Pore Pathway Research Articles

Membrane Permeant Inhibitor of Myosin Light Chain Kinase Worsens Survival in Murine Polymicrobial Sepsis.

Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Genetic deletion of myosin light chain kinase (MLCK) reverses intestinal hyperpermeability and improves survival in a murine model of intra-abdominal sepsis. In an attempt to determine whether these findings could be translated using a more clinically relevant strategy, this study aimed to determine if pharmacologic inhibition of MLCK using the membrane permeant inhibitor of MLCK (PIK) improved gut barrier function and survival following sepsis. C57BL/6 mice underwent cecal ligation and puncture to induce sepsis and were then randomized to receive either PIK or vehicle. Unexpectedly, PIK significantly worsened 7-day survival following sepsis (24% vs. 62%). The three pathways of intestinal permeability were then interrogated by orally gavaging septic mice with creatinine (6Å), FD-4 (28Å), and rhodamine70 (120Å) and assaying their appearance in the bloodstream. PIK led to increased permeability in the leak pathway with higher levels of FD-4 in the bloodstream compared to septic mice given vehicle. In contrast, no differences were detected in the pore or unrestricted pathways of permeability. Examination of jejunal tight junctions for potential mechanisms underlying increased leak permeability revealed that mice that received PIK had increased phosphorylated MLC without alterations in occludin, ZO-1, or JAM-A. PIK administration was not associated with significant differences in systemic or peritoneal bacterial burden, cytokines, splenic or Peyer's Patches immune cells or intestinal integrity. These results demonstrate that pharmacologic inhibition of MLCK unexpectedly increases mortality, associated with worsened intestinal permeability through the leak pathway, and suggest caution is required in targeting the gut barrier as a potential therapy in sepsis.

Chronic opioid use modulates human enteric microbiota and intestinal barrier integrity

ABSTRACT Over the past three decades the United States has experienced a devastating opioid epidemic. One of the many debilitating side effects of chronic opioid use is opioid-induced bowel dysfunction. We investigated the impact of methadone maintenance treatment (MMT) on the gut microbiome, the gut bacterial metabolite profile, and intestinal barrier integrity. An imbalance in key bacterial communities required for production of short-chain fatty acids (SCFAs), mucus degradation, and maintenance of barrier integrity was identified. Consistent with dysbiosis, levels of fecal SCFAs were reduced in MMT. We demonstrated that metabolites synthesized by Akkermansia muciniphila modulate intestinal barrier integrity in vitro by strengthening the pore pathway and regulating tight junction protein expression. This study provides essential information about the therapeutic potential of A. muciniphila and warrants development of new clinical strategies that aim to normalize the gut microbiome in individuals affected by chronic opioid use.

Rapid Evaluation of Intestinal Paracellular Permeability Using the Human Enterocytic-Like Caco-2/TC7 Cell Line.

Paracellular permeability of the intestinal epithelium is a feature of the intestinal barrier, which plays an important role in the physiology of gut and the whole organism. Intestinal paracellular permeability is controlled by complex processes and is involved in the passage of ions and fluids (called pore pathway) and macromolecules (called leak pathway) through tight junctions, which seal the intercellular space. Impairment of intestinal paracellular permeability is associated with several diseases. The identification of a defect in intestinal paracellular permeability may help to understand the implication of gut barrier as a cause or a consequence in human pathology. Here we describe two complementary methods to evaluate alteration of paracellular permeability in cell culture, using the human intestinal cell line Caco-2 and its clone Caco-2/TC7.

Differentiating Between Tight Junction-Dependent and Tight Junction-Independent Intestinal Barrier Loss In Vivo.

The intestinal barrier is an essential component of innate host defense. The single layer of epithelial cells that line the intestine must balance barrier function with both active, transcellular and diffusive, paracellular transport. Tight junctions, which link adjacent cells, form a selectively permeable seal that defines both paracellular transport and barrier properties. Molecules can cross tight junctions by either of two distinct routes, termed pore and the leak pathways, that differ in capacity, charge-selectivity, size-selectivity, and responses to physiological and pathophysiological stimuli. A third intestinal permeability route, the unrestricted pathway, reflects loss of the epithelial barrier, as occurs with mucosal damage, is independent of paracellular and transcellular pathways, and is neither charge- nor size-selective.The most commonly used approach for measuring intestinal permeability in vivo involves gavage of FITC-4kDa dextran and analysis of the quantity recovered in serum. Unfortunately, this method cannot distinguish between leak and unrestricted pathways, as 4kDa dextran can cross both. Moreover, 4kDa dextran is too large to cross the pore pathway and, therefore, provides no information regarding this paracellular flux route. Here we describe a multiplex method that allows simultaneous, independent analysis of each pathway.

Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity.

Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction-independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage. Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay.

Claudin-2 pore causes leak that breaches the dam in intestinal inflammation.

The tight junction protein claudin-2 is upregulated in inflammatory bowel disease, and yet its deficit worsens infectious and chemical colitis. In this issue of the JCI, Raju and Shashikanth et al. examined the contribution of claudin-2 to immune-mediated colitis. The authors used transgenic mouse models to show that claudin-2 deficiency attenuated colitis progression as well as a leak barrier defect, albeit at the risk of intestinal obstruction. Further, inhibition of claudin-2 by targeting casein kinase 2 (CK2) also ameliorated colitis. The findings reveal unsuspected links between the pore and leak pathways of intestinal permeability and immune responses leading to colitis. They additionally suggest potential targets for therapeutic intervention in inflammatory bowel disease.

Dimer interaction in the Hv1 proton channel

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: It is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute, but the nature of transmembrane packing is unclear. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, we find that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. Our results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.

Regulation of Intestinal Epithelial Barrier and Immune Function by Activated T Cells.

Background & AimsCommunication between T cells and the intestinal epithelium is altered in many diseases, causing T-cell activation, depletion, or recruitment, and disruption of the epithelium. We hypothesize that activation of T cells regulates epithelial barrier function by targeting the assembly of the tight junction complex.MethodsIn a 3-dimensional and 2-dimensional co-culture model of activated T cells subjacent to the basolateral surface of an epithelial monolayer, the pore, leak, and unrestricted pathways were evaluated using transepithelial resistance and flux of fluorescently labeled tracers. T cells were acutely and chronically activated by cross-linking the T-cell receptor. Tight junction assembly and expression were measured using quantitative polymerase chain reaction, immunoblot, and immunofluorescence confocal microscopy.ResultsCo-culture with acutely and chronically activated T cells decreased the magnitude of ion flux through the pore pathway, which was maintained in the presence of acutely activated T cells. Chronically activated T cells after 30 hours induced a precipitous increase in the magnitude of both ion and molecular flux, resulting in an increase in the unrestricted pathway, destruction of microvilli, expansion in cell surface area, and cell death. These fluctuations in permeability were the result of changes in the assembly and expression of tight junction proteins, cell morphology, and viability. Co-culture modulated the expression of immune mediators in the epithelium and T cells.ConclusionsBidirectional communication between T cells and epithelium mediates a biphasic response in barrier integrity that is facilitated by the balance between structural proteins partitioning in the mobile lateral phase vs the tight junction complex and cell morphology.

The Effects of Prebiotic Supplementation with OMNi-LOGiC® FIBRE on Fecal Microbiome, Fecal Volatile Organic Compounds, and Gut Permeability in Murine Neuroblastoma-Induced Tumor-Associated Cachexia.

Malignant diseases can cause tumor-associated cachexia (TAC). Supplementation with prebiotic non-digestible carbohydrates exerts positive metabolic effects in experimental oncologic diseases. The aim of this project was to assess the effect of prebiotic supplementation with OMNi-LOGiC® FIBRE on intestinal microbiome, bacterial metabolism, gut permeability, and inflammation in a murine model of neuroblastoma (NB)-associated TAC. For this study, 2,000,000 NB cells (MHH-NB11) were implanted into athymic mice followed by daily supplementation with water or 200 mg prebiotic oligosaccharide (POS) OMNi-LOGiC® FIBRE (NB-Aqua, n = 12; NB-POS, n = 12). Three animals of each tumor group did not develop NB. The median time of tumor growth (first visibility to euthanasia) was 37 days (IQR 12.5 days) in the NB-Aqua group and 37 days (IQR 36.5 days) in the NB-POS group (p = 0.791). At euthanasia, fecal microbiome and volatile organic compounds (VOCs), gut permeability (fluorescein isothiocyanate-dextran (FITC-dextran), and gut barrier markers were measured. Values were compared to sham animals following injection of culture medium and gavage of either water or OMNi-LOGiC® FIBRE (SH-Aqua, n = 10; SH-POS, n = 10). Alpha diversity did not differ significantly between the groups. Principal coordinate analysis (PCoA) revealed clustering differences between Aqua and POS animals. Both NB and POS supplementation led to taxonomic alterations of the fecal microbiome. Of 49 VOCs, 22 showed significant differences between the groups. NB animals had significantly higher gut permeability than Aqua animals; POS did not ameliorate these changes. The pore and leak pathways of tight junctions did not differ between groups. In conclusion, our results suggest that NB-induced TAC causes increased gut permeability coupled with compositional changes in the fecal microbiome and VOC profile. Prebiotic supplementation with OMNi-LOGiC® FIBRE seemed to induce modifications of the fecal microbiome and VOC profile but did not improve gut permeability.

A Disposable Paper-Based Urea Sensor Using Metal Oxides

The level of urea in urine can be a crucial biomarker to detect kidney failure, dehydration, renal function, and hepatic failure. [1] Thus, rapid and accurate sensing of urea is essential in a diagnostic clinic, especially for point-of-care testing devices (POCT). Paper-based analytical devices have been demonstrated as POCT due to its disposability, portability, low cost, and environmental-friendly quality. [2] In order to measure urea, electrochemical biosensors based on the enzyme were explored, but the application of enzyme has its limitations due to low shelf-life, complicated immobilization procedure, high cost, and complicate operating condition which is not fit to POCT application. [3] To overcome the limitations of enzyme, non-enzymatic urea sensor have attracted from researchers due to advantages such as good stability, re-usability, high sensitivity, redox flexibility, simplicity, and low cost. [4]As a non-enzymatic catalyst, nano metal oxides such as NiO, SnO2, and Fe2O3, exhibited catalytic activity on urea by the formation of the redox couple on the sensor surface.[5] [6] [7] To utilize non-enzymatic catalysts with comparable properties of enzyme, the development to increase reaction surface area has been emphasized by using nanomaterial, hierarchical structure, and support matrix.[8] [9] However, those processes involve complicate fabrication steps and often need additional post-process to remove the template leading to poisoning of the catalyst. As a potential approach for template-free process, paper can be used for an electrochemical sensor substrate. Paper has a complex fiber matrix that can provide pore pathways for the electrolyte to infiltrate inside, and by distributing catalyst uniformly within the paper, which formsmore reaction sites. Therefore, enhancement of performance and simplification of the fabrication can be achieved with the use of paper.In this study, the simple, disposable paper-based device was developed for urea electro-oxidation by using paper matrix as support. The paper-based electrode was fabricated by a screen printing method with filter paper, and different combinations of nano-metal oxides, NiO, SnO2, and Fe2O3. The metal oxide catalysts were loaded on the working electrode area and compared the performance. Structure and morphology of paper-based electrode were characterized by XRD and SEM. Investigation of selectivity and LOD was conducted by potentiostat. A detailed description of the fabrication of paper-based electrode and sensing performances will be presented.

Effect of ZO Protein Knockdown on Leak Pathway Pore Size in MDCK Renal Epithelial Cell Populations

The tight junction is the primary barrier to the paracellular movement of water and solutes across epithelial cells (paracellular permeability). Paracellular permeability is divided into two pathways: the Pore Pathway (small solute size, high capacity, charge‐selective) and the Leak Pathway (large solute size, low capacity, charge‐nonselective). Much has been learned about the molecular basis, properties, and regulation of the Pore Pathway. The Leak Pathway is much less well understood. Two basic questions about the Leak Pathway that remain controversial are: 1) does the Leak Pathway have a size limit; and 2) if so, how is the Leak Pathway size limit affected by manipulating specific tight junction proteins. We have initiated an investigation of these two questions by measuring the paracellular permeability of a size range of fluorescein dextrans (4 kDa to 70 kDa) and calculating pore size using the mathematical model, the Renkin sieving equation. In this mathematical model, calculation of pore size is independent of total flux rate, which is also affected by the pore volume fraction and pore length. We first used this approach to examine Leak Pathway permeability in the wild type MDCK renal epithelial cell line. The paracellular movement of all fluorescein dextrans across MDCK cell monolayers was linear with time. The apparent permeability (Papp) decreased with increasing solute Stokes radius. Using the Renkin sieving equation, the Leak Pathway pore size in wild type MDCK cell monolayers was calculated to be ~423 Angstroms. We then examined the effect of knockdown of either of the tight junction‐associated cytoplasmic proteins, ZO‐1 or ZO‐2, on pore size. The paracellular flux rates for all fluorescein dextrans were higher across ZO‐1 knockdown MDCK (ZO‐1 KD) cell populations than across either wild type MDCK cell populations or ZO‐2 knockdown MDCK (ZO‐2 KD) cell populations, which were similar. The paracellular flux of each fluorescein dextran was linear with time across populations of either ZO‐1 KD cells or ZO‐2 KD cells. Papp decreased with increasing Stokes radius for both knockdown cell lines. The calculated pore size for ZO‐2 KD cell populations was similar to that calculated for wild type MDCK cell populations. In contrast, the pore size for ZO‐1 KD cell populations was substantially smaller (~171 Angstroms) than was calculated for wild type MDCK cell populations despite the fact that the total flux rate for all fluorescein dextrans was significantly higher across ZO‐1 KD cell populations. These results indicate that knockdown of ZO‐1 in MDCK cells decreases the Leak Pathway pore size, whereas, knockdown of ZO‐2 does not alter Leak Pathway pore size. In addition, since the flux rate across ZO‐1 KD cell monolayers is higher than across monolayers of the other two cell lines, these results suggest ZO‐1 KD cell populations exhibit either a greater pore volume fraction and/or a shorter pore length than do either wild type MDCK cell populations or ZO‐2 KD cell populations.Support or Funding InformationNYITCOM internal funds

Dimer Interaction in the Hv1 Proton Channel

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: it is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4) and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s, which are thought to couple S4 activation motions.

Function and three-dimensional structure of intervessel pit membranes in angiosperms: a review

ABSTRACT Pit membranes in bordered pits of tracheary elements of angiosperm xylem represent primary cell walls that undergo structural and chemical modifications, not only during cell death but also during and after their role as safety valves for water transport between conduits. Cellulose microfibrils, which are typically grouped in aggregates with a diameter between 20 to 30 nm, make up their main component. While it is clear that pectins and hemicellulose are removed from immature pit membranes during hydrolysis, recent observations of amphiphilic lipids and proteins associated with pit membranes raise important questions about drought-induced embolism formation and spread via air-seeding from gas-filled conduits. Indeed, mechanisms behind air-seeding remain poorly understood, which is due in part to little attention paid to the three-dimensional structure of pit membranes in earlier studies. Based on perfusion experiments and modelling, pore constrictions in fibrous pit membranes are estimated to be well below 50 nm, and typically smaller than 20 nm. Together with the low dynamic surface tensions of amphiphilic lipids at air-water interfaces in pit membranes, 5 to 20 nm pore constrictions are in line with the observed xylem water potentials values that generally induce spread of embolism. Moreover, pit membranes appear to show ideal porous medium properties for sap flow to promote hydraulic efficiency and safety due to their very high porosity (pore volume fraction), with highly interconnected, non-tortuous pore pathways, and the occurrence of multiple pore constrictions within a single pore. This three-dimensional view of pit membranes as mesoporous media may explain the relationship between pit membrane thickness and embolism resistance, but is largely incompatible with earlier, two-dimensional views on air-seeding. It is hypothesised that pit membranes enable water transport under negative pressure by producing stable, surfactant coated nanobubbles while preventing the entry of large bubbles that would cause embolism.

Nano-viscosimetry analysis of the membrane disrupting action of the bee venom peptide melittin

Melittin is one of the most studied α-helical cationic membrane disrupting peptides. It is the main component of bee venom, however it is considered an antimicrobial peptide for its ability to kill bacteria. Melittin is believed to act by opening large toroidal pores in the plasma membrane of the targeted cells/bacteria, although this is questioned by some authors. Little is known, however, about the molecular mechanism leading to this activity. In this study the mechanism of action of melittin was studied by dye leakage and quartz crystal microbalance fingerprinting analysis in biomimetic model membranes. The results revealed the existence of multiple stages in the membrane disrupting action with characteristic differences between different membrane types. In bacterial-mimetic (charged) lipid mixtures the viscoelastic fingerprints suggest a surface-acting mechanism, whereas in mammalian-mimetic (neutral) membranes melittin appears to penetrate the bilayer already at low concentrations. In domain-forming mixed membranes melittin shows a preference for the domain containing predominantly zwitterionic lipids. The results confirm membrane poration but are inconsistent with the insertion-to-toroidal pore pathway. Therefore hypotheses of the two membrane disrupting pathways were developed, describing the membrane disruption as either surface tension modulation leading to toroidal pore formation, or linear aggregation leading to fissure formation in the membrane.

Tortuous Pore Path Through the Glaucomatous Lamina Cribrosa

The lamina cribrosa is a primary site of damage in glaucoma. While mechanical distortion is hypothesized to cause reduction of axoplasmic flow, little is known about how the pores, which contains the retinal ganglion cell axons, traverse the lamina cribrosa. We investigated lamina cribrosa pore paths in vivo to quantify differences in tortuosity of pore paths between healthy and glaucomatous eyes. We imaged 16 healthy, 23 glaucoma suspect and 48 glaucomatous eyes from 70 subjects using a swept source optical coherence tomography system. The lamina cribrosa pores were automatically segmented using a previously described segmentation algorithm. Individual pore paths were automatically tracked through the depth of the lamina cribrosa using custom software. Pore path convergence to the optic nerve center and tortuosity was quantified for each eye. We found that lamina cribrosa pore pathways traverse the lamina cribrosa closer to the optic nerve center along the depth of the lamina cribrosa regardless of disease severity or diagnostic category. In addition, pores of glaucoma eyes take a more tortuous path through the lamina cribrosa compared to those of healthy eyes, suggesting a potential mechanism for reduction of axoplasmic flow in glaucoma.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

BackgroundMammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia.ResultsHere we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired.ConclusionsDifferentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.

Tight junctions of the proximal tubule and their channel proteins.

The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na+ and K+. Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na+ and water share the same pore. Already solved claudin crystal structures reveal a characteristic β-sheet, comprising β-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.

NOD2 Deficiency Impairs the Intestinal Tight Junction Barrier in a Murine Short Bowel Model.

Introduction: Short bowel syndrome with chronic intestinal failure occurs after extensive small bowel resection. Despite intestinal adaptation, protein, energy, fluid and micronutrient balance cannot be maintained by a conventional diet. NOD2 mutations are a risk factor for chronic intestinal failure independent of the underlying disease. The mechanisms are not well understood. In a murine short bowel model the epithelial barrier function in the adapted jejunum was studied in the presence and absence of NOD2. Methods: Male C57BL6/J NOD2 knockout (−/−) and wild type (+/+) mice underwent 40% ileocecal resection (ICR). Sham control mice received transsection of the ileum. Mice were followed up for 14 days. At baseline (d0) 1cm of the most proximal resected ileum was studied in a vertical Ussing chamber and histologically. After 14d 1cm of the most distal remaining small intestine was studied and compared to baseline. In order to study the paracellular leak pathway, flux of 4kDa FITC-dextran was measured. The pore pathway was characterized by transepithelial resistance (TER) and NaCl diultion potentials. Results: Both +/+ and −/− animals showed similar morphologic adaptation 7 days after ICR such as significantly increased villus height (+/+ 229% P=0.028 vs. d0 and −/− 191% P=0.028 vs. d0, n=6-7 each) and crypt depth (+/+ 153% P=0.028 vs. d0 and −/− 129% P=0.028 vs. d0, n=6-7 each). The barrier to uncharged macromolecules (4 kDa FITC-dextran flux) was largely unaffected by NOD2 status. TER was not different in jejunal segments of +/+ and −/− mice at baseline levels (d0) and increased in the adapted jejunum. TER increase at d14 was significantly higher in −/− mice (74.5±6.0 Ω*cm2 +/+ ICR vs. 105.0±10.6 Ω*cm2 −/− ICR, n=5). In line with this, relative permeability to sodium was more severely impaired in the adapted tissue of −/− mice (+/+ ICR d0 7.7±0.4 vs. 14d 6.9±0.3 P=0.35, −/− ICR d0 6.3±0.1 vs. 14d 5.7±0.1 P=0.0157, n=5 each). Conclusion: Profound morphologic adaptation occurs after extensive ileocecal resection. The paracellular leak flux across the adapted epithelium is unaffected by NOD2 status. In contrast, TER is increased after adaptation and more so in absence of NOD2. This may in part be explained by an altered internal electric field in the structurally altered mucosa. However, adaptation of the tight junction barrier occurs as well. Our studies indicate that NOD2 function specifically contributes to the regulation of tight junction mediated charge selective ion flux in the adapted jejunum.

Dynamic modeling of the tight junction pore pathway.

Claudins define paracellular permeability to small molecules by forming ion-selective pores within the tight junction. We recently demonstrated that claudin-2 channels are gated and open and close on a submillisecond timescale. To determine if and how the ensemble behavior of this unique class of entirely extracellular gated ion channels could define global epithelial barrier function, we have developed an in silico model of local claudin-2 behavior. This model considers the complex anastomosing ultrastructure of tight junction strands and can be scaled to show that local behavior defines global epithelial barrier function of epithelial monolayers expressing different levels of claudin-2. This is the first mathematical model to describe global epithelial barrier function in terms of the dynamic behavior of single tight junction channels and establishes a framework to consider gating kinetics as a means to regulate barrier function.

Reactive oxygen species (ROS) Play a Key Role in Bile Acid (BA)‐Induced Barrier Dysfunction And Alters the Leak Function of Tight Junctions (TJ) in Human Colon Carcinoma T84 Cells

Dysfunction of the pore and leak pathways of TJ plays a vital role in the pathogenesis of inflammatory and diarrheal diseases. Excess colonic BAs are known to alter mucosal permeability. We have shown that chenodeoxycholic acid (CDCA; 500μM) and its derivative, lithocholic acid (LCA; 50μM) have opposing effects on the barrier integrity of T84 cells (FASEB J 2016 30:1223.29). While CDCA‐induced changes in pore and leak function of TJs are further enhanced by pro‐inflammatory cytokines (PiC [ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), LCA reversed CDCA± PiC's effects only on TJ leak function. As ROS is known to increase paracellular permeability, we examined its role in BA regulation of TJ dysfunction.Confluent T84 cells (TransEpithelial Resistance; TER >1000Ωcm2) were treated apically with DMSO, 500μM CDCA, 50μM LCA alone, in combination, or ± PiCs for 0.5–18 hours (H). Mitochondrial ROS (mROS, CellRox, flow cytometry), H2O2 release (colorimetry) and apoptosis (Annexin V, flow cytometry) were measured. ROS contribution to BA regulation of TJ was assessed by examining the effect of ROS scavenger, N‐acetyl cysteine (NAC; 1mM)±BA±PiC on i. Oxidative stress; ii. Pore function measured as TER or dilution potential (Dψ, upon 50% reduction in apical [NaCl]); iii. Leak function measured as fluorescein isothiocyanate‐10 kDa dextran flux (F10D).CDCA increased mROS and this was unaltered by PiC (CellRox+ cells, in %, DMSO: 8±1; CDCA: 24±3; PiC: 11±1; PiC+CDCA: 25±3, n=3). Similarly, LCA did not alter CDCA‐induced mROS changes. CDCA ± PiC increased H2O2 release over time (4 H: fold–change, DMSO: 1; PiC: 2, CDCA: 1.5, CDCA+PiC: 2; 18 H: PiC: 4.5, CDCA: 17, CDCA+PiC: 14; n=6). LCA reversed H2O2 release by CDCA or PiC at 4 and 18H or CDCA+PiC at 4H (p<0.05). However, at 18H LCA only inhibited ~50% of CDCA+PiC‐induced H2O2 release. NAC inhibited ROS production in all of the above conditions. In contrast, NAC had no significant effect on BA‐induced apoptosis (18 H: DMSO 8±0.3%, CDCA: 18±1%; LCA: 8±0.2%; CDCA+LCA: 12±0.3%; NAC: 9±0.4%; CDCA+NAC: 15±1%; LCA+NAC: 7±0.1%; CDCA+LCA+NAC: 14±0.2%, p>0.05, n=6).In terms of pore function, NAC neither altered CDCA±LCA‐induced decreases in TER (% decrease compared to DMSO; 1H CDCA: 83±7; CDCA+NAC: 84±5; CDCA+LCA: 80±3; CDCA+LCA+NAC: 81±7, n=3) nor ion selectivity (DΨ, 25 min, in mV; DMSO: 8 ± 1, CDCA: − 7 ± 2, LCA: 8 ± 1; CDCA+LCA: −5 ± 2, NAC: 7±1, CDCA+NAC: −6±1, LCA+NAC: 9±2, CDCA+LCA+NAC: −5±0.4, n≥3). In contrast, NAC altered the leak function over time causing ~65% reduction in CDCA‐induced F10D flux (18 H, μg of F10D: Control: 8±1; CDCA: 120±2; NAC: 7±0.1; CDCA+NAC: 41±4; p<0.05, n=3). LCA decreased CDCA's effect by 60%, which was further attenuated by NAC (18 H (μg): LCA: 13±2; NAC+LCA: 6±0.2; CDCA+LCA: 50±3; NAC+CDCA+LCA: 17±1; p<0.05, n≥3). We demonstrate that ROS plays a role in CDCA‐induced TJ dysfunction by altering leak, but not pore function in T84 cells. Equally important, LCA may be acting as an antioxidant as it reverses CDCA action on H2O2 release and the leak function which restores barrier integrity. Understanding the role of ROS in BA action can lead to novel therapeutic strategies for inflammation‐associated diarrheas.Support or Funding InformationThis was supported by APS‐UGSRF to HS, NSF‐MRI: DBI‐1427937 and Institutional Funds: BU to JS, UIC to MR.