Epithelial IL-8 Research Articles

Suppression of type-2 mediated allergic diseases by dectin-1

Abstract Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental proteins. Arthropods, such as mites, cockroaches, and crustaceans, are a major source of allergens, yet the majority of us live with or consume these invertebrates with no ill effects. Recent discoveries have raised the possibility that altered innate immune recognition of allergens, through pattern recognition receptors (PRR), may underlie disease susceptibility. Here, we identify the PRR, dectin-1 (CLEC7A), as a key receptor in mediating protection against dust mite-driven type 2 immune responses through the inhibition of epithelial cell IL-33 production, and the downstream recruitment of IL-13-producing innate lymphoid 2 cells (ILC2). Surprisingly, we observed that this effect was not mediated by the prototypical dectin-1 ligand, β-glucan, within allergen extracts. We discovered a novel protein ligand conserved across the arthropod phyla, sensing of which confers protection against aberrant type 2 responses that drive respiratory allergy and food anaphylaxis in mice. While this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, dectin-1 is repressed in asthmatics and patients with chronic-rhinosinusitis. Importantly, we report a novel SNP in CLEC7A in the SAGE and GALAII GWAS studies, associated with lung function. Collectively, our findings reveal a previously unrecognized negative regulatory pathway in which ligation of dectin-1 results in reduction of type 2 immune responses, while disruption of this critical regulatory circuit is associated with susceptibility to a spectrum of allergic disorders.

MiR-200b inhibits TNF-α-induced IL-8 secretion and tight junction disruption of intestinal epithelial cells in vitro.

This was the first time that the inhibitory role of miR-200b on intestinal epithelial inflammation and paracellular permeability has been reported. Moreover, we further divided the intestinal epithelial cells (IECs) into two differentiated conditions and investigated the distinct impacts of miR-200b. Finally, we put forward and proved that myosin light chain kinase (MLCK) was a novel target of miR-200b.

Innate Immune Responses after Airway Epithelial Stimulation with Mycobacterium bovis Bacille-Calmette Guérin.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.

IL-33 promotes food anaphylaxis in epicutaneously sensitized mice by targeting mast cells

Cutaneous exposure to food allergens predisposes to food allergy, which is commonly associated with atopic dermatitis (AD). Levels of the epithelial cytokine IL-33 are increased in skin lesions and serum of patients with AD. Mast cells (MCs) play a critical role in food-induced anaphylaxis and express the IL-33 receptor ST2. The role of IL-33 in patients with MC-dependent food anaphylaxis is unknown. We sought to determine the role and mechanism of action of IL-33 in patients with food-induced anaphylaxis in a model of IgE-dependent food anaphylaxis elicited by oral challenge of epicutaneously sensitized mice. Wild-type, ST2-deficient, and MC-deficient KitW-sh/W-sh mice were epicutaneously sensitized with ovalbumin (OVA) and then challenged orally with OVA. Body temperature was measured by means of telemetry, Il33 mRNA by means of quantitative PCR, and IL-33, OVA-specific IgE, and mouse mast cell protease 1 by means of ELISA. Bone marrow-derived mast cell (BMMC) degranulation was assessed by using flow cytometry. Il33 mRNA expression was upregulated in tape-stripped mouse skin and scratched human skin. Tape stripping caused local and systemic IL-33 release in mice. ST2 deficiency, as well as ST2 blockade before oral challenge, significantly reduced the severity of oral anaphylaxis without affecting the systemic TH2 response to the allergen. Oral anaphylaxis was abrogated in KitW-sh/W-sh mice and restored by means of reconstitution with wild-type but not ST2-deficient BMMCs. IL-33 significantly enhanced IgE-mediated degranulation of BMMCs invitro. IL-33 is released after mechanical skin injury, enhances IgE-mediated MC degranulation, and promotes oral anaphylaxis after epicutaneous sensitization by targeting MCs. IL-33 neutralization might be useful in treating food-induced anaphylaxis in patients with AD.

Asthma inflammatory phenotypes show differential microRNA expression in sputum

Asthma is classified according to severity and inflammatory phenotype and is likely to be distinguished by specific microRNA (miRNA) expression profiles. We sought to associate miRNA expression in sputum supernatants with the inflammatory cell profile and disease severity in asthmatic patients. We investigated miRNA expression in sputum supernatants of 10 healthy subjects, 17 patients with mild-to-moderate asthma, and 9 patients with severe asthma using high-throughput, stem-loop, reverse transcriptase quantitative real-time PCR miRNA expression profiling (screening cohort, n=36). Differentially expressed miRNAs were validated in an independent cohort (n=60; 10 healthy subjects and 50 asthmatic patients). Cellular miRNA origin was examined by using in situ hybridization and reverse transcriptase quantitative real-time PCR. The functional role of miRNAs was assessed by using in silico analysis and invitro transfecting miRNA mimics in human bronchial epithelial cells. In 2 independent cohorts expression of miR-629-3p, miR-223-3p, and miR-142-3p was significantly upregulated in sputum of patients with severe asthma compared with that in healthy control subjects and was highest in patients with neutrophilic asthma. Expression of the 3 miRNAs was associated with sputum neutrophilia, and miR-223-3p and miR-142-3p expression was associated also with airway obstruction (FEV1/forced vital capacity). Expression of miR-629-3p was localized in the bronchial epithelium, whereas miR-223-3p and miR-142-3p were expressed in neutrophils, monocytes, and macrophages. Transfecting human bronchial epithelial cells with miR-629-3p mimic induced epithelial IL-8 mRNA and protein expression. IL-1β and IL-8 protein levels were significantly increased in sputum of patients with severe asthma and were positively associated with sputum neutrophilia. Expression of miR-223-3p, miR-142-3p, and miR-629-3p is increased in sputum of patients with severe asthma and is linked to neutrophilic airway inflammation, suggesting that these miRNAs contribute to this asthma inflammatory phenotype.

CD146 promotes metastasis and predicts poor prognosis of hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Recurrence and metastasis after curative resection remain critical obstacles in HCC treatment. CD146 predicted poor prognosis of a variety of cancers including melanoma, breast tumors, prostate cancer, and gastric cancer. However, the role of CD146 in HCC has not yet been systematically explored.MethodsTo investigate the role of CD146 in HCC, we evaluated its expression in HCC tissues and HCC cell lines using real-time PCR and western blotting (WB). Second, we established HCC cell lines that stably overexpressed and interfered CD146 and explored the function of CD146 in HCC in vitro and in vivo. Third, we conducted microarray analysis to investigate the potential mechanism by identifying differentially expressed genes. Last, follow ups were conducted to help uncover the connection of CD146 expression and the prognosis of HCC patients.ResultsWe found that CD146 was overexpressed in HCC tissues and that high CD146 expression predicted poor overall survival time and shorter recurrence period in HCC patients. In vitro and in vivo experiments indicated that CD146 promoted migration and invasion of HCC cell lines. Further study indicated that CD146 promoted epithelial mesenchymal transition (EMT), IL-8 upregulation, and STAT1 downregulation. CD146 was upregulated in HCC tissues and cell lines.ConclusionsCD146 promoted metastasis of HCC cells and predicted poor prognosis of HCC patients. CD146 induced EMT, and IL-8 upregulation and STAT1 downregulation may be the potential underlying mechanism. The exact mechanism still needs further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0313-3) contains supplementary material, which is available to authorized users.

IL-33 Promotes Food Anaphylaxis in Epicutaneously-Sensitized Mice By Targeting Mast Cells

Cutaneous exposure to food allergens predisposes to food allergy, which is commonly associated with atopic dermatitis (AD). The levels of the epithelial cytokine IL-33 are increased in skin lesions and serum of AD patients. Mast cells (MC) play a critical role in food anaphylaxis and express the IL-33 receptor ST2. The role of IL-33 in MC-dependent food anaphylaxis is unknown.We aim to determine the role and mechanism of action of IL-33 in food anaphylaxis in a model of IgE-dependent food anaphylaxis elicited by oral challenge of epicutaneously (EC)-sensitized mice. WT, ST2-deficient and MC-deficient KitW-sh/W-sh mice were EC sensitized with ovalbumin (OVA) then challenged orally with OVA. Body temperature was measured by telemetry, Il33 mRNA by qPCR, and IL-33, OVA-specific IgE and mouse mast cell protease 1 (mMCP-1) by ELISA. Bone marrow-derived MCs (BMMCs) degranulation was assessed by flow cytometry. Il33 mRNA expression was upregulated in tape-stripped mouse skin and scratched human skin, and tape stripping caused local and systemic IL-33 release in mice. ST2 deficiency, as well as ST2 blockade prior to oral challenge, significantly reduced the severity of oral anaphylaxis without affecting antigen-specific IgE or Th2 responses. Oral anaphylaxis was abrogated in KitW-sh/W-sh mice, and restored by reconstitution with WT, but not ST2-deficient, BMMCs. IL-33 significantly enhanced IgE-mediated degranulation of BMMCs in vitro. IL-33 is released following mechanical skin injury, enhances IgE-mediated MC degranulation, and promotes oral anaphylaxis following EC sensitization by targeting MCs. IL-33 neutralization may be useful in treating food anaphylaxis in AD patients.

The effects of smoking and smoking cessation on nasal mucociliary clearance, mucus properties and inflammation

OBJECTIVE:The aim of the present study was to assess nasal mucociliary clearance, mucus properties and inflammation in smokers and subjects enrolled in a Smoking Cessation Program (referred to as quitters).METHOD:A total of 33 subjects with a median (IQR) smoking history of 34 (20-58) pack years were examined for nasal mucociliary clearance using a saccharine transit test, mucus properties using contact angle and sneeze clearability tests, and quantification of inflammatory and epithelial cells, IL-6 and IL-8 concentrations in nasal lavage fluid. Twenty quitters (mean age: 51 years, 9 male) were assessed at baseline, 1 month, 3 months and 12 months after smoking cessation, and 13 smokers (mean age: 52 years, 6 male) were assessed at baseline and after 12 months. Clinicaltrials.gov: NCT02136550.RESULTS:Smokers and quitters showed similar demographic characteristics and morbidities. At baseline, all subjects showed impaired nasal mucociliary clearance (mean 17.6 min), although 63% and 85% of the quitters demonstrated significant nasal mucociliary clearance improvement at 1 month and 12 months, respectively. At 12 months, quitters also showed mucus sneeze clearability improvement (∼26%), an increased number of macrophages (2-fold) and no changes in mucus contact angle or cytokine concentrations.CONCLUSION:This study showed that smoking cessation induced early improvements in nasal mucociliary clearance independent of mucus properties and inflammation. Changes in mucus properties were observed after only 12 months of smoking cessation.

Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit.

Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells and smooth muscle hypercontractility. This response, known as 'weep and sweep', requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells). Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells.

Airway epithelial dual oxidase 1 mediates allergen-induced IL-33 secretion and activation of type 2 immune responses

The IL-1 family member IL-33 plays a critical role in type 2 innate immune responses to allergens and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood. Based on previous findings indicating involvement ofthe NADPH oxidase dual oxidase 1 (DUOX1) in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of asthmatic patients. Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite, and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored by using small interfering RNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy subjects or asthmatic patients were evaluated for DUOX1 expression and allergen-induced responses. Invitro or invivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which depended critically on DUOX1-mediated activation of epithelial epidermal growth factor receptor and the protease calpain-2 through a redox-dependent mechanism involving cysteine oxidation within epidermal growth factor receptor and the tyrosine kinase Src. Primary nasal epithelial cells from patients with allergic asthma were found to express increased DUOX1 and IL-33 levels and demonstrated enhanced IL-33 secretion in response to allergen challenge compared with values seen in nasal epithelial cells from nonasthmatic subjects. Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens and indicate that enhanced DUOX1 expression and IL-33 secretion might present important contributing features of allergic asthma.

Eosinophils and IL-33 Perpetuate Chronic Inflammation and Fibrosis in a Pediatric Population with Stricturing Crohn's Ileitis.

Fibrostenosis and stricture are well-recognized endpoints in Crohn's disease (CD). We hypothesized that stricturing CD is characterized by eosinophilia and epithelial IL-33. We proposed that eosinophil exposure to IL-33 would perpetuate inflammatory chronicity and subsequent fibrostenosis. We performed a retrospective study of 74 children with inflammatory and stricturing ileal CD comparing clinicopathological features to immunohistochemical measures of eosinophilia and IL-33. To scrutinize eosinophil patterns, we developed a novel eosinophil peroxidase score encompassing number, distribution, and degranulation. Human eosinophils and intestinal fibroblasts were cultured with IL-33 and IL-13, and inflammatory and remodeling parameters were assessed. Antieosinophil therapy was also administered to the Crohn's-like ileitis model (SAMP1/SkuSlc). Our novel eosinophil peroxidase score was more sensitive than H&E staining, revealing significant differences in eosinophil patterns, comparing inflammatory and stricturing pediatric CD. A significant relationship between ileal eosinophilia and complicated clinical/histopathological phenotype including fibrosis was determined. IL-33 induced significant eosinophil peroxidase secretion and IL-13 production. Exposure to eosinophils in the presence of IL-33, "primed" fibroblasts to increase proinflammatory cytokines (TNF-α, IL-1β, and IL-6), eosinophil-associated chemokines (CCL24 and CCL26), and IL-13Rα2 production. Production of fibrogenic molecules (collagen 1A2, fibronectin, and periostin) increased after exposure of "primed" fibroblasts to IL-13. Epithelial-IL-33 was increased in pediatric Crohn's ileitis and strongly associated with clinical and histopathological activity, ileal eosinophilia, and complicated fibrostenotic disease. SAMP1/SkuSlc eosinophil-targeted treatment resulted in significant improvements in inflammation and remodeling. Our study of specimens from pediatric patients with ileal CD linked eosinophil patterns and IL-33 to fibrosis and suggested that these may contribute to the perpetuation of inflammation and subsequent stricture in pediatric CD.

Properdin provides protection from Citrobacter rodentium-induced intestinal inflammation in a C5a/IL-6-dependent manner.

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium-induced colitis. Following infection, properdin knockout (P(KO)) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell-derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to P(KO) mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of P(KO) mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

IL-25 as a novel therapeutic target in nasal polyps of patients with chronic rhinosinusitis

Chronic rhinosinusitis (CRS) with nasal polyps (NPs) in Western populations is associated with TH2 cytokine polarization. IL-25, an IL-17 family cytokine, was recently reported to induce TH2-type immune responses and to contribute to several allergic diseases, such as atopic dermatitis and asthma. However, the role of IL-25 in Asian patients with nasal polyposis remains unclear. We sought to determine the role of IL-25 in Asian patients with nasal polyposis and CRS. We investigated IL-25 expression and its cellular origins in NPs of human subjects using immunohistochemistry (IHC), quantitative RT-PCR, and ELISA of NP tissues. Correlations between IL-25 expression and expression of other inflammatory markers in NP tissues were also explored. Anti-IL-25 neutralizing antibody was administered in an ovalbumin- and staphylococcal enterotoxin B-induced murine NP model to confirm the function of IL-25 during nasal polypogenesis. IL-25 expression was upregulated in NP mucosa from patients with CRS with NPs compared with uncinate process tissue from control subjects and those with CRS without NPs. Overexpression of epithelial IL-25 was confirmed by using IHC, and double IHC staining showed that tryptase-positive cells were one of the main sources of IL-25 among immune cells. Furthermore, IL-17 receptor B levels were also increased in immune cells of patients with NPs compared with those in control subjects. In NPs IL-25 mRNA expression positively correlated with the expression of several inflammatory markers, including T-box transcription factor, RAR-related orphan receptor C, GATA3, eosinophil cationic protein, TGF-β1, and TGF-β2. IL-25 was more abundant in the murine NP model compared with control mice, and similar correlations between IL-25 and inflammatory markers were observed in murine models. Anti-IL-25 treatment reduced the number of polyps, mucosal edema thickness, collagen deposition, and infiltration of inflammatory cells, such as eosinophils and neutrophils. This treatment also inhibited expression of local inflammatory cytokines, such as IL-4 and IFN-γ. Furthermore, expression of CCL11, CXCL2, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in the nasal mucosa was suppressed in the anti-IL-25-treated group. Our results suggest that IL-25 secreted from the sinonasal epithelia and infiltrating mast cells plays a crucial role in the pathogenesis of CRS with NPs in Asian patients. In addition, our results suggest the novel possibility of treating nasal polyposis with anti-IL-25 therapy.

Epithelial IL-33 and TSLP Elicit Innate Lymphoid Cell Responses to Mediate Ozone-Induced Airway Inflammation and Hyperresponsiveness

Exposure to the common air pollutant ozone is a global respiratory health problem. The mechanisms by which ozone impairs lung function and exacerbates respiratory diseases remain unclear. Pulmonary group-2 innate lymphoid cells (ILC2) are a prominent source of IL-5 and IL13 in responses to epithelium-derived cytokines IL-33 and TSLP.

Staphyloccoccus Aureus Induces a Th2 Response Via TSLP and IL-33 Release in Human Airway Mucosa

S A T U R D A Y 259 Staphyloccoccus Aureus Induces a Th2 Response Via TSLP and IL-33 Release in Human Airway Mucosa Feng Lan, MD, Nan Zhang, Gabriele Holtappels, Natalie De Ruyck, Nikolaos G. Papadopoulos, MD, FAAAAI, EAACI President, Sebastian L. Johnston, MD, PhD, Claus Bachert, MD, PhD; Upper Airway Research Laboratory (URL), Ghent University Hospital, Ghent, Belgium, Allergy Research Center, Athens, Greece, Imperial College London, London, United Kingdom, Division of ENT Diseases, Karolinska Institute, Stockholm, Sweden. RATIONALE: Chronic rhinosinusitis with nasal polyps (CRSwNP) is mainly characterized by a Th2-skewed inflammation. We hypothesized that S. aureus induces Th2 response in CRSwNP via epithelial derived cytokine production. METHODS: The levels of epithelial derived cytokines TSLP and IL-33 and consecutively Th2 cytokines were assessed in human mucosal S. aureus infection model. The localization of IL-33 and cell death in human CRSwNP tissue after S. aureus infection was evaluated by immunofluroscence staining. Human bronchial epithelial cell line BEAS-2B with S. aureus infection was examined for the epithelial cell-derived cytokine production pathway. RESULTS: The levels of TSLP and IL-5 significantly increased in supernatants of CRSwNP tissue after S. aureus infection. Even though an increased protein level of TSLP was found in control tissue after S. aureus infection, no change of IL-5 cytokine was observed. IL-33 was released into the extracellular space between epithelial cells, where TUNEL-positive cells were localized after S. aureus stimulation in CRSwNP tissue. At the same time, IL-33 cytokine was over-expressed in CRSwNP tissue homogenates after S. aureus infection. Increased levels of IL-33 and TSLP were induced by S. aureus in a dose-dependent manner in BEAS-2B cells with an increase phosphorylation process of p50, p65 and p38. CONCLUSIONS:Wehere demonstrate for the first time that S. aureuscan directly induces epithelial cell-derived cytokine in human nasal tissue, and propagates Th2 response only in CRSwNP tissue, not in controls. The NFkB and MAPK pathway might be involved in the production of TSLP and IL-33 after S. aureus infection.

Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33

Asthma in a mouse model spontaneously resolves after cessation of allergen exposure. We developed a mouse model in which asthma features persisted for 6 months after cessation of allergen exposure. We sought to elucidate factors contributing to the persistence of asthma. We used a combination of immunologic, genetic, microarray, and pharmacologic approaches to dissect the mechanism of asthma persistence. Elimination of T cells though antibody-mediated depletion or lethal irradiation and transplantation of recombination-activating gene (Rag1)(-/-) bone marrow in mice with chronic asthma resulted in resolution of airway inflammation but not airway hyperreactivity or remodeling. Elimination of T cells and type 2 innate lymphoid cells (ILC2s) through lethal irradiation and transplantation of Rag2(-/-)γc(-/-) bone marrow or blockade of IL-33 resulted in resolution of airway inflammation and hyperreactivity. Persistence of asthma required multiple interconnected feedback and feed-forward circuits between ILC2s and epithelial cells. Epithelial IL-33 induced ILC2s, a rich source of IL-13. The latter directly induced epithelial IL-33, establishing a positive feedback circuit. IL-33 autoinduced, generating another feedback circuit. IL-13 upregulated IL-33 receptors and facilitated IL-33 autoinduction, thus establishing a feed-forward circuit. Elimination of any component of these circuits resulted in resolution of chronic asthma. In agreement with the foregoing, IL-33 and ILC2 levels were increased in the airways of asthmatic patients. IL-33 levels correlated with disease severity. We present a critical network of feedback and feed-forward interactions between epithelial cells and ILC2s involved in maintaining chronic asthma. Although T cells contributed to the severity of chronic asthma, they were redundant in maintaining airway hyperreactivity and remodeling.

PS-084 Differential Regulation Of Interleukin-8 And Human Beta-defensin-2 In Intestinal Epithelial Cells To Pseudomonas Infection

<h3>Background and aims</h3> Pseudomonas aeruginosa (P.aeruginosa), carriers the highest case fatality rate of all gram-negative infections and antimicrobial therapy has not been demonstrated to improve clinical outcome. Moreover, the emergence of multidrug resistant P. aeruginosa has become a major concern in the hospital setting. Fever and diarrhoea were the2 most common initial symptoms in P. aeruginosa sepsis in previously healthy infants and children. This implied that intestinal epithelial cells (IECs) contacting with the pathogen may play an important role on innate immunity to P.aeruginosa infection. Therefore, we aim to investigate the intestinal epithelial IL-8 and hBD-2 expression to P. aeruginosa infection and its regulators. <h3>Methods</h3> We applied ELISA for IL-8 andhBD-2 protein secretion, RT-PCR for IL-8 and hBD-2 mRNA expression, Western blot for signal pathway as well as inhibitors and siRNA to investigate the involved proteins in P. aeruginosa-induced IL-8 and hBD-2 expression in SW480 cells. <h3>Results</h3> We demonstrated after prolonged infection by P. aeruginosa, secreted IL-8 protein was suppressed but hBD-2protein was enhanced though both mRNAs were increased in SW480 cells. Interactions between the PI3K/Akt pathway and ERK kinase result ultimately in post-transcriptional effects that decrease P. aeruginosa-inducedIL-8 production while NOD1 protein is involved in Pseudomonas-induced hBD-2 expression in SW480 cells. <h3>Conclusions</h3> P. aeruginosa induced pro inflammatory response and antimicrobial peptide in IECs. The antimicrobial peptide in IECs has been shown to continuously protect the host against prolonged infection while modulation of pro inflammatory response prevents the host from the detrimental effects of overwhelming inflammation.

Chronic low dose chlorine exposure aggravates allergic inflammation and airway hyperresponsiveness and activates inflammasome pathway.

BackgroundEpidemiologic clinical studies suggested that chronic exposure to chlorine products is associated with development of asthma and aggravation of asthmatic symptoms. However, its underlying mechanism was not clearly understood. Studies were undertaken to define the effects and mechanisms of chronic low-dose chlorine exposure in the pathogenesis of airway inflammation and airway hyperresponsiveness (AHR).MethodsSix week-old female BALB/c mice were sensitized and challenged with OVA in the presence and absence of chronic low dose chlorine exposure of naturally vaporized gas of 5% sodium hypochlorite solution. Airway inflammation and AHR were evaluated by bronchoalveolar lavage (BAL) cell recovery and non-invasive phlethysmography, respectively. Real-time qPCR, Western blot assay, and ELISA were used to evaluate the mRNA and protein expressions of cytokines and other inflammatory mediators. Human A549 and murine epithelial (A549 and MLE12) and macrophage (AMJ2-C11) cells were used to define the responses to low dose chlorine exposure in vitro.ResultsChronic low dose chlorine exposure significantly augmented airway inflammation and AHR in OVA-sensitized and challenged mice. The expression of Th2 cytokines IL-4 and IL-5 and proinflammatory cytokine IL-1β and IL-33 were significantly increased in OVA/Cl group compared with OVA group. The chlorine exposure also activates the major molecules associated with inflammasome pathway in the macrophages with increased expression of epithelial alarmins IL-33 and TSLP in vitro.ConclusionChronic low dose exposure of chlorine aggravates allergic Th2 inflammation and AHR potentially through activation of inflammasome danger signaling pathways.

From phyto-chemoprevention to discovery of novel markers and targets for therapy of breast cancer

s of Pharma Nutrition 2013 / PharmaNutrition 2 (2014) 75–119 81 functions. Using the human epithelial intestinal cell line Caco-2 in a transwell system as an in vitromodel, we studied the effect of DON in the presence and absence of galacto-oligosaccharides (scGOS). Parameters assessed included transepithelial electrical resistance (TEER), paracellular tracer flux, expression of tight junction proteins, cytokine release and overall cell viability. Interestingly, when a DON challenged non-permeable Caco-2 monolayer was preincubated with scGOS for 24h, TEER and paracellular transport of the marker lucifer yellow remained unaffected. In parallel, the levels of claudin-3 mRNA expression were not affected by DON exposure in the presence of scGOS. Furthermore, the increase in epithelial IL-8 mRNA expression and in IL-8 secretion, observed after DON exposure, was counteracted by scGOS. In conclusion, we demonstrated with this in vitro model that scGOS protects and restores the epithelial barrier dysfunction exerted by the food contaminant DON. In addition, the hypothesis that scGOS has antiinflammatory properties was confirmed by demonstrating that the DON-induced production of IL-8was counteracted by scGOS. Taken together, these findings indicate that dietary oligosaccharides may be a promising approach to mitigate the effects of undesirable dietary mycotoxin exposure.

Chitin Activates Parallel Immune Modules that Direct Distinct Inflammatory Responses via Innate Lymphoid Type 2 and γδ T Cells

Chitin, a polysaccharide constituent of many allergens and parasites, initiates innate type 2 lung inflammation through incompletely defined pathways. We show that inhaled chitin induced expression of three epithelial cytokines, interleukin-25 (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP), which nonredundantly activated resident innate lymphoid type 2 cells (ILC2s) to express IL-5 and IL-13 necessary for accumulation of eosinophils and alternatively activated macrophages (AAMs). In the absence of all three epithelial cytokines, ILC2s normally populated the lung but failed to increase IL-5 and IL-13. Although eosinophils and AAMs were attenuated, neutrophil influx remained normal without these epithelial cytokines. Genetic ablation of ILC2s, however, enhanced IL-1β, TNFα, and IL-23 expression, increased activation of IL-17A-producing γδ T cells, and prolonged neutrophil influx. Thus, chitin elicited patterns of innate cytokines that targeted distinct populations of resident lymphoid cells, revealing divergent but interacting pathways underlying the tissue accumulation of specific types of inflammatory myeloid cells.