Jon Meddings

Epithelial myosin light chain kinase–dependent barrier dysfunction mediates T cell activation–induced diarrhea in vivo

Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell-mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

Targeted Epithelial Tight Junction Dysfunction Causes Immune Activation and Contributes to Development of Experimental Colitis

BACKGROUND & AIMS Inflammatory bowel disease (IBD) is a multifactorial disease thought to be caused by alterations in epithelial function, innate and adaptive immunity, and luminal microbiota. The specific role of epithelial barrier function remains undefined, although increased activity of intestinal epithelial myosin light chain kinase (MLCK), which is the primary mechanism of tumor necrosis factor-induced barrier dysfunction, occurs in human IBD. Our aim was to determine whether, in an intact epithelium, primary dysregulation of the intestinal epithelial barrier by pathophysiologically relevant mechanisms can contribute to development of colitis. METHODS We developed transgenic (Tg) mice that express constitutively active MLCK (CA-MLCK) specifically within intestinal epithelia. Their physiology, immune status, and susceptibility to disease were assessed and compared with non-Tg littermate controls. RESULTS CA-MLCK Tg mice demonstrated significant barrier loss but grew and gained weight normally and did not develop spontaneous disease. CA-MLCK Tg mice did, however, develop mucosal immune activation demonstrated by increased numbers of lamina propria CD4(+)lymphocytes, redistribution of CD11c+cells, increased production of interferon-gamma and tumor necrosis factor, as well as increased expression of epithelial major histocompatibility complex class I. When challenged with CD4+CD45+Rb(hi) lymphocytes, Tg mice developed an accelerated and more severe form of colitis and had shorter survival times than non-Tg littermates. CONCLUSIONS Primary pathophysiologically relevant intestinal epithelial barrier dysfunction is insufficient to cause experimental intestinal disease but can broadly activate mucosal immune responses and accelerate the onset and severity of immune-mediated colitis.

Is small intestinal permeability really increased in relatives of patients with Crohn's disease?

BACKGROUND Patients with Crohns disease have increased intestinal permeability, which may precede the development of clinical disease and be involved in disease pathogenesis. Subsequent studies have suggested that, as a group, first-degree relatives of patients with Crohns disease do not have significantly increased small intestinal permeability rates. The present study proposes that conventional data analysis, used in these studies, may be inappropriate and has overlooked an important observation. METHODS Lactulose and mannitol permeabilities were defined in healthy controls and in patients with Crohns disease and their first-degree relatives. RESULTS Intestinal permeability in relatives was similar to that in the control group, but a subpopulation had abnormally high permeability rates in the absence of clinical evidence for disease. Raw data from another investigator confirmed this finding in an additional study; consequently, it is concluded that the original hypothesis is still viable. A small proportion of individuals, at high risk of developing Crohns disease, have increased intestinal permeability. CONCLUSIONS Increased intestinal permeability may precede clinical manifestations of Crohns disease.

The safety, tolerance, pharmacokinetic and pharmacodynamic effects of single doses of AT-1001 in coeliac disease subjects: a proof of concept study

Lifelong adherence to a strict gluten‐free diet is the cornerstone of coeliac disease treatment. Elucidation of disease pathogenesis has created opportunities for novel therapeutic approaches to coeliac disease. AT‐1001 is an inhibitor of paracellular permeability whose structure is derived from a protein secreted by Vibrio cholerae.

Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse

Background: Defects in the small intestinal epithelial barrier have been associated with inflammatory bowel disease but their role in the causation of disease is still a matter of debate. In some models of disease increased permeability appears to be a very early event. The interleukin 10 (IL10) gene-deficient mouse spontaneously develops colitis after 12 weeks of age. These mice have been shown to have increased small intestinal permeability that appears early in life. Furthermore, the development of colitis is dependent upon luminal agents, as animals do not develop disease if raised under germ-free conditions. Aims: To determine if the elevated small bowel permeability can be prevented, and if by doing so colonic disease is prevented or attenuated. Methods: IL10 gene-deficient (IL10−/−) mice) were treated with AT-1001 (a zonulin peptide inhibitor), a small peptide previously demonstrated to reduce small intestinal permeability. Small intestinal permeability was measured, in vivo, weekly from 4 to 17 weeks of age. Colonic disease was assessed at 8 weeks in Ussing chambers, and at 17 weeks of age inflammatory cytokines and myeloperoxidase were measured in the colon. Colonic permeability and histology were also endpoints. Results: Treated animals showed a marked reduction in small intestinal permeability. Average area under the lactulose/mannitol time curve: 5.36 (SE 0.08) in controls vs 3.97 (SE 0.07) in the high-dose AT-1001 group, p<0.05. At 8 weeks of age there was a significant reduction of colonic mucosal permeability and increased electrical resistance. By 17 weeks of age, secretion of tumour necrosis factor α (TNFα) from a colonic explant was significantly lower in the treated group (25.33 (SE 4.30) pg/mg vs 106.93 (SE 17.51) pg/ml in controls, p<0.01). All other markers also demonstrated a clear reduction of colitis in the treated animals. Additional experiments were performed which demonstrated that AT-1001 was functionally active only in the small intestine. Conclusions: This work suggests that increased intestinal permeability may be an important aetiological event in the development of colitis in IL10−/− mice.

Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2

Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR2) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR2 or the use of PAR2−/− mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its α2- and β-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.

Sucrose: A novel permeability marker for gastroduodenal disease

BACKGROUND Nonsteroidal anti-inflammatory agents (NSAIDs) commonly cause asymptomatic gastroduodenal damage that may be clinically severe. At present the only method to determine the presence or absence of such damage is by invasive techniques such as endoscopy. Because distal small intestinal damage can be noninvasively detected with oral permeability tests, the hypothesis that gastroduodenal damage could be detected using similar methods was examined in the present study. RESULTS Animal data are presented suggesting that sucrose represents an ideal probe molecule to detect increased gastroduodenal permeability in a site-specific manner. With gastroduodenal damage, caused by either ethanol or NSAIDs, sucrose permeability is increased. Furthermore, because sucrose is rapidly degraded within the small intestine, this probe does not detect small intestinal damage, making it specific for the upper gastrointestinal tract. Finally, a pilot study in humans is presented to show the use of this technique in evaluating human gastric permeability. CONCLUSIONS Sucrose represents a novel permeability probe with specificity for damage of the upper gastrointestinal tract. In animals and humans it appears useful to noninvasively detect gastroduodenal injury caused by several agents.

Discrimination of site-specific alterations in gastrointestinal permeability in the rat ☆ ☆☆

BACKGROUND & AIMS Detection of gastrointestinal damage can be accomplished by permeability testing. However, current methodology presents several problems, including lack of site specificity and an inability to easily measure colonic damage. We hypothesized that probes of similar size could be used to simultaneously assess permeability along the length of the gastrointestinal tract. Differentiation of damage sites could be made, provided that probes were selectively destroyed at different levels. Class 1 probes should be destroyed after leaving the stomach, class 2 after leaving the small intestine, and class 3 not at all. In this manner, class 1 probes would report damage to the proximal gut, class 2 to the small intestine, and class 3 to the gut as a whole. METHODS We defined saccharide probes with these characteristics and observed their permeability patterns in defined models of localized gastrointestinal damage. RESULTS With gastric damage, permeability of only class 1 probes increased, whereas with colonic damage only permeation rates of class 3 probes was increased. Furthermore, the permeation rates of both class 2 and 3 probes increased in the presence of small intestinal disease. CONCLUSIONS These techniques allow a single screening test that is sensitive to damage at any level of the gastrointestinal tract and may be used in either animals or humans.

CD45RO expression on circulating CD19+ B cells in Crohn's disease correlates with intestinal permeability☆

BACKGROUND/AIMS Increased intestinal permeability is observed in Crohns disease and a subset of first-degree relatives. An alteration in isoform expression of the common leukocyte antigen (CD45) is also found in a significant fraction of patients. Because this alteration may be a measure of antigen exposure, the hypothesis of the study was that this alteration would be observed in both patients and relatives of patients with Crohns disease and that this would correlate with increased intestinal permeability. METHODS Lactulose and mannitol permeability were defined in healthy controls, patients with Crohns disease, and their first-degree relatives. Simultaneously, peripheral blood was assayed using flow cytometry for CD45RO expression on CD19+ B cells. RESULTS A subset of relatives had significantly increased permeability, as did the majority of patients with Crohns disease. A small fraction of peripheral B cells from controls expressed the CD45 isoform (< 6%). This fraction was significantly increased for patients with Crohns disease and their relatives. Relatives with no clinical evidence of Crohns disease were only found to have increased CD45RO expression if they had increased permeability. CONCLUSIONS Individuals at risk for developing Crohns disease include a subset with increased intestinal permeability. These people have an associated phenotypic alteration of circulating B cells that is not observed in controls or relatives with normal intestinal permeability.

A Key Claudin Extracellular Loop Domain is Critical for Epithelial Barrier Integrity

Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-1(53-80)) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-1(53-80) associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-1(53-80), because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-1(53-80) to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-1(53-80), capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier.