[Show abstract][Hide abstract] ABSTRACT: Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation.
[Show abstract][Hide abstract] ABSTRACT: Background & aims:
The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy.
We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy.
IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors.
A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.
[Show abstract][Hide abstract] ABSTRACT: Background & aims:
Substance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions.
Mesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients.
Tacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients.
Preadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.
[Show abstract][Hide abstract] ABSTRACT: Tight junctions create a paracellular barrier that is essential for survival of complex organisms. In many cases tight junctions define separate, generally sterile, tissue compartments. In the skin and gut, tight junctions must also seal the paracellular space to prevent microbiota from accessing the internal milieu. This is a relatively simple task in the integument, where an absolute barrier is effective. However, intestinal epithelial tight junctions are charged with the far more complex task of supporting paracellular transport of water, ions, and nutrients while providing a barrier to microbial translocation. The delicate nature of this balance, which is disrupted in disease, makes the intestine a unique organ in which to explore the complexities of tight junction permeability and barrier regulation. Here we review recent progress in understanding the molecular determinants of barrier function and events responsible for regulation, and dysregulation, of tight junction permeability.
Seminars in Cell and Developmental Biology 09/2014; 36. DOI:10.1016/j.semcdb.2014.09.022 · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Intestinal barrier dysfunction is an important contributor to alcoholic liver disease (ALD). Translocated microbial products trigger an inflammatory response in the liver and contribute to steatohepatitis. Our aim was to investigate mechanisms of barrier disruption after chronic alcohol feeding. A Lieber-DeCarli model was used to induce intestinal dysbiosis, increased intestinal permeability, and liver disease in mice. Alcohol feeding for 8 weeks induced intestinal inflammation in the jejunum, which is characterized by an increased number of tumor necrosis factor alpha (TNF-α)-producing monocytes and macrophages. These findings were confirmed in duodenal biopsies from patients with chronic alcohol abuse. Intestinal decontamination with nonabsorbable antibiotics restored eubiosis, decreased intestinal inflammation and permeability, and reduced ALD in mice. TNF-receptor I (TNFRI) mutant mice were protected from intestinal barrier dysfunction and ALD. To investigate whether TNFRI on intestinal epithelial cells mediates intestinal barrier dysfunction and ALD, we used TNFRI mutant mice carrying a conditional gain-of-function allele for this receptor. Reactivation of TNFRI on intestinal epithelial cells resulted in increased intestinal permeability and liver disease that is similar to wild-type mice after alcohol feeding, suggesting that enteric TNFRI promotes intestinal barrier dysfunction. Myosin light-chain kinase (MLCK) is a downstream target of TNF-α and was phosphorylated in intestinal epithelial cells after alcohol administration. Using MLCK-deficient mice, we further demonstrate a partial contribution of MLCK to intestinal barrier dysfunction and liver disease after chronic alcohol feeding.
Dysbiosis-induced intestinal inflammation and TNFRI signaling in intestinal epithelial cells mediate a disruption of the intestinal barrier. Therefore, intestinal TNFRI is a crucial mediator of ALD.
[Show abstract][Hide abstract] ABSTRACT: Approximately half of all adult burn patients are intoxicated at the time of their injury and have worsened clinical outcomes compared to those without prior alcohol exposure. This study tested the hypothesis that intoxication alters the gut-liver axis leading to increased pulmonary inflammation mediated by burn-induced IL-6 in the liver. To this end, C57BL/6 mice were given 1.2 g/kg ethanol 30 minutes prior to a 15% total body surface area burn. To restore gut barrier function, a specific myosin light chain kinase inhibitor [membrane-permeant inhibitor of kinase (PIK)] was administered 30 minutes after injury which we have demonstrated to reduce bacterial translocation from the gut. Limiting bacterial translocation with PIK attenuated hepatic damage as measured by a 47% reduction in serum alanine aminotransferase (p<0.05) as well as a 33% reduction in hepatic IL-6 mRNA expression (p<0.05) compared to intoxicated burned mice without PIK. This mitigation of hepatic damage was associated with a 49% decline in pulmonary neutrophil infiltration (p<0.05) and decreased alveolar wall thickening compared to matched controls. These results were reproduced by prophylactically reducing the bacterial load in the intestines with oral antibiotics before intoxication and burn. Overall these data suggest the gut-liver axis is deranged when intoxication precedes burn and that limiting bacterial translocation in this setting attenuates hepatic damage and pulmonary inflammation.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)-matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.
Science translational medicine 07/2014; 6(243):243ra87. DOI:10.1126/scitranslmed.3008941 · 15.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.
American Journal Of Pathology 06/2014; 184(8). DOI:10.1016/j.ajpath.2014.05.003 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND & AIMS: Expression of the tight junction protein claudin-2 (cldn2), which forms a pore that mediates paracellular Na+ and water flux, is increased in colitis. We have shown that cldn2-/- mice are partially protected from adoptive transfer (AT) colitis, as demonstrated by reductions in stool Na+ and water content as well as mucosal cytokines. In vitro studies have shown that dephosphorylation of S408 within the occludin C-terminus drives assembly of a protein complex that includes cldn2 and disrupts pore function. CK2 is the primary kinase that phosphorylates S408, and in vitro studies have shown that CK2 inhibition can restore epithelial barrier function following IL-13-induced cldn2 expression in epithelial monolayers. However, some data suggest that CK2 may contribute to epithelial homeostasis in DSS colitis. Our aim was to determine whether in vivo CK2 inhibition could be beneficial in colitis. METHODS: Colitis was induced by AT or DSS using established methods in wild type (WT), cldn2-/-, and Rag1-/- mice. The orally-bioavailable CK2 inhibitor CX4945 was delivered daily by gavage. RESULTS: CX4945 markedly improved distal colonic histopathology in DSS-treated mice. DSS treatment resulted in complete loss of surface epithelium, mucin depletion, and diffuse ulceration within the distal colon of mice treated with daily saline gavage, while CX4945-treated mice demonstrated intact surface epithelium, normal goblet cell maturation, and only minor damage. Consistent with this, colonic permeability to 4kD dextran was 1.9±0.2-fold less in CX4945 treated mice (P<0.05). CX4945 also delayed DSS-induced weight loss and disease progression. Effects of CX4945 were similar in WT and cldn2-/- mice, indicating that CK2 inhibition limits DSS colitis by a cldn2- independent mechanism and that CK2 activity is not essential for epithelial homeostasis. To assess the effect of CK2 inhibition in an IBD-relevant model of immune mediated colitis, Rag1-/- and cldn2 -/-Rag1-/- mice were treated with CX4945 beginning 10 days after CD4+CD45Rbhi AT. CX4945 reduced increases in stool Na+ and water content, colonic 4kD dextran permeability (2.1±0.5-fold less in CX4945-treated mice) , mucosal cytokine production, weight loss, histopathology in Rag1-/- mice (P<0.05), but had no benefit in cldn2 -/-Rag1-/- mice; parameters were similar in CX4945-treated Rag1-/- mice and either CX4945-treated or saline-treated cldn2 -/-Rag1-/- mice. CONCLUSIONS: In vivo cldn2- mediated pore function can be inhibited by CK2 inhibition and that this can be an effective therapeutic target in experimental IBD. The data also remove concerns that CK2 activity is essential for mucosal homeostasis in colitis and, conversely, indicate that CK2 inhibition can limit intestinal damage by both cldn2-dependent and cldn2-independent mechanisms. Clinical trials of CK2 inhibitors are indicated in IBD.