altered in endothelial cells, hinting for a function of this pathway during inflammation. Therefore, we hypothesize that Notch signaling is involved in BBB function during homeostasis and during inflammatory processes. Here we show that inflammation alters Notch receptors and ligand expression in brain endothelial cells. Furthermore, when Notch signaling is inhibited, brain endothelial cells show a decrease in barrier function as demonstrated by decreased resistance and increased permeability of endothelial monolayers to fluorescent molecules. During inflammatory conditions we observe an even more pronounced effect, demonstrating the importance of Notch signaling in coping with inflammation. These results were also confirmed by the Notch receptors knockdown in brain endothelial cells. In addition, our results show that Notch ligands are important during migration processes of T cells through the brain vasculature. Taken together our results demonstrate the importance of Notch signaling in BBB homeostasis and function. We believe that by understanding how BBB function is regulated, we can develop targeted therapies to the brain vasculature and restore its function during disease.

Skin aging, gene expression and calcium

The human epidermis provides a very effective barrier function against chemical, physical and microbial insults from the environment. This is only possible as the epidermis renews itself constantly. Stem cells located at the basal lamina which forms the dermoepidermal junction provide an almost inexhaustible source of keratinocytes which differentiate and die during their journey to the surface where they are shed off as scales. Despite the continuous renewal of the epidermis it nevertheless succumbs to aging as the turnover rate of the keratinocytes is slowing down dramatically. Aging is associated with such hallmarks as thinning of the epidermis, elastosis, loss of melanocytes associated with an increased paleness and lucency of the skin and a decreased barrier function. As the differentiation of keratinocytes is strictly calcium dependent, calcium also plays an important role in the aging epidermis.Just recently it was shown that the epidermal calcium gradient in the skin that facilitates the proliferation of keratinocytes in the stratum basale and enables differentiation in the stratum granulosum is lost in the process of skin aging. In the course of this review we try to explain how this calcium gradient is built up on the one hand and is lost during aging on the other hand. How this disturbed calcium homeostasis is affecting the gene expression in aged skin and is leading to dramatic changes in the composition of the cornified envelope will also be discussed. This loss of the epidermal calcium gradient is not only specific for skin aging but can also be found in skin diseases such as Darier disease, Hailey–Hailey disease, psoriasis and atopic dermatitis, which might be very helpful to get a deeper insight in skin aging.

An in vitro Skin Penetration Model for Compromised Skin: Estimating Penetration of Polyethylene Glycol [14C]-PEG-7 Phosphate

Background/Aims: Establishing dermal penetration rates is important to better understand the safety of topically applied materials, especially for premature infant skin with compromised skin barrier function. Skin prematurity involves thinner stratum corneum and underdeveloped epidermis/dermis resulting in decreased barrier function, higher transepidermal water loss and greater chemical penetration, when compared to healthy full-term neonate/adult skin. Methods: We developed an in vitro skin penetration model using human ex vivo skin to estimate penetration for premature/compromised skin barrier conditions by tape stripping. Skin barrier deficiency was characterized by transepidermal water loss. Baby wipe lotion containing 5 mg/cm² [¹⁴C]-PEG-7 phosphate was applied 5 times to human skin samples of intact, moderately or highly compromised skin barrier and once at 25 mg/cm² over 24 h. Results: Overall penetration of [¹⁴C]-PEG-7 phosphate was low (<5%) even for highly compromised skin. The absorption rate was higher (p < 0.001) for compromised skin versus intact skin. No significant difference was seen between moderately and highly compromised skin by repeated dosing. Under single-dose conditions, penetration through highly compromised skin was significantly higher compared to intact skin (p = 0.001). Conclusion: Our model demonstrates that even under highly compromised skin conditions, penetration of [¹⁴C]-PEG-7 phosphate is low (<5%) and only 4-6 times higher compared to mature/intact skin and does not approach 100%. Penetration was unaffected by single or multiple dosing conditions.

TNF-α and Th2 Cytokines Induce Atopic Dermatitis–Like Features on Epidermal Differentiation Proteins and Stratum Corneum Lipids in Human Skin Equivalents

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.

IL-4 induces columnar-like differentiation of esophageal squamous epithelium through JAK/PI3K pathway: possible role in pathogenesis of Barrett's esophagus

Barrett's esophagus is characterized by a distinct Th2-predominant cytokine profile (IL-4) from in vivo or ex vivo evidence. The detailed role of cytokines in Barrett's esophagus, particularly whether Th2 cytokines are causative factors driving metaplastic processes, remains unknown. In this study, air-liquid interface-cultured human esophageal epithelial cells were stimulated by a Th2 cytokine, IL-4, and Th1 cytokines, TNF-α and IL-1β, continuously for 10 days. Barrier function was determined by transepithelial electrical resistance. Morphological changes were investigated by hematoxylin and eosin staining. Keratin profile (keratin 7, 8, 13, and 14) and squamous differentiation markers (involucrin) were investigated by RT-quantitative PCR, Western blotting, and immunohistochemical staining. Pharmacological inhibitors were used to identify the underlying cellular signaling. We report that IL-4, TNF-α, and IL-1β decrease barrier function, but only IL-4 significantly increases cell layers and changes cell morphology. IL-4 time dependently downregulates the expression levels of the squamous cell markers involucrin and keratin 13 and upregulates the expression levels of the columnar cell markers keratin 7 and 8. Neither TNF-α nor IL-1β shows any effect on these indexes. JAK inhibitor I and PI3K inhibitors significantly block the IL-4-induced changes in the levels of keratin 8 and 13. In conclusion, IL-4 inhibits squamous differentiation program of esophageal epithelial cells and induces differentiation toward columnar cells through the JAK/PI3K pathway. Thus IL-4 may be involved in the early stages of Barrett's esophagus development.

Oncostatin M Is Elevated In Chronic Rhinosinusitis and Decreases Barrier Function In Human Airway Epithelium

Oncostatin M Is Elevated In Chronic Rhinosinusitis and Decreases Barrier Function In Human Airway Epithelium

P-212 A Role for Hypoxia-Inducible Factor (HIF) in Intestinal Epithelial Junctional Assembly and Barrier Function

The gastrointestinal mucosa constitutes the largest mucosal surface found in multicellular organisms, and provides the primary physical barrier against gut luminal contents. Intestinal epithelial cells that line the GI tract selectively permit flux of nutrients and water, while protecting against omnipresent luminal antigens. In mucosal diseases such as inflammatory bowel disease (IBD), hypoxia and inflammation occur coincidentally at the level of the epithelium. Adaptive transcriptional responses to oxygen deprivation are mediated primarily through the hypoxia-inducible factor complex (HIF), comprising an oxygen-labile “alpha” subunit and constitutively expressed “beta” subunit. A role for HIF has been implicated in orchestrating a signaling program that promotes intestinal epithelial function and homeostasis. Disruption of the epithelial barrier by proinflammatory cytokines such as IFN-gamma defines a major pathophysiological consequence of intestinal inflammation in IBD. We previously demonstrated that HIF activity and expression in intestinal epithelial cells (IECs) is prominently repressed at the transcriptional level by IFN-gamma. Given the concurrent expression of both HIF-1alpha and HIF-2alpha isoforms of the HIF transcriptional complex in IECs, we sought to define the relative contribution of HIF-1 and HIF-2 to epithelial barrier function. Human intestinal epithelial cell lines T84 and Caco-2 were used throughout. Stable knockdown lines targeting individual HIF subunits were generated using shRNA lentiviral transduction particles (MISSION TRC). To monitor apical junction assembly after calcium switch, T84 cells plated on transwell inserts were incubated for 16h in low Ca2+ medium (LCM) or 5 mins in HBSS with 2mM EDTA before switching to normal Ca2+ conditions (1.8 mM). Transepithelial resistance (TER) was measured over time using a voltohmeter, and expressed in ohm.cm2. Stable shRNA-mediated knockdown of HIF-1alpha, HIF-2alpha and HIF-1beta in model T84 epithelial cells resulted in significantly increased transcellular and paracellular permeability, as measured by transepithelial resistance and FITC-dextran flux assays. Immunolocalization studies of tight junction and adherens junction markers ZO-1 and E-cadherin, respectively, revealed non-uniform, undulating junctions in HIF-1alpha depleted T84 monolayers, consistent with attenuated intercellular tension and apical junction formation under conditions of altered HIF signaling. IFNgamma treatment of shControl IECs resulted in decreased barrier function and a junctional morphology similar to that observed in HIF-1alpha knockdown epithelia. Moreover, western blot analyses of shControl and knockdown lysates revealed increased myosin light chain (MLC) phosphorylation in HIF-1alpha knockdown IECs, indicative of an activated myosin II motor and increased perijunctional actomyosin contractility. Finally, inhibition of myosin light chain kinase (MLCK) or the Rho-associated protein kinases (ROCK) in HIF knockdown epithelia attenuated barrier defects and improved junctional morphology. These results define a role for HIF signaling in maintenance of apical IEC junctions at the level of the perijunctional actomyosin ring, and may in part explain the pathomechanism of IFN-gamma mediated loss of junctional integrity.

Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism.

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C

The enteric epithelium must absorb nutrients and water and act as a barrier to the entry of luminal material into the body; this barrier function is a key component of innate immunity. Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy occurs via inhibition of prostaglandin synthesis and perturbed epithelial mitochondrial activity. Here, the direct effect of NSAIDs [indomethacin, piroxicam (cyclooxygenase 1 and 2 inhibitors), and SC-560 (a cyclooxygenase 1 inhibitor)] on the barrier function of human T84 epithelial cell line monolayers was assessed by transepithelial electrical resistance (TER) and internalization and translocation of a commensal Escherichia coli. Exposure to E. coli in the presence and absence of drugs for 16 h reduced TER; however, monolayers cotreated with E. coli and indomethacin, but not piroxicam or SC-560, displayed significant increases in internalization and translocation of the bacteria. This was accompanied by increased reactive oxygen species (ROS) production, which was also increased in epithelia treated with E. coli only. Colocalization revealed upregulation of superoxide synthesis by mitochondria in epithelia treated with E. coli + indomethacin. Addition of antioxidants (vitamin C or a green tea polyphenol, epigallocathechin gallate) quenched the ROS and prevented the increase in E. coli internalization and translocation evoked by indomethacin, but not the drop in TER. Evidence of increased apoptosis was not observed in this model. The data implicate epithelial-derived ROS in indomethacin-induced barrier dysfunction and show that a portion of the bacteria likely cross the epithelium via a transcellular pathway. We speculate that addition of antioxidants as dietary supplements to NSAID treatment regimens would reduce the magnitude of decreased barrier function, specifically the transepithelial passage of bacteria.

Effect of neutrophils from septic patients on endothelial barrier function

BACKGROUNDSepsis is marked by systemic activation of the inflammatory system. With ongoing inflammation, integrity of the endothelium is compromised, leading to vascular leak, hypotension, and organ hypoperfusion. The effect of neutrophils obtained from septic patients on the integrity of the endothelial monolayer was examined and compared to naïve and fMLP‐activated cells.METHODSBlood was obtained from septic patients and healthy volunteers. Neutrophils were purified from whole blood using anti‐CD15 affinity bead purification. Human umbilical vein endothelial cells were grown to confluence and activated with TNFα. The relative effect of neutrophils on endothelial barrier function was determined by endothelial cell‐substrate impedance sensing.RESULTSAll neutrophil populations decreased monolayer resistance; however, the kinetics and extent of effect varied according to cell source and treatment. Neutrophils from septic patients caused less decrease in barrier function and exhibited a more biphasic response over time, compared to naïve and activated neutrophils.CONCLUSIONSNeutrophils from septic patients manifest a distinctly different effect on endothelial barrier function when compared to fully activated or naïve cells. Supported by NIHGM066194.

Hyperoxia Increases Elastic Modulus of Alveolar Epithelial Cells Through Rho Kinase

Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and both hyperoxia and mechanical ventilation can independently cause injury. However, the precise mechanisms that lead to epithelial injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be more stiff due to increased f‐action formation via activation of the GTPase RhoA and its downstream effecter Rho kinase (ROCK). We used fluorescence microscopy to examine cytoskeletal structures in murine lung alveolar epithelial cells. We measured cell elasticity (E) using atomic force microscopy in the indenter mode. Hyperoxia caused increased f‐actin stress fibers formation, and cells became stiffer (higher E). Treatment with an inhibitor of ROCK significantly decreased E and prevented the cytoskeletal changes. Pre‐exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched using the Flexercell device, but the ROCK inhibitor prevented this effect. Loss of cell adhesion would result in decreased barrier function. In summary, hyperoxia caused cytoskeletal remodeling that resulted in increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK. Supported by NIH grant HL094366 (CMW).

Dissecting the Causes of Atopic Dermatitis in Children: Less Foods, More Mites

Atopic dermatitis (AD) is a common, chronic or chronically relapsing, multifactorial skin disease that mainly occurs in children but affects also adults. AD usually begins early in life and often concerns people with a personal or family history of asthma and allergic rhinitis. AD is characterized by eczematous changes in the epidermis and originates from a late, T-cell mediated reaction associated to the formation and production of memory T-cell of TH2 type, occurrence of homing receptor at skin level and cutaneous lymphocyte-associated (CLA) antigens. Extrinsic or allergic AD, but not intrinsic AD, shows high total serum IgE levels and the presence of specific IgE for environmental and food allergens. A pivotal role in the pathogenesis of AD is played by filaggrin, a protein contained in the granular layer of the epidermis regulating the aggregation of keratin filaments. Mutation in the filaggrin gene causes decreased barrier function of the corny layers of the epidermis. This favours the enter through the skin of environmental allergens, especially the house dust mite, that further facilitates such entering by the proteolytic activity of its major allergen Der p 1. In fact, recent advances suggest that the dust mite, more than foods, is the major cause of allergic AD. As far as the causal diagnosis of AD is concerned, there is notable evidence supporting the capacity of the atopy patch test (APT) to reproduce the pathophysiologic events of AD. This makes APT a valuable diagnostic tool for AD.