Epithelial Paracellular Permeability Research Articles

Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer’s patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Simple graphical approach to investigate differences in transepithelial paracellular leak pathway permeability.

Although many studies have reported differences in epithelial paracellular Leak Pathway permeability following genetic manipulations and treatment with various agents, the basis for these differences remains mostly unclear. Two primary mechanisms which could underlie differences in Leak Pathway permeability are differences in the density of Leak Pathway openings and differences in the opening size. Using a computational approach, we demonstrate that these two possibilities can be readily distinguished graphically by comparing the apparent paracellular permeabilities of a size panel of solutes measured across different cell layers. Using this approach, we demonstrated that depletion of ZO‐1 protein in MDCK Type II renal epithelial cells decreased Leak Pathway opening size and increased opening density. Depletion of ZO‐2 protein either had no effect or minimally decreased opening size and did not markedly change opening density. Comparison of MDCK Type II cells with MDCK Type I cells revealed that Type I cells exhibited a substantially smaller Leak Pathway permeability than did Type II cells. This lower permeability was due to a decrease in opening density with little or no change in opening size. These results demonstrate the utility of this approach to provide insights into the basis for observed differences in epithelial Leak Pathway permeability. This approach has wide applications including analysis of the molecular basis for Leak Pathway permeability, the effects of specific manipulations on Leak Pathway permeability properties, and the effects of permeation enhancers on Leak Pathway permeability properties.

NOVEL CONTRIBUTIONS OF CLAUDIN-4 TO EPITHELIAL BARRIER REGULATION AND COLITIS

Abstract Intestinal barrier dysfunction is linked to human IBD and contributes to progression of experimental, immune-mediated colitis. Claudin family tight junction proteins are critical determinants of epithelial paracellular permeability and barrier function. Expression of one of these, claudin-2, a pore-forming claudin, is specifically upregulated in IBD. We have shown that claudin-2 overexpression or, conversely, channel inhibition, augment or attenuate, respectively, immune-mediated colitis severity. We sought to understand claudin contributions to barrier function using in vitro and in vivo models. Claudin-4 overexpression has been shown to reduce cation permeability in vitro, leading to the conclusion that this protein is a barrier-forming claudin. We found, however, that, in the absence of claudin-2 expression, neither knockout nor overexpression of claudin-4 affected barrier function. Instead, claudin-4 specifically suppressed claudin-2 pore function by destabilizing claudin-2 polymers and disrupting claudin-2 anchoring at the tight junction. Thus, claudin-4 is not barrier-forming but is the first pore-regulating claudin. Together with observations that claudin-2 exacerbates colitis in vivo and that claudin-4 antagonizes claudin-2 function in vitro led us to hypothesize that intestinal epithelial claudin-4 knockout would exacerbate colitis. Knockout mice were, however, protected from both immune-mediated and DSS-induced chemical colitis. Conversely, transgenic claudin-4 overexpression exacerbated colitis. Preliminary in vitro studies suggest that claudin-4 may limit collective migration and epithelial wound repair. As a whole, our studies require a reconsideration of claudin functional classifications and reveal unanticipated contributions of claudin-4 to barrier regulation and epithelial wound repair. The relationship between these functions and claudin-4 downregulation in human IBD remains to be explored.

NOVEL CONTRIBUTIONS OF CLAUDIN-4 TO EPITHELIAL BARRIER REGULATION AND COLITIS

Intestinal barrier dysfunction is linked to human IBD and contributes to progression of experimental, immune-mediated colitis. Claudin family tight junction proteins are critical determinants of epithelial paracellular permeability and barrier function. Expression of one of these, claudin-2, a pore-forming claudin, is specifically upregulated in IBD. We have shown that claudin-2 overexpression or, conversely, channel inhibition, augment or attenuate, respectively, immune-mediated colitis severity.

Insulin resistance per se drives early and reversible dysbiosis-mediated gut barrier impairment and bactericidal dysfunction

ObjectiveA common feature of metabolic diseases is their association with chronic low-grade inflammation. While enhanced gut permeability and systemic bacterial endotoxin translocation have been suggested as key players of this metaflammation, the mechanistic bases underlying these features upon the diabesity cascade remain partly understood. MethodsHere, we show in mice that, independently of obesity, the induction of acute and global insulin resistance and associated hyperglycemia, upon treatment with an insulin receptor (IR) antagonist (S961), elicits gut hyperpermeability without triggering systemic inflammatory response. ResultsOf note, S961-treated diabetic mice display major defects of gut barrier epithelial functions, such as increased epithelial paracellular permeability and impaired cell-cell junction integrity. We also observed in these mice the early onset of a severe gut dysbiosis, as characterized by the bloom of pro-inflammatory Proteobacteria, and the later collapse of Paneth cells antimicrobial defense. Interestingly, S961 treatment discontinuation is sufficient to promptly restore both the gut microbial balance and the intestinal barrier integrity. Moreover, fecal transplant approaches further confirm that S961-mediated dybiosis contributes at least partly to the disruption of the gut selective epithelial permeability upon diabetic states. ConclusionsTogether, our results highlight that insulin signaling is an indispensable gatekeeper of intestinal barrier integrity, acting as a safeguard against microbial imbalance and acute infections by enteropathogens.

Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact invitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Short peptide sequence enhances epithelial permeability through interaction with protein kinase C

We have identified a short peptide sequence (L-R5) acting as partial inhibitor of intracellular protein kinase C, capable of tight junction modulation in terms of reversible and non-toxic drug permeation enhancement. L-R5 is a pentapeptide with a cell-penetrating group at the N-terminus and of the sequence myristoyl-ARRWR. Apically applied in vitro, L-R5 transiently increased epithelial permeability within minutes, enhancing apical-to-basolateral (AB) transport of 4-kDa dextran and BCS class III drug naloxone. L-R5 was shown to be stable and effective at 37°C over a period of 24 hours. L-R5 was shown to be non-cytotoxic in consecutive exposure studies on primary human nasal epithelial cells by LDH release assay and ciliary beating frequency test. Finally, L-R5 by itself showed very low diffusion across epithelial monolayers, which is of advantage with regard to its expected negligible systemic bioavailability and side effects. Taken together, these data demonstrate the potential of short peptide partial inhibitor L-R5 to enhance the epithelial paracellular permeability via a reversible mechanism, and in a non-toxic manner.

Campylobacter jejuni increases the paracellular permeability of broiler chickens in a dose-dependent manner

In recent years, several studies emphasize the deleterious effects of Campylobacter jejuni on the chicken intestine. In this context, it was shown that C. jejuni, contrary to the general belief, has a negative influence on the gut barrier in chickens. More precisely, we demonstrated that C. jejuni affects gut physiology characterized by changes in ion transport and transepithelial ion conductance, but the underlying mechanism is yet to be investigated. In the actual study, to determine epithelial paracellular permeability, the mucosal to serosal flux of 14C-mannitol in the small and large intestine was measured applying Ussing chamber. A total of seventy-five 1-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to 3 different groups (n = 25 with 5 replicates/group) and infected at 14 d of age with a high (108 colony forming units [CFU]) or a low (104 CFU) dose of C. jejuni and a third group kept as noninfected control. Infection with the low dose of C. jejuni resulted in delayed cecal colonization but equalized at 21 d postinfection, independent of the dose. Invasion of liver and spleen with C. jejuni was only noticed in birds infected with 108 (CFU). Body weight (BW) and body weight gain of all birds infected with C. jejuni were lower than in the control group and varied with the dose of infection, confirming a negative correlation between the infection dose and birds BW. Mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in all C. jejuni infected birds compared with control birds. Likewise, significant differences in mannitol flux of both jejunum and cecum were detected depending on the infection dose of C. jejuni. The correlation analyses revealed a positive relationship between Campylobacter dose and mannitol flux of both jejunum and cecum. Altogether, the actual results emphasize that the adverse effect of C. jejuni on gut permeability arises in a dose-dependent manner.

α-Tocopherol Attenuates the Severity of Pseudomonas aeruginosa-induced Pneumonia.

Pseudomonas aeruginosa is a lethal pathogen that causes high mortality and morbidity in immunocompromised and critically ill patients. The type III secretion system (T3SS) of P. aeruginosa mediates many of the adverse effects of infection with this pathogen, including increased lung permeability in a Toll-like receptor 4/RhoA/PAI-1 (plasminogen activator inhibitor-1)-dependent manner. α-Tocopherol has antiinflammatory properties that may make it a useful adjunct in treatment of this moribund infection. We measured transendothelial and transepithelial resistance, RhoA and PAI-1 activation, stress fiber formation, P. aeruginosa T3SS exoenzyme (ExoY) intoxication into host cells, and survival in a murine model of pneumonia in the presence of P. aeruginosa and pretreatment with α-tocopherol. We found that α-tocopherol alleviated P. aeruginosa-mediated alveolar endothelial and epithelial paracellular permeability by inhibiting RhoA, in part, via PAI-1 activation, and increased survival in a mouse model of P. aeruginosa pneumonia. Furthermore, we found that α-tocopherol decreased the activation of RhoA and PAI-1 by blocking the injection of T3SS exoenzymes into alveolar epithelial cells. P. aeruginosa is becoming increasingly antibiotic resistant. We provide evidence that α-tocopherol could be a useful therapeutic agent for individuals who are susceptible to infection with P. aeruginosa, such as those who are immunocompromised or critically ill.

Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis.

Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1β (IL-1β), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease.

Validation of an Ex Vivo Permeation Method for the Intestinal Permeability of Different BCS Drugs and Its Correlation with Caco-2 In Vitro Experiments.

The absorption study of drugs through different biological membranes constitutes an essential step in the development of new pharmaceutical dosage forms. Concerning orally administered forms, methods based on monolayer cell culture of Caco-2 (Caucasian colon adenocarcinoma) have been developed to emulate intestinal mucosa in permeability studies. Although it is widely accepted, it has disadvantages, such as high costs or high technical complexity, and limitations related to the simplified structure of the monolayer or the class of molecules that can be permeated according to the transport mechanisms. The aim of this work was to develop a new ex vivo methodology which allows the evaluation of the intestinal apparent permeability coefficient (Papp) while using fewer resources and to assess the correlation with Caco-2. To this end, pig (Sus scrofa) duodenum segments were mounted in Franz diffusion cells and used to permeate four different drugs: ketorolac tromethamine (Kt), melatonin (Mel), hydrochlorothiazide (Htz), and furosemide (Fur). No statistically significant differences (p > 0.05) were observed corelating Papp values from Franz diffusion cells and Caco-2 cell experiments for Kt, Htz, and Fur. However, there were statistically significant differences (p < 0.05) correlating Papp values and Mel. The difference is explained by the role of Mel in the duodenal epithelial paracellular permeability reduction. Ex vivo permeation may be an equivalent method to Caco-2 for drugs that do not produce intestinal membrane phenomena that could affect absorption.

Feeding of deoxynivalenol increases the intestinal paracellular permeability of broiler chickens

In recent years, the deleterious effects attributed to mycotoxins, in particular on the intestine, faced increased attention and it was shown that deoxynivalenol (DON) causes adverse effects on gut health. In this context, it has been repeatedly reported that DON can alter the intestinal morphology, disrupt the intestinal barrier and reduce nutrient absorption. The underlying mechanism of a compromised intestinal barrier caused by DON in chickens has yet to be illustrated. Although, DON is rapidly absorbed from the upper parts of the small intestine, the effects on the large intestine cannot be excluded. Additionally, a damaging effect of DON on the gut epithelium might decrease the resistance of the gut against infectious agents. Consequently, the objectives of the present studies were: (1) to investigate the impact of DON on the epithelial paracellular permeability by demonstrating the mucosal to serosal flux of 14C-mannitol in the small and large intestine applying Ussing chambers and (2) to delineate the effects of DON on the colonization and translocation of Escherichia coli. Both parameters are well suited as potential indicators for gut barrier failure. For this, a total of 75 one-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to three different groups (n = 25 with 5 replicates/group) and were fed for 5 weeks with either contaminated diets (5 or 10 mg DON/kg feed) or basal diets (control). Body weight (BW) and BW gain of birds in the group fed with 10 mg/kg DON were significantly lower than in group with 5 mg/kg DON and the control group. Moreover, the mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in DON-fed groups compared to control birds. Consistent with this, DON enhanced the translocation of E. coli with a higher number of bacteria encountered in the spleen and liver. Altogether, the actual results verified that DON can alter the intestinal paracellular permeability in broiler chickens and facilitates the translocation of enteric microorganisms such as E. coli to extra-intestinal organs. Considering that moderate levels of DON are present in feed, the consumption of DON-contaminated feed can induce an intestinal breakdown with negative consequences on broiler health.

A Fixed Combination of Probiotics and Herbal Extracts Attenuates Intestinal Barrier Dysfunction from Inflammatory Stress in an In vitro Model Using Caco-2 Cells

Inflammatory Bowel Diseases (IBD), are considered a growing global disease, with about ten million people being affected worldwide. Maintenance of intestinal barrier integrity is crucial for preventing IBD onset and exacerbations. Some recent patents regarding oily formulations containing probiotics (WO2010122107A1 and WO2010103374A9) and the use of probiotics for gastrointestinal complaints (US20110110905A1 and US9057112B2) exist, or are pending application. In this work, we studied the effect of a fixed combination of registered Lactobacillus reuteri and Lactobacillus acidophilus strains and herbal extracts in an in vitro inflammation experimental model. Caco-2 cell monolayer was exposed to INF-γ+TNF-α or to LPS; Trans Epithelial Electrical Resistance (TEER) and paracellular permeability were investigated. ZO-1 and occludin Tight Junctions (TJs) were also investigated by mean of immunofluorescence. Pre-treatment with the fixed combination of probiotics and herbal extracts prevented the inflammation-induced TEER decrease, paracellular permeability increase and TJs translocation. In summary, the fixed combination of probiotics and herbal extracts investigated in this research was found to be an interesting candidate for targeting the re-establishment of intestinal barrier function in IBD conditions.

Chronic stress-associated visceral hyperalgesia correlates with severity of intestinal barrier dysfunction.

In humans, chronic psychological stress is associated with increased intestinal paracellular permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. However, it is unknown whether enhanced visceral pain and permeability are intrinsically linked and correlate. Treatment of rats with lubiprostone during WAS significantly reduced WAS-induced changes in intestinal epithelial paracellular permeability and visceral hyperalgesia in a subpopulation of rats. Lubiprostone also prevented WAS-induced decreases in the epithelial tight junction protein, occludin (Ocln). To address the question of whether the magnitude of visceral pain correlates with the extent of altered intestinal permeability, we measured both end points in the same animal because of well-described individual differences in pain response. Our studies demonstrate that visceral pain and increased colon permeability positively correlate (0.6008, P = 0.0084). Finally, exposure of the distal colon in control animals to Ocln siRNA in vivo revealed that knockdown of Ocln protein inversely correlated with increased paracellular permeability and enhanced visceral pain similar to the levels observed in WAS-responsive rats. These data support that Ocln plays a potentially significant role in the development of stress-induced increased colon permeability. We believe this is the first demonstration that the level of chronic stress-associated visceral hyperalgesia directly correlates with the magnitude of altered colon epithelial paracellular permeability.

Manipulation of Actin Cytoskeleton and Tight Junction Protein Knockdown Differentially Interact to Modulate Basal and H2O2‐Induced Paracellular Permeability of Renal Epithelial Cells

Renal epithelial tight junction permeability to large molecules (leak pathway) is increased by hydrogen peroxide (H2O2). Previous studies have suggested a role for the actin cytoskeleton in regulation of epithelial paracellular permeability but its possible involvement in H2O2‐induced regulation of renal epithelial paracellular permeability is unknown. To assess the role of the actin cytoskeleton, we examined the effect of two agents that manipulate differentially the actin cytoskeleton, blebbistatin (inhibits myosin II ATPase) and jasplakinolide (enhances F‐actin nucleation), on basal and H2O2‐induced renal epithelial leak pathway permeability, measured as calcein flux rate. The effects of these compounds was examined in wild type MDCK cells and in MDCK cells in which either occludin protein or ZO‐1 protein has been knocked down to determine if either of these tight junction proteins might be important in mediating actin network actions. Under basal conditions, treatment with 40 mM blebbistatin produced little or no change in calcein flux rate in wild type MDCK cells. H2O2 increased calcein flux rate, as expected. The combination of H2O2 and blebbistatin produced a synergistic increase in calcein flux rate. Jasplakinolide (200 nM) produced a marked increase in calcein flux rate. H2O2 and jasplakinolide did not increase calcein flux rate beyond the rate produced by jasplakinolide alone. Knockdown of occludin protein in MDCK cells did not affect basal calcein flux rate but enhanced the H2O2‐induced increase. In the absence of H‐2O2, blebbistatin produced little or no change in calcein flux rate. H2O2 and blebbistatin did not increase calcein flux rate further than that produced by H2O2 alone. Jasplakinolide increased the calcein flux rate but attenuated the increase produced by H2O2 when cells were incubated with both compounds. Knockdown of ZO‐1 protein increased basal calcein flux rate approximately two‐fold. Treatment with H2O2 did not further increase flux rate. Treatment of ZO‐1 knockdown cells with blebbistatin increased the calcein flux rate, in contrast to its lack of effect on wild type MDCK cells. Treatment with the combination of H2O2 and blebbistatin did not increase flux rate beyond that produced by blebbistatin alone. Jasplakinolide alone increased calcein flux rate in ZO‐1‐knockdown cells. The combination of H2O2 and jasplakinolide did not increase flux rate beyond that produced by jasplakinolide alone. These results suggest a complex role of the actin cytoskeleton in regulation of basal and H2O2‐stimulated leak pathway permeability in renal epithelial cells. First, they suggest that an intact F‐actin network is necessary to maintain low basal leak pathway permeability. Second, they suggest the tight junction is under inward actomyosin‐mediated tension, via the ZO‐1 protein, which contributes to maintenance of low leak pathway permeability, particularly in stress conditions. Third, they suggest that H2O2 treatment produces a state in MDCK cells that is functionally similar to knockdown of ZO‐1 protein. Fourth, they suggest that the effect of blebbistatin is mediated via occludin protein.Support or Funding InformationNIDDK and institutional fundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Colon dysregulation in methamphetamine self-administering HIV-1 transgenic rats.

The integrity and function of the gut is impaired in HIV-infected individuals, and gut pathogenesis may play a role in several HIV-associated disorders. Methamphetamine is a popular illicit drug abused by HIV-infected individuals. However, the effect of methamphetamine on the gut and its potential to exacerbate HIV-associated gut pathology is not known. To shed light on this scenario, we evaluated colon barrier pathology in a rat model of the human comorbid condition. Intestinal barrier integrity and permeability were assessed in drug-naïve Fischer 344 HIV-1 transgenic (Tg) and non-Tg rats, and in Tg and non-Tg rats instrumented with jugular cannulae trained to self-administer methamphetamine or serving as saline-yoked controls. Intestinal permeability was determined by measuring the urine content of orally gavaged sugars. Intestinal barrier integrity was evaluated by immunoblotting or immunofluorescence of colon claudin-1 and zonula occludens-1 (ZO-1), two major tight junction proteins that regulate gut epithelial paracellular permeability. Both non-Tg and Tg rats self-administered moderate amounts of methamphetamine. These amounts were sufficient to increase colon permeability, reduce protein level of claudin-1, and reduce claudin-1 and ZO-1 immunofluorescence in Tg rats relative to non-Tg rats. Methamphetamine decreased tight junction immunofluorescence in non-Tg rats, with a similar, but non-significant trend observed in Tg rats. However, the effect of methamphetamine on tight junction proteins was subthreshold to gut leakiness. These findings reveal that both HIV-1 proteins and methamphetamine alter colon barrier integrity, and indicate that the gut may be a pathogenic site for these insults.

Acrolein Disrupts Tight Junction Proteins and Causes Endoplasmic Reticulum Stress-Mediated Epithelial Cell Death Leading to Intestinal Barrier Dysfunction and Permeability

Increasing evidence suggests that environmental and dietary factors can affect intestinal epithelial integrity leading to gut permeability and bacterial translocation. Intestinal barrier dysfunction is a pathogenic process associated with many chronic disorders. Acrolein is an environmental and dietary pollutant and a lipid-derived endogenous metabolite. The impact of acrolein on the intestine has not been investigated before and is evaluated in this study, both invitro and invivo. Our data demonstrate that oral acrolein exposure in mice caused damage to the intestinal epithelial barrier, resulting in increased permeability and subsequently translocation of bacterial endotoxin-lipopolysaccharide into the blood. Similar results were seen invitro using established Caco-2 cell monolayers wherein acrolein decreased barrier function and increased permeability. Acrolein also caused the down-regulation and/or redistribution of three representative tight junction proteins (ie, zonula occludens-1, Occludin, Claudin-1) that critically regulate epithelial paracellular permeability. In addition, acrolein induced endoplasmic reticulum stress-mediated death of epithelial cells, which is an important mechanism contributing to intestinal barrier damage/dysfunction, and gut permeability. Overall, we demonstrate that exposure to acrolein affects the intestinal epithelium by decrease/redistribution of tight junction proteins and endoplasmic reticulum stress-mediated epithelial cell death, thereby resulting in loss of barrier integrity and function. Our findings highlight the adverse consequences of environmental and dietary pollutants on intestinal barrier integrity/function with relevance to gut permeability and the development of disease.

Complementary Roles of Nod2 in Hematopoietic and Nonhematopoietic Cells in Preventing Gut Barrier Dysfunction Dependent on MLCK Activity

Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2 mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. Depletion of CD4 T cells in Nod2 mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-α and interferon-γ secretion by CD4 T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis.

Comparison of electrogenic glucose transport processes and permeability between proximal and distal jejunum of laying hens

ABSTRACT1. The current objective was to assess (1) differences in mucosal transepithelial short-circuit current (Isc) and tissue conductance (GT), (2) the effect of a glucose stimulus and (3) epithelial paracellular permeability in the proximal and distal jejunum of laying hens.2. Proximal and distal jejunal segments used in the Ussing chambers were collected at 9 ± 0.5 and 73 ± 3.4% (SEM) of jejunal length, respectively. The proximal jejunal mucosa showed a small negative Isc (−1.3 µA/cm2), whereas the distal jejunum had a higher Isc (32.9 µA/cm2). Similarly, GT was 2.5-fold greater in the distal compared to the proximal jejunum.3. Increased paracellular permeability in the distal jejunum was displayed as demonstrated by a 5-fold higher mucosal to serosal flux of fluorescein isothiocyanate and horseradish peroxidase, representing molecules of low and high molecular weight, respectively.4. Addition of glucose to the mucosal side (5 mmol/l, final concentration in the chamber) to stimulate an absorptive effect caused 3-fold greater GT in the distal compared to the proximal jejunum.5. In conclusion, the present results supported site-specific electrogenic transport processes for the jejunal mucosa of laying hens. Therefore, precise description of the jejunal site may contribute to an improved comparability of electrophysiological data.

Real-time monitoring of trans-epithelial electrical resistance in cultured intestinal epithelial cells: the barrier protection of water-soluble dietary fiber.

In this study we aimed to verify a real-time trans-epithelial electrical resistance (TEER) monitoring system in a Caco-2 monolayer and to investigate the therapeutic effect of partially hydrolyzed guar gum (PHGG), a dietary fiber, against interferon (IFN)-γ-induced intestinal barrier dysfunction using this monitoring system. We measured TEER using a real-time monitoring system and evaluated epithelial paracellular permeability using fluorescein isothiocyanate-conjugated dextran (4 kDa; FD4) in Caco-2 monolayers treated with IFN-γ for 48 h. The expression and distribution of tight junction (TJ)-associated proteins, ZO-1 and occludin, were analyzed by Western blot and immunocytochemistry, respectively. In some experiments PHGG was added prior to IFN-γ treatment in order to investigate its protective effect on barrier function. IFN-γ treatment significantly decreased TEER and increased FD4 flux across Caco-2 monolayers, indicating a great influence of IFN-γ on the intestinal epithelial paracellular permeability. In contrast, the pretreatment of PHGG significantly reduced the IFN-γ-induced increment of FD4 flux without affecting TEER. Neither IFN-γ nor PHGG treatment affected the expressions of TJ-associated proteins, while immunocytochemistry showed that IFN-γ-induced redistribution of occludin was clearly restored by PHGG. Real-time TEER monitoring enabled us to evaluate the dynamic changes of intestinal epithelial barrier function. PHGG may have a protective effect against IFN-γ-induced barrier dysfunction by attenuating the paracellular hyperpermeability; thus, its promotion as a functional food is anticipated.