Impaired Epithelial Barrier Function Research Articles

Rebamipide ameliorates radiation-induced intestinal injury in a mouse model

Radiation-induced enteritis is a major side effect in cancer patients undergoing abdominopelvic radiotherapy. Radiation exposure produces an uncontrolled inflammatory cascade and epithelial cell loss leading to impaired epithelial barrier function. The goal of this study was to determine the effect of rebamipide on regeneration of the intestinal epithelia after radiation injury. The abdomens of C57BL/6 mice were exposed to 13Gy of irradiation (IR) and then the mice were treated with rebamipide. Upon IR, intestinal epithelia were destroyed structurally at the microscopic level and bacterial translocation was increased. The intestinal damage reached a maximum level on day 6 post-IR and intestinal regeneration occurred thereafter. We found that rebamipide significantly ameliorated radiation-induced intestinal injury. In mice treated with rebamipide after IR, intestinal barrier function recovered and expression of the tight junction components of the intestinal barrier were upregulated. Rebamipide administration reduced radiation-induced intestinal mucosal injury. The levels of proinflammatory cytokines and matrix metallopeptidase 9 (MMP9) were significantly reduced upon rebamipide administration. Intestinal cell proliferation and β-catenin expression also increased upon rebamipide administration. These data demonstrate that rebamipide reverses impairment of the intestinal barrier by increasing intestinal cell proliferation and attenuating the inflammatory response by inhibiting MMP9 and proinflammatory cytokine expression in a murine model of radiation-induced enteritis.

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi—a process termed ‘cell shedding’. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes.

Oak and chestnut wood extracts enhance the intestinal gut barrier function in a porcine in vitro cell model

The protective effect of oak wood and chestnut wood extracts on the gut barrier function of a porcine intestinal epithelial cell line (IPEC-J2) was investigated. Tannins, polyphenolic compounds, are likely to improve the impaired epithelial barrier function by inhibiting the disruptive effect of TNF-α [Ren et al., 2012]. Therefore, extracts from oak and chestnut wood were prepared and tested in an in vitro cell culture model, using non-transformed, differentiated IPEC-J2. Cells were seeded in non-coated Transwell® membrane inserts (1.12 cm2; 0.4 µm pore size) for 7 days, allowing the formation of tight junctions (TJs) and the build-up of a representative intestinal layer, which was observed by an increase of transepithelial electrical resistance (TEER). Cells were pre-treated with a previously selected non-toxic concentration of the respective extracts. On day 8, a stressor was additionally added to the pre-treated cells and to the control. TEER was measured after 24, 48, and 72h. Following the final TEER measurement, a neutral red cytotoxicity assay was performed.

Altered Prostasin (CAP1/Prss8) Expression Favors Inflammation and Tissue Remodeling in DSS-induced Colitis.

Inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease are diseases with impaired epithelial barrier function. We aimed to investigate whether mutated prostasin and thus, reduced colonic epithelial sodium channel activity predisposes to develop an experimentally dextran sodium sulfate (DSS)-induced colitis. Wildtype, heterozygous (fr/+), and homozygous (fr/fr) prostasin-mutant rats were treated 7 days with DSS followed by 7 days of recovery and analyzed with respect to histology, clinicopathological parameters, inflammatory marker mRNA transcript expression, and sodium transporter protein expression. In this study, a more detailed analysis on rat fr/fr colons revealed reduced numbers of crypt and goblet cells, and local angiodysplasia, as compared with heterozygous (fr/+) and wildtype littermates. Following 2% DSS treatment for 7 days followed by 7 days recovery, fr/fr animals lost body weight, and reached maximal diarrhea score and highest disease activity after only 3 days, and strongly increased cytokine levels. The histology score significantly increased in all groups, but fr/fr colons further displayed pronounced histological alterations with near absence of goblet cells, rearrangement of the lamina propria, and presence of neutrophils, eosinophils, and macrophages. Additionally, fr/fr colons showed ulcerations and edemas that were absent in fr/+ and wildtype littermates. Following recovery, fr/fr rats reached, although significantly delayed, near-normal diarrhea score and disease activity, but exhibited severe architectural remodeling, despite unchanged sodium transporter protein expression. In summary, our results demonstrate a protective role of colonic prostasin expression against experimental colitis, and thus represent a susceptibility gene in the development of inflammatory bowel disease.

Probiotic mixture VSL#3 reduces colonic inflammation and improves intestinal barrier function in Muc2 mucin-deficient mice.

It is unclear whether probiotics require an intact mucin barrier to first colonize and/or exert their protective functions. In this study we used mucin-deficient (Muc2-/-) mice to interrogate if the multispecies probiotic mixture VSL#3 could enhance epithelial barrier function. In the absence of a mucus bilayer, VSL#3 dampened proinflammatory and chemokine production, accelerated restitution, and markedly improved gut permeability mediated by the short-chain fatty acid acetate in the colon.

Protein Kinase D2 Protects against Acute Colitis Induced by Dextran Sulfate Sodium in Mice.

Inflammatory bowel disease is characterized by dysregulation of the mucosal immune system resulting from impaired intestinal epithelial barrier function. Protein kinase D2 has been implicated in the regulation of immune responses. The present study was to define PKD2 might affect murine colitis. Colitis was induced in wild-type mice (PKD2WT/WT) and PKD2 catalytic activity deficient mice (PKD2SSAA/SSAA) with dextran sulfate sodium. PKD2SSAA-knockin mice displayed catalytic activity deficiency and increased susceptibility to DSS-induced colitis with enhanced weight loss, colonic inflammation compared with PKD2WT/WT mice. Furthermore, crucial inflammatory cytokines mRNA levels in PKD2SSAA-knockin mice were higher than controls accompanied with down-regulation of ZO-1, MUC2 and intestinal barrier dysfunction. However, there were no differences in the proliferation or apoptosis of intestinal epithelial cells in PKD2SSAA-knockin mice compared with wild-type controls. In addition, PKD2 expression was repressed in patients with IBD compared with healthy controls. These studies suggested that activation of PKD2 in the colonic epithelium microenvironment may contribute to protect against DSS-induced colitis through regulation of intestinal mucosal immunity and barrier function.

Correction to 'The zinc sensing receptor, ZnR/GPR39, triggers metabotropic calcium signalling in colonocytes and regulates occludin recovery in experimental colitis'.

Impaired epithelial barrier function is a hallmark of inflammatory bowel diseases, such as colitis, contributing to diarrhoea and perpetuating inflammation. We show that the zinc sensing receptor, ZnR/GPR39, triggers intracellular Ca(2+) signalling in colonocytes thereby inducing occludin expression. Moreover, ZnR/GPR39 is essential for epithelial barrier recovery in the dextran sodium sulfate (DSS) ulcerative colitis model. Loss of ZnR/GPR39 results in increased susceptibility to DSS-induced inflammation, owing to low expression of the tight junction protein occludin and impaired epithelial barrier. Recovery of wild-type (WT) mice from the DSS insult was faster than that of ZnR/GPR39 knockout (KO) mice. Enhanced recovery of the epithelial layer and increased crypt regeneration were observed in WT mice compared with ZnR/GPR39 KO, suggesting that ZnR/GPR39 is promoting epithelial barrier integrity following DSS insult. Indeed, cell proliferation and apical expression of occludin, following the DSS-induced epithelial erosion, were increased in WT tissue but not in ZnR/GPR39 KO tissue. Importantly, survival following DSS treatment was higher in WT mice compared with ZnR/GPR39 KO mice. Our results support a direct role for ZnR/GPR39 in promoting epithelial renewal and barrier function following DSS treatment, thereby affecting the severity of the disease. We suggest ZnR/GPR39 as a novel therapeutic target that can improve epithelial barrier function in colitis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Reversal of cigarette smoke extract-induced sinonasal epithelial cell barrier dysfunction through Nrf2 Activation.

Environmental factors such as inhaled pollutants like cigarette smoke may play a significant role in diseases of the upper airway including chronic rhinosinusitis (CRS). Recent studies have shown that cigarette smoke causes impaired airway epithelial cell barrier function likely through environmental oxidative stress related pathways. The purpose of this study is to explore whether enhancing nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2], the body's master antioxidant system, can ameliorate cigarette smoke-induced sinonasal epithelial cell (SNEC) barrier dysfunction. Human SNECs (HSNECs) were grown from control patients at the air-liquid interface (ALI). HSNECs were stimulated with cigarette smoke extract (CSE) with and without pharmacologic activation of Nrf2. HSNECs were then stained for the epithelial cell junctional proteins zonula occludens 1 (ZO-1) and junctional adhesion molecule A (JAM-A) using confocal microscopy. In addition, transepithelial electrical resistance (TER) was measured in cultures before and after stimulation with CSE. CSE stimulation caused a global disruption of the epithelial junctional proteins ZO-1 and JAM-A along with an associated decrease in TER levels. Enhancing Nrf2 levels prior to stimulation with CSE was associated with increased localization of ZO-1 and JAM-A levels at the cell surface and statistically significant increases in TER levels. This is the first study to demonstrate that cigarette smoke induced SNEC barrier dysfunction is reversible by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for cigarette smoke-associated sinonasal inflammation.

The zinc sensing receptor, ZnR/GPR39, triggers metabotropic calcium signalling in colonocytes and regulates occludin recovery in experimental colitis.

Impaired epithelial barrier function is a hallmark of inflammatory bowel diseases, such as colitis, contributing to diarrhoea and perpetuating inflammation. We show that the zinc sensing receptor, ZnR/GPR39, triggers intracellular Ca(2+) signalling in colonocytes thereby inducing occludin expression. Moreover, ZnR/GPR39 is essential for epithelial barrier recovery in the dextran sodium sulfate (DSS) ulcerative colitis model. Loss of ZnR/GPR39 results in increased susceptibility to DSS-induced inflammation, owing to low expression of the tight junction protein occludin and impaired epithelial barrier. Recovery of wild-type (WT) mice from the DSS insult was faster than that of ZnR/GPR39 knockout (KO) mice. Enhanced recovery of the epithelial layer and increased crypt regeneration were observed in WT mice compared with ZnR/GPR39 KO, suggesting that ZnR/GPR39 is promoting epithelial barrier integrity following DSS insult. Indeed, cell proliferation and apical expression of occludin, following the DSS-induced epithelial erosion, were increased in WT tissue but not in ZnR/GPR39 KO tissue. Importantly, survival following DSS treatment was higher in WT mice compared with ZnR/GPR39 KO mice. Our results support a direct role for ZnR/GPR39 in promoting epithelial renewal and barrier function following DSS treatment, thereby affecting the severity of the disease. We suggest ZnR/GPR39 as a novel therapeutic target that can improve epithelial barrier function in colitis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Impaired barrier function in patients with house dust mite–induced allergic rhinitis is accompanied by decreased occludin and zonula occludens-1 expression

Tight junction (TJ) defects have recently been associated with asthma and chronic rhinosinusitis. The expression, function, and regulation of nasal epithelial TJs remain unknown in patients with allergic rhinitis (AR). We investigated the expression, function, and regulation of TJs in the nasal epithelium of patients with house dust mite (HDM)-induced AR and in an HDM-induced murine model of allergic airway disease. Air-liquid interface cultures of primary nasal epithelial cells of control subjects and patients with HDM-induced AR were used for measuring transepithelial resistance and passage to fluorescein isothiocyanate-dextran 4 kDa (FD4). Ex vivo transtissue resistance and FD4 permeability of nasal mucosal explants were measured. TJ expression was evaluated by using real-time quantitative PCR and immunofluorescence. In addition, the effects of IL-4, IFN-γ, and fluticasone propionate (FP) on nasal epithelial cells were investigated in vitro. An HDM murine model was used to study the effects of allergic inflammation and FP treatment on transmucosal passage of FD4 in vivo. A decreased resistance in vitro and ex vivo was found in patients with HDM-induced AR, with increased FD4 permeability and reduced occludin and zonula occludens-1 expression. AR symptoms correlated inversely with resistance in patients with HDM-induced AR. In vitro IL-4 decreased transepithelial resistance and increased FD4 permeability, whereas IFN-γ had no effect. FP prevented IL-4-induced barrier dysfunction in vitro. In an HDM murine model FP prevented the allergen-induced increased mucosal permeability. We found impaired nasal epithelial barrier function in patients with HDM-induced AR, with lower occludin and zonula occludens-1 expression. IL-4 disrupted epithelial integrity in vitro, and FP restored barrier function. Better understanding of nasal barrier regulation might lead to a better understanding and treatment of AR.

Nlrp3 inflammasome activation by microbial products leads to Il-18 synthesis and impaired epithelial barrier function in cholangiocytes

Nlrp3 inflammasome activation by microbial products leads to Il-18 synthesis and impaired epithelial barrier function in cholangiocytes

Protocadherin-1 is a glucocorticoid-responsive critical regulator of airway epithelial barrier function

BackgroundImpaired epithelial barrier function renders the airway vulnerable to environmental triggers associated with the pathogenesis of bronchial asthma. We investigated the influence of protocadherin-1 (PCDH1), a susceptibility gene for bronchial hyperresponsiveness, on airway epithelial barrier function.MethodsWe applied transepithelial electric resistance and dextran permeability testing to evaluate the barrier function of cultured airway epithelial cells. We studied PCDH1 function by siRNA-mediated knockdown and analyzed nasal or bronchial tissues from 16 patients with chronic rhinosinusitis (CRS) and nine patients with bronchial asthma for PCDH1 expression.ResultsPCDH1 was upregulated with the development of epithelial barrier function in cultured airway epithelial cells. Immunocytochemical analysis revealed that PCDH localized to cell-cell contact sites and colocalized with E-cadherin at the apical site of airway epithelial cells. PCDH1 gene knockdown disrupted both tight and adhesion junctions. Immunohistochemical analysis revealed strong PCDH1 expression in nasal and bronchial epithelial cells; however, expression decreased in inflamed tissues sampled from patients with CRS or bronchial asthma. Dexamethasone (Dex) increased the barrier function of airway epithelial cells and increased PCDH1 expression. PCDH1 gene knockdown eradicated the effect of Dex on barrier function.ConclusionThese results suggest that PCDH1 is important for airway function as a physical barrier, and its dysfunction is involved in the pathogenesis of allergic airway inflammation. We also suggest that glucocorticoids promotes epithelial barrier integrity by inducing PCDH1.

Altered intestinal microbial flora and impaired epithelial barrier structure and function in CKD: the nature, mechanisms, consequences and potential treatment.

Chronic kidney disease (CKD) results in systemic inflammation and oxidative stress which play a central role in CKD progression and its adverse consequences. Although many of the causes and consequences of oxidative stress and inflammation in CKD have been extensively explored, little attention had been paid to the intestine and its microbial flora as a potential source of these problems. Our recent studies have revealed significant disruption of the colonic, ileal, jejunal and gastric epithelial tight junction in different models of CKD in rats. Moreover, the disruption of the epithelial barrier structure and function found in uremic animals was replicated in cultured human colonocytes exposed to uremic human plasma in vitro We have further found significant changes in the composition and function of colonic bacterial flora in humans and animals with advanced CKD. Together, uremia-induced impairment of the intestinal epithelial barrier structure and function and changes in composition of the gut microbiome contribute to the systemic inflammation and uremic toxicity by accommodating the translocation of endotoxin, microbial fragments and other noxious luminal products in the circulation. In addition, colonic bacteria are the main source of several well-known pro-inflammatory uremic toxins such as indoxyl sulfate, p-cresol sulfate, trimethylamine-N-oxide and many as-yet unidentified retained compounds in end-stage renal disease patients. This review is intended to provide an overview of the effects of CKD on the gut microbiome and intestinal epithelial barrier structure and their role in the pathogenesis of systemic inflammation and uremic toxicity. In addition, potential interventions aimed at mitigating these abnormalities are briefly discussed.

Mo1824 miR-511-3p, Embedded in the Macrophage Mannose Receptor Gene, Contributes to Experimental Colitis

Approximately 40% of inflammatory bowel diseases (IBD) are associated with thromboembolic events causing significant rate of morbidity and mortality. These extra intestinal events of IBD are initiated by the qualitative and quantitative abnormalities in both coagulation and fibrinolytic factors producing states of hypercoagulability. Nevertheless, the interrelationship between IBD pathogenesis and hypercoagulability condition is not well understood (Figure 1). Here we examine the role of a coagulable state triggered by impaired fibrinolysis in the development of chronic colonic inflammation. Plasminogen (Plg) is the primary fibrinolytic enzyme, becomes activated to serine protease plasmin to dissolve the fibrin clot and to counterbalance hypercoagulability. The rapidity of IBD is caused by combining the effects of genetic and environmental factors. Our data show that when fed with a high fat diet (HFD, 45% milk fat), mouse knockout of Plg (Plg-/mice) developed continuous lesions in the colon with multiple inflammatory polyps (as of 8 weeks, n=15) bearing ulcers. This pathophysiology was not observed in HFD fed wild type (Plg+/+) mice and standard chow diet fed Plg+/+ and Plg-/mice. The increased inflammatory cell infiltration in the colon of HFD fed Plg-/mice was corroborated with increased levels of pro-inflammatory cytokines (IL-6, IL-17 & TNF alpha). In addition, HFD-fed Plg-/mice expressed reduced levels of colonic epithelial tight junctions (Claudin1, Occludin and ZO1), a finding compatible with an impaired epithelial barrier function, compared to Plg+/+ mice. Moreover, HFD fed Plg-/mice showed increased levels of plasma endotoxin, an indicator of impaired intestinal barrier function, compared with HFD-fed Plg+/+ mice. All these pathologies of HFD fed Plg-/mice were associated with excessive fibrin deposition in both vascular and extravascular space in the colon compared with HFD fed Plg+/+ mice providing a link between fibrin deposition and inflammation. Altogether, our present data imply that fibrinolytic pathway plays a crucial role in colon homeostasis and defects in this pathway can cause impaired colon barrier function and sustained colonic inflammation. In depth exploration of this pathway will provide a clue towards the potential role of Plg in colon homeostasis. This study will also challenge current paradigms on the relationship between coagulation abnormalities and IBD, which presume that these abnormalities are a consequence rather than a cause of the disease.

Mo1825 The Interaction of Enteric Bacterial Effectors With Host Engulfment Pathway in Phagocytes Regulates the Pathogenesis of Infection

Approximately 40% of inflammatory bowel diseases (IBD) are associated with thromboembolic events causing significant rate of morbidity and mortality. These extra intestinal events of IBD are initiated by the qualitative and quantitative abnormalities in both coagulation and fibrinolytic factors producing states of hypercoagulability. Nevertheless, the interrelationship between IBD pathogenesis and hypercoagulability condition is not well understood (Figure 1). Here we examine the role of a coagulable state triggered by impaired fibrinolysis in the development of chronic colonic inflammation. Plasminogen (Plg) is the primary fibrinolytic enzyme, becomes activated to serine protease plasmin to dissolve the fibrin clot and to counterbalance hypercoagulability. The rapidity of IBD is caused by combining the effects of genetic and environmental factors. Our data show that when fed with a high fat diet (HFD, 45% milk fat), mouse knockout of Plg (Plg-/mice) developed continuous lesions in the colon with multiple inflammatory polyps (as of 8 weeks, n=15) bearing ulcers. This pathophysiology was not observed in HFD fed wild type (Plg+/+) mice and standard chow diet fed Plg+/+ and Plg-/mice. The increased inflammatory cell infiltration in the colon of HFD fed Plg-/mice was corroborated with increased levels of pro-inflammatory cytokines (IL-6, IL-17 & TNF alpha). In addition, HFD-fed Plg-/mice expressed reduced levels of colonic epithelial tight junctions (Claudin1, Occludin and ZO1), a finding compatible with an impaired epithelial barrier function, compared to Plg+/+ mice. Moreover, HFD fed Plg-/mice showed increased levels of plasma endotoxin, an indicator of impaired intestinal barrier function, compared with HFD-fed Plg+/+ mice. All these pathologies of HFD fed Plg-/mice were associated with excessive fibrin deposition in both vascular and extravascular space in the colon compared with HFD fed Plg+/+ mice providing a link between fibrin deposition and inflammation. Altogether, our present data imply that fibrinolytic pathway plays a crucial role in colon homeostasis and defects in this pathway can cause impaired colon barrier function and sustained colonic inflammation. In depth exploration of this pathway will provide a clue towards the potential role of Plg in colon homeostasis. This study will also challenge current paradigms on the relationship between coagulation abnormalities and IBD, which presume that these abnormalities are a consequence rather than a cause of the disease.

Tight junction disruption by cadmium in an in vitro human airway tissue model.

BackgroundThe cadmium (Cd) present in air pollutants and cigarette smoke has the potential of causing multiple adverse health outcomes involving damage to pulmonary and cardiovascular tissue. Injury to pulmonary epithelium may include alterations in tight junction (TJ) integrity, resulting in impaired epithelial barrier function and enhanced penetration of chemicals and biomolecules. Herein, we investigated mechanisms involved in the disruption of TJ integrity by Cd exposure using an in vitro human air-liquid-interface (ALI) airway tissue model derived from normal primary human bronchial epithelial cells.MethodsALI cultures were exposed to noncytotoxic doses of CdCl2 basolaterally and TJ integrity was measured by Trans-Epithelial Electrical Resistance (TEER) and immunofluorescence staining with TJ markers. PCR array analysis was used to identify genes involved with TJ collapse. To explore the involvement of kinase signaling pathways, cultures were treated with CdCl2 in the presence of kinase inhibitors specific for cellular Src or Protein Kinase C (PKC).ResultsNoncytotoxic doses of CdCl2 resulted in the collapse of barrier function, as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining. CdCl2 exposure altered the expression of several groups of genes encoding proteins involved in TJ homeostasis. In particular, down-regulation of select junction-interacting proteins suggested that a possible mechanism for Cd toxicity involves disruption of the peripheral junctional complexes implicated in connecting membrane-bound TJ components to the actin cytoskeleton. Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs, possibly by preventing occludin tyrosine hyperphosphorylation, rather than reversing the down-regulation of the junction-interacting proteins.ConclusionsOur findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex.

Glycoprotein A33 deficiency: a new mouse model of impaired intestinal epithelial barrier function and inflammatory disease.

ABSTRACTThe cells of the intestinal epithelium provide a selectively permeable barrier between the external environment and internal tissues. The integrity of this barrier is maintained by tight junctions, specialised cell-cell contacts that permit the absorption of water and nutrients while excluding microbes, toxins and dietary antigens. Impairment of intestinal barrier function contributes to multiple gastrointestinal disorders, including food hypersensitivity, inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Glycoprotein A33 (GPA33) is an intestinal epithelium-specific cell surface marker and member of the CTX group of transmembrane proteins. Roles in cell-cell adhesion have been demonstrated for multiple CTX family members, suggesting a similar function for GPA33 within the gastrointestinal tract. To test a potential requirement for GPA33 in intestinal barrier function, we generated Gpa33−/− mice and subjected them to experimental regimens designed to produce food hypersensitivity, colitis and CAC. Gpa33−/− mice exhibited impaired intestinal barrier function. This was shown by elevated steady-state immunosurveillance in the colonic mucosa and leakiness to oral TRITC-labelled dextran after short-term exposure to dextran sodium sulphate (DSS) to injure the intestinal epithelium. Gpa33−/− mice also exhibited rapid onset and reduced resolution of DSS-induced colitis, and a striking increase in the number of colitis-associated tumours produced by treatment with the colon-specific mutagen azoxymethane (AOM) followed by two cycles of DSS. In contrast, Gpa33−/− mice treated with AOM alone showed no increase in sporadic tumour formation, indicating that their increased tumour susceptibility is dependent on inflammatory stimuli. Finally, Gpa33−/− mice displayed hypersensitivity to food allergens, a common co-morbidity in humans with IBD. We propose that Gpa33−/− mice provide a valuable model to study the mechanisms linking intestinal permeability and multiple inflammatory pathologies. Moreover, this model could facilitate preclinical studies aimed at identifying drugs that restore barrier function.

Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice.

Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.

Mast cell deficiency exacerbates inflammatory bowel symptoms in interleukin-10-deficient mice.

To test the role of mast cells in gut inflammation and colitis using interleukin (IL)-10-deficient mice as an experimental model. Mast cell-deficient (Kit (W-sh/W-sh) ) mice were crossbred with IL-10-deficient mice to obtain double knockout (DKO) mice. The growth, mucosal damage and colitis status of DKO mice were compared with their IL-10-deficient littermates. DKO mice exhibited exacerbated colitis compared with their IL-10-deficient littermates, as shown by increased pathological score, higher myeloperoxidase content, enhanced Th1 type pro-inflammatory cytokines and inflammatory signaling, elevated oxidative stress, as well as pronounced goblet cell loss. In addition, deficiency in mast cells resulted in enhanced mucosal damage, increased gut permeability, and impaired epithelial tight junctions. Mast cell deficiency was also linked to systemic inflammation, as demonstrated by higher serum levels of tumor necrosis factor α and interferon γ in DKO mice than that in IL-10-deficient mice. Mast cell deficiency in IL-10-deficient mice resulted in systematic and gut inflammation, impaired gut barrier function, and severer Th1-mediated colitis when compared to mice with only IL-10-deficiency. Inflammation and impaired gut epithelial barrier function likely form a vicious cycle to worsen colitis in the DKO mice.

Mucosal antimicrobial protection toward enteroaggregative Escherichia coli infection is modulated by dietary tryptophan through an IL17-dependent mechanism (MUC4P.846)

Abstract Enteroaggregative E. coli (EAEC) is a predominant extracellular pathogen that causes inflammatory diarrhea in individuals worldwide, especially in persons suffering from malnutrition. We previously reported that protein-energy malnutrition abrogates protective Th17-dependent antimicrobial immunity in a murine model of EAEC infection. However, mechanisms whereby malnutrition enhances host-susceptibility to EAEC colonization remain unreported. This study presents evidence that tryptophan is crucial for antibacterial protection against EAEC infection. Mice fed tryptophan-free diet had reduced antimicrobial peptide production coinciding with significantly high pathogen levels. Likewise, bacterial burden occurred in a dose dependent manner when mice were supplemented with glycly-L-tryptophan dipeptide. Low serum tryptophan directly correlated with hampered gene expression levels of cytokines regulating effector IL-17A responses (e.g. IL-23 and IL-6). Mechanistically, orally administered tryptophan enhanced IL-17A and IL-17C production at the colonic and ileal mucosa facilitating antimicrobial defense and protection against EAEC colonization. IL17a-/- and IL17ra-/- mice receiving nourished diets or tryptophan supplements were unable to recover from disease due to impaired intestinal epithelial barrier function. Thus, our data suggests that tryptophan modulates IL17-dependent antimicrobial responses and is required for preventing EAEC colonization and pathogenic disease.