Search

A high fruit and vegetable (FV) diet reduces asthma exacerbations in adults; this has not been examined in children to date.To investigate the effect of a 6-month, high FV diet on the time to first asthma exacerbation in children with asthma, in a parallel-group, randomized, controlled trial.Children (aged 3-11 years) with asthma, history of exacerbations and usual low FV intake (≤3 serves/day) were randomized to the intervention (high FV diet) or control group (usual diet) for 6 months. The primary outcome was time to first exacerbation requiring medical intervention. Secondary outcomes included exacerbation rate, lung function, plasma TNF-α, CRP, and IL-6, faecal microbiota and peripheral blood mononuclear cell (PBMC) histone deacetylase (HDAC) activity and G-protein coupled receptor (GPR) 41/43 and HDAC (1-11) expression.67 children were randomized between September 2015 and July 2018. FV intake (difference in change (∆): 3.5 serves/day, 95% CI: [2.6, 4.4] p 0.001) and plasma total carotenoids (∆: 0.44 µg/ml [0.19, 0.70] p = 0.001) increased after 6 months (intervention vs control). Time to first exacerbation (HR: 0.81, 95% CI: [0.38, 1.69], p = 0.569; control vs. intervention) and exacerbation rate (IRR: 0.84, [0.47, 1.49], p = 0.553; control vs. intervention) were similar between groups. In per-protocol analysis, airway reactance z-scores increased in the intervention versus control group (XA high FV diet did not affect asthma exacerbations over the 6-month intervention, though warrants further investigation as a strategy for improving lung function and protecting against systemic inflammation in children with asthma.

Rhinovirus (RV) infections in asthmatic patients are often associated with asthma exacerbation, characterized by worsened airways hyperreactivity and increased immune cell infiltration to the airways. The C-X-C chemokines, CXCL3 and CXCL5, regulate neutrophil trafficking to the lung via CXCR2, and their expression in the asthmatic lung is associated with steroid-insensitive type 2 inflammatory signatures. Currently, the role of CXCL3 and CXCL5 in regulating neutrophilic and type 2 responses in viral-induced asthma exacerbation is unknown. Inhibition of CXCL3 or CXCL5 with silencing RNAs in a mouse model of RV-induced exacerbation of asthma attenuated the accumulation of CXCR2+ neutrophils, eosinophils, and innate lymphoid cells in the lung and decreased production of type 2 regulatory factors IL-25, IL-33, IL-5, IL-13, CCL11, and CCL24. Suppression of inflammation was associated with decreased airways hyperreactivity, mucus hypersecretion, and collagen deposition. Similar results were obtained by employing RC-3095, which has been shown to bind to CXCR2, or by depletion of neutrophils. Our data demonstrate that CXCL3 and CXCL5 may be critical in the perpetuation of RV-induced exacerbation of asthma through the recruitment of CXCR2-positive neutrophils and by promoting type 2 inflammation. Targeting the CXCL3/CXCL5/CXCR2 axis may provide a new therapeutic approach to attenuating RV-induced exacerbations of asthma.

BackgroundAsthma is the most frequent cause of hospitalisation among children; however, little is known regarding the effects of asthma on immune responses in children.ObjectiveThe present study aimed to evaluate cytokine responses of peripheral blood mononuclear cells (PBMCs), PBMC composition and lung function in children with and without asthma.MethodsUsing a case-control design, we compared 48 children with asthma aged 3-11 years with 14 age-matched healthy controls. PBMC composition and cytokine production including interferon (IFN)-γ, interleukin (IL)-1β, IL-5 and lL-6 following stimulation with rhinovirus-1B (RV1B), house dust mite (HDM) and lipopolysaccharide (LPS) were measured. Lung function was assessed using impulse oscillometry and nitrogen multiple breath washout.ResultsThe frequency of group 2 innate lymphoid cells were significantly higher in asthmatics and PBMCs from asthmatics had deficient IFN-γ production in response to both RV1B and LPS compared with controls (PConclusionOur study indicates that children with asthma have a reduced lung function in concert with impaired immune responses and altered immune cell subsets. Improving our understanding of immune responses to viral and bacterial infection in childhood asthma can help to tailor management of the disease.

Virus-induced respiratory tract infections are a major health burden in childhood, and available treatments are supportive rather than disease modifying. Rhinoviruses (RVs), the cause of approximately 80% of common colds, are detected in nearly half of all infants with bronchiolitis and the majority of children with an asthma exacerbation. Bronchiolitis in early life is a strong risk factor for the development of asthma. Here, we found that RV infection induced the expression of miRNA 122 (miR-122) in mouse lungs and in human airway epithelial cells. In vivo inhibition specifically in the lung reduced neutrophilic inflammation and CXCL2 expression, boosted innate IFN responses, and ameliorated airway hyperreactivity in the absence and in the presence of allergic lung inflammation. Inhibition of miR-122 in the lung increased the levels of suppressor of cytokine signaling 1 (SOCS1), which is an in vitro–validated target of miR-122. Importantly, gene silencing of SOCS1 in vivo completely reversed the protective effects of miR-122 inhibition on RV-induced lung disease. Higher miR-122 expression in nasopharyngeal aspirates was associated with a longer time on oxygen therapy and a higher rate of treatment failure in 87 infants hospitalized with moderately severe bronchiolitis. These results suggest that miR-122 promotes RV-induced lung disease via suppression of its target SOCS1 in vivo. Higher miR-122 expression was associated with worse clinical outcomes, highlighting the potential use of anti-miR-122 oligonucleotides, successfully trialed for treatment of hepatitis C, as potential therapeutics for RV-induced bronchiolitis and asthma exacerbations.

Objectives Eosinophilic oesophagitis (EoE) is characterised by oesophageal inflammation, fibrosis and dysfunction. Micro (mi)‐RNAs interfere with pro‐inflammatory and pro‐fibrotic transcriptional programs, and miR‐223 was upregulated in oesophageal mucosal biopsy specimens from EoE patients. The therapeutic potential of modulating miR‐223 expression in vivo has not been determined. We aimed to elucidate the relevance of oesophageal miR‐223 expression in an in vivo model of EoE by inhibiting miR‐223 tissue expression. Methods The expression of miR‐223 and the validated miR‐223 target insulin‐like growth factor receptor 1 (IGF1R) protein was determined in our paediatric cohort of EoE patients. A murine model of Aspergillus fumigatus‐induced EoE was employed, and oesophagi were assessed for miR‐233, IGF1R, T lymphocyte type 2 (T2) cytokine expression and eosinophil infiltration. Mice were treated with antagomirs targeting miR‐223 or resveratrol targeting its upstream regulator Midline‐1(MID‐1). Results There was an inverse relationship between an increased expression of miR‐223 and a decreased IGF1R protein concentration in biopsy specimens from EoE patients. TNF‐related apoptosis‐inducing ligand deficiency, MID‐1 inhibition and resveratrol treatment suppressed miR‐223 expression. Furthermore, inhibition of miR‐223 and treatment with resveratrol in the oesophagus resulted in an amelioration of EoE hallmark features including eosinophilic infiltration, oesophageal circumference and a reduction in T2 cytokine expression. Conclusion miR‐223 has a key role in the perpetuation of EoE hallmark features downstream of TNF‐related apoptosis‐inducing ligand and MID‐1 in an experimental model. These studies highlight a potentially critical role of miRNA function in EoE aetiology. miR‐223 expression in the oesophagus may be therapeutically modulated by resveratrol, providing a potential new therapeutic option to be explored in EoE patients for this increasingly prevalent condition., miR‐223 has a key role in the perpetuation of eosinophilic oesophagitis hallmark features downstream of TNF‐related apoptosis‐inducing ligand and MID‐1. These studies highlight a critical role of miRNA function in eosinophilic oesophagitis aetiology and suggest that miR‐223 expression in the oesophagus can be therapeutically modulated by resveratrol, providing a potential new therapy for this increasingly prevalent condition with limited current treatment options.

Objective Offspring born to mothers with asthma in pregnancy are known to have lower lung function which tracks with age. Human group 2 innate lymphoid cells (ILC2) accumulate in foetal lungs, at 10‐fold higher levels compared to adult lungs. However, there are no data on foetal ILC2 numbers and the association with respiratory health outcomes such as lung function in early life. We aimed to investigate cord blood immune cell populations from babies born to mothers with asthma in pregnancy. Methods Cord blood from babies born to asthmatic mothers was collected, and cells were stained in whole cord blood. Analyses were done using traditional gating approaches and computational methodologies (t‐distributed stochastic neighbour embedding and PhenoGraph algorithms). At 6 weeks of age, the time to peak tidal expiratory flow as a percentage of total expiratory flow time (tPTEF/tE%) was determined as well as Lung Clearance Index (LCI), during quiet natural sleep. Results Of 110 eligible infants (March 2017 to November 2019), 91 were successfully immunophenotyped (82.7%). Lung function was attempted in 61 infants (67.0%), and 43 of those infants (70.5% of attempted) had technically acceptable tPTEF/tE% measurements. Thirty‐four infants (55.7% of attempted) had acceptable LCI measurements. Foetal ILC2 numbers with increased expression of chemoattractant receptor‐homologous molecule (CRTh2), characterised by two distinct analysis methodologies, were associated with poorer infant lung function at 6 weeks of age.” Conclusion Foetal immune responses may be a surrogate variable for or directly influence lung function outcomes in early life., Cord blood CRTh2high ILC2 populations from infants born to asthmatic mothers may influence lung function later in life. The association between cord blood CRTh2high ILC2 and foetal eosinophils numbers may indicate a role of foetal ILC2 in promoting eosinophilia, which warrants further investigation.

BackgroundPeanut allergy affects 3% of Australian children and has a higher risk of anaphylaxis than most food allergies. Predicting who is likely to develop anaphylaxis is still an inexact science. The fraction of exhaled nitric oxide (FeNO) shows promise as a biomarker involved in peanut allergy, as nitric oxide plays a role in inhibiting mast cell degranulation which is relevant in anaphylaxis, where mast cell degranulation plays a mediator role. The aim of this study was to assess the change in FeNO in children during peanut challenge.MethodsThirty-six children aged from 5 to 17 years were recruited for open-labelled peanut challenge. Participants had skin prick test to peanut performed, and serum collected for Ara h2 specific IgE and peanut specific IgE. FeNO was measured by portable device (NIOX VERO) prior to and throughout the peanut challenge.ResultsWhen grouped according to reaction type at peanut challenge (anaphylaxis, clinical allergy not anaphylaxis and tolerant), there were significant differences in the mean change in FeNO measurement between the anaphylaxis group and the clinical allergy, not anaphylaxis group (p = 0.005), and between the anaphylaxis group and tolerant group (p ConclusionsFeNO decreased more significantly in those who subsequently developed anaphylaxis than in those with clinical allergy, not anaphylaxis or negative peanut challenge (tolerance). As a bedside test that can be used in children, it has potential for further research into mechanisms of anaphylaxis in food allergy and potentially assists in predicting an imminent anaphylactic reaction in some patients.Trial registrationClinicalTrials.gov: PEAnut Anaphylaxis Predictors (PEAAP) NCT02424136.

Asthma is characterized by airway hyperreactivity and inflammation. In the lungs, parasympathetic and sensory nerves control airway tone and induce bronchoconstriction. Dysregulation of these nerves results in airway hyperreactivity. Humans with eosinophilic asthma have significantly increased sensory nerve density in airway epithelium, suggesting that type 2 cytokines and inflammatory cells promote nerve growth. Similarly, mice with congenital airway eosinophilia also have airway hyperreactivity and increased airway sensory nerve density. Here, we tested whether this occurs during development. We show that transgenic mice that overexpress IL-5, a cytokine required for eosinophil hematopoiesis, give birth to wild-type offspring that have significantly increased airway epithelial nerve density and airway hyperreactivity that persists into adulthood. These effects are caused by in utero exposure to maternal IL-5 and resulting fetal eosinophilia. Allergen exposure of these adult wild-type offspring results in severe airway hyperreactivity, leading to fatal reflex bronchoconstriction. Our results demonstrate that fetal exposure to IL-5 is a developmental origin of airway hyperreactivity, mediated by hyperinnervation of airway epithelium.

Summary In this review, we highlight experiments conducted in our laboratories that have elucidated functional roles for CD4+ T-helper type-2 lymphocytes (TH2 cells), their associated cytokines, and eosinophils in the regulation of hallmark features of allergic asthma. Notably, we consider the complexity of type-2 responses and studies that have explored integrated signaling among classical TH2 cytokines (IL-4, IL-5, and IL-13), which together with CCL11 (eotaxin-1) regulate critical aspects of eosinophil recruitment, allergic inflammation, and airway hyper-responsiveness (AHR). Among our most important findings, we have provided evidence that the initiation of TH2 responses is regulated by airway epithelial cell-derived factors, including TRAIL and MID1, which promote TH2 cell development via STAT6-dependent pathways. Further, we highlight studies demonstrating that microRNAs are key regulators of allergic inflammation and potential targets for anti-inflammatory therapy. On the background of TH2 inflammation, we have demonstrated that innate immune cells (notably, airway macrophages) play essential roles in the generation of steroid-resistant inflammation and AHR secondary to allergen- and pathogen-induced exacerbations. Our work clearly indicates that understanding the diversity and spatiotemporal role of the inflammatory response and its interactions with resident airway cells is critical to advancing knowledge on asthma pathogenesis and the development of new therapeutic approaches.

Summary Clinical and epidemiological studies indicate that obesity affects the development and phenotype of asthma by inducing inflammatory mechanisms in addition to eosinophilic inflammation. The aim of this study was to assess the effect of obesity on allergic airway inflammation and T helper type 2 (Th2) immune responses using an experimental model of asthma in BALB/c mice. Mice fed a high-fat diet (HFD) for 10 weeks were sensitized and challenged with ovalbumin (OVA), and analyses were performed at 24 and 48 h after the last OVA challenge. Obesity induced an increase of inducible nitric oxide synthase (iNOS)-expressing macrophages and neutrophils which peaked at 48 h after the last OVA challenge, and was associated with higher levels of interleukin (IL)-4, IL-9, IL-17A, leptin and interferon (IFN)-γ in the lungs. Higher goblet cell hyperplasia was associated with elevated mast cell influx into the lungs and trachea in the obese allergic mice. In contrast, early eosinophil influx and lower levels of IL-25, thymic stromal lymphopoietin (TSLP), CCL11 and OVA-specific immunoglobulin (IgE) were observed in the obese allergic mice in comparison to non-obese allergic mice. Moreover, obese mice showed higher numbers of mast cells regardless of OVA challenge. These results indicate that obesity affects allergic airway inflammation through mechanisms involving mast cell influx and the release of TSLP and IL-25, which favoured a delayed immune response with an exacerbated Th1, Th2 and Th17 profile. In this scenario, an intense mixed inflammatory granulocyte influx, classically activated macrophage accumulation and intense mucus production may contribute to a refractory therapeutic response and exacerbate asthma severity.

the aim of this study is to elucidate the role of TRAIL during rhinovirus (RV) infection in vivo. Naïve wild-type and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-deficient ( Tnfsf10 −/−) BALB/c mice were infected intranasally with RV1B. In separate experiments, Tnfsf10 −/− mice were sensitized and challenged via the airway route with house dust mite (HDM) to induce allergic airways disease and then challenged with RVIB or UV-RVIB. Airway hyperreactivity (AHR) was invasively assessed as total airways resistance in response to increasing methacholine challenge and inflammation was assessed in bronchoalveolar lavage fluid at multiple time points postinfection. Chemokines were quantified by ELISA of whole lung lysates and viral load was determined by quantitative RT-PCR and tissue culture infective dose (TCID50). Human airway epithelial cells (BEAS2B) were infected with RV1B and stimulated with recombinant TRAIL or neutralizing anti-TRAIL antibodies and viral titer assessed by TCID50. HDM-challenged Tnfsf10 −/− mice were protected against RV-induced AHR and had suppressed cellular infiltration in the airways upon RV infection. Chemokine C-X-C-motif ligand 2 (CXCL2) production was suppressed in naïve Tnfsf10 −/− mice infected with RV1B, with less RV1B detected 24 h postinfection. This was associated with reduced apoptotic cell death and a reduction of interferon (IFN)-λ2/3 but not IFN-α or IFN-β. TRAIL stimulation increased, whereas anti-TRAIL antibodies reduced viral replication in RV1B-infected BEAS2B cells in vitro. In conclusion, TRAIL promotes RV-induced AHR, inflammation and RV1B replication, implicating this molecule and its downstream signaling pathways as a possible target for the amelioration of RV1B-induced allergic and nonallergic lung inflammation and AHR.

Food antigens are common inflammatory triggers in pediatric eosinophilic esophagitis (EoE). TNF-related apoptosis-inducing ligand (TRAIL) promotes eosinophilic inflammation through the upregulation of the E3 ubiquitin ligase Midline (MID)-1 and subsequent downregulation of protein phosphatase 2A (PP2A), but the role of this pathway in EoE that is experimentally induced by repeated food antigen challenges has not been investigated. Esophageal mucosal biopsies were collected from children with EoE and controls and assessed for TRAIL and MID-1 protein and mRNA transcript levels. Wild-type and TRAIL-deficient (Tnfsf10−/−) mice were administered subcutaneous ovalbumin (OVA) followed by oral OVA challenges. In separate experiments, OVA-challenged mice were intraperitoneally administered salmeterol or dexamethasone. Esophageal biopsies from children with EoE revealed increased levels of TRAIL and MID-1 and reduced PP2A activation compared with controls. Tnfsf10−/− mice were largely protected from esophageal fibrosis, eosinophilic inflammation, and the upregulation of TSLP, IL-5, IL-13, and CCL11 when compared with wild-type mice. Salmeterol administration to wild-type mice with experimental EoE restored PP2A activity and also prevented esophageal eosinophilia, inflammatory cytokine expression, and remodeling, which was comparable to the treatment effect of dexamethasone. TRAIL and PP2A regulate inflammation and fibrosis in experimental EoE, which can be therapeutically modulated by salmeterol.

Introduction and Aim: Severe asthma is characterised by frequent exacerbations with the majority triggered by viral infections. Treatment of severe allergic asthma with Omalizumab reduces exacerbation risk. We sought to determine how Omalizumab improved antiviral innate immune responses to influenza A (IAV) and rhinovirus (RV). Methods: We recruited 10 adults, with severe allergic asthma and 10 healthy controls. Participants were assessed at basdeline 4 and 24 weeks after treatment. Peripheral blood monocytes (PBMCs) were isolated, exposed to IAV, RV. Monocyte cell types and FceI expression was identified by flow cytometry, Response was assessed by ELISA/RNA for FceI, interferon (IFN)-a, IFN-l, IFN-g, IL-6, IL-10. Results: At base line visit subjects with severe allergic asthma compared to healthy controls demonstrated impaired IFN-α, and IFN-λ release in response to IAV (p Conclusion: Adults with severe allergic asthma demonstrate impaired systemic DC immune responses to IAV and RV. Treatment with Omalizumab, that results in reduced exacerbations is associated with reduced DC FceI expression, enhanced DC IFN-α/λ responses to viruses and this effect is seen within 4 weeks treatment.

Background Eosinophilic oesophagitis (EoE) is an emergent chronic immune-mediated disease of the oesophagus, which affects both children and adults. It is clinically characterized by dysphagia, food impaction and oesophageal eosinophilia. Epidemiological studies indicate that obesity can worsen allergic symptoms; however, its effect on EoE immunopathological response has not been evaluated yet. This study aimed to assess the effect of obesity on allergic inflammation and T helper-2 profile in an EoE experimental model. Methods Obesity was induced by high-fat feeding. After 7 weeks of diet, male BALB/c mice were subcutaneously sensitized and orally challenged with OVA. Results Obesity itself induced a significant mast cell and eosinophil accumulation in the oesophagus, trachea, gut and lung. After allergy induction, this number was higher, when compared to lean-allergic mice. Moreover, obese-allergic mice showed higher remodelling area, in the oesophagus, associated with higher IL-5 and TSLP mRNA expression. In contrast, FoxP3 and IL-10 were less expressed in comparison with lean-allergic mice. In addition, the amount of CD11c+ MHCII+ PDL1+ dendritic cells was reduced, while the number of CD11c+ MHCII+ CD80+ DCs and CD3+ CD4+ GATA3 + IL-4+ cells was increased in obese-allergic mice in the spleen and lymph nodes when compared to lean-allergic mice. Conclusion Obesity aggravated the immune histopathological characteristics in the EoE experimental model, which was associated with the reduction in the regulatory profile, and the increased inflammatory cells influx, related to the TH 2 profile. Altogether, the data provide new knowledge about obesity as a risk factor, worsening EoE symptoms, and contribute for future treatment strategies for this specific profile.

Objective Chronic obstructive pulmonary disease (COPD) is a progressive disease that causes significant mortality and morbidity worldwide and is primarily caused by the inhalation of cigarette smoke (CS). Lack of effective treatments for COPD means there is an urgent need to identify new therapeutic strategies for the underlying mechanisms of pathogenesis. Tristetraprolin (TTP) encoded by the Zfp36 gene is an anti‐inflammatory protein that induces mRNA decay, especially of transcripts encoding inflammatory cytokines, including those implicated in COPD. Methods Here, we identify a novel protective role for TTP in CS‐induced experimental COPD using Zfp36aa/aa mice, a genetically modified mouse strain in which endogenous TTP cannot be phosphorylated, rendering it constitutively active as an mRNA‐destabilising factor. TTP wild‐type (Zfp36 +/+) and Zfp36aa/aa active C57BL/6J mice were exposed to CS for four days or eight weeks, and the impact on acute inflammatory responses or chronic features of COPD, respectively, was assessed. Results After four days of CS exposure, Zfp36aa/aa mice had reduced numbers of airway neutrophils and lymphocytes and mRNA expression levels of cytokines compared to wild‐type controls. After eight weeks, Zfp36aa/aa mice had reduced pulmonary inflammation, airway remodelling and emphysema‐like alveolar enlargement, and lung function was improved. We then used pharmacological treatments in vivo (protein phosphatase 2A activator, AAL(S), and the proteasome inhibitor, bortezomib) to promote the activation and stabilisation of TTP and show that hallmark features of CS‐induced experimental COPD were ameliorated. Conclusion Collectively, our study provides the first evidence for the therapeutic potential of inducing TTP as a treatment for COPD., Cigarette smoke (CS) exposure increases the mRNA expression of inflammatory mediators resulting in pulmonary inflammation. Ongoing inflammation drives the development of airway remodelling and emphysema, subsequently leading to lung function decline. This study highlights the importance of having active TTP to control CS‐induced inflammation, pulmonary remodelling, emphysema and impaired lung function in mice.

We tested whether the T helper (Th) type 2 (Th2) cell agonist and allergenic ligand IL-33 was associated with eosinophilic esophagitis (EoE) development in a pediatric cohort and whether IL-33 protein could induce disease symptoms in mice. Biopsies from EoE patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast cells, and epithelial cell markers near blood vessels. Circulating levels of IL-33 were not increased. The time course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model and chronicity of IL-33 expression has been demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 wk and esophageal pathology was evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and upregulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, along with innate lymphoid cell group 2, Th1/17, and M2 macrophage marker genes, were increased after IL-33 application. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with pediatric EoE development and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.

Introduction: Maternal asthma has been linked to detectable alterations in cord blood immune cell populations. Innate lymphoid cells (ILCs) have been implicated in the development of allergic diseases. ILC subsets have potential roles in the early development of asthma but this remains poorly understood. Aim: To evaluate the cord blood immunophenotype in infants born to mothers with asthma during pregnancy taking part in the Breathing for Life Trial (BLT). Methods: Cord blood from 32 BLT participants was collected into EDTA tubes and processed within 6 hours of birth. Cells were stained in whole cord blood: Eosinophils (CD45+CD16-CD193+), ILC1(Lin–CD127+CD161+CD117–CD294–), ILC2(Lin–CD127+CD161+CD294+), ILC3 natural cytotoxic receptor (NCR)– (Lin–CD127+CD161+CD294–CD117+NKp44–), ILC3 NCR+ (Lin– CD127+CD161+CD294–CD117+NKp44+). After being fixed, cells were acquired on a BD LSRFortessa X-20 flow cytometer and analysed using FlowJo software. Tidal breathing flow-volume loop (TBFVL) measurements during quiet sleep were performed in infants at 6 weeks of age. Results: This preliminary analysis has shown correlations between ILC1 and ILC2 (n=32) and ILC3s NCR- (r=0.64-0.68). Eosinophils correlated with ILC2 (r=0.40) and were inversely correlated with lung function (tPTEF/tE%) at 6 weeks (r=−0.58, n=13). Conclusion: The study of ILC subtypes in cord blood is feasible and ILC1, ILC2 and ILC3 NCR+ numbers in cord blood show strong correlations. The correlation between ILC2s and eosinophils in cord blood and the potential association with lung function at 6 weeks of age warrants further investigation in this ongoing study.

Background Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. Objective We sought to elucidate the role of TRAIL in EoE. Methods We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL −/− ) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. Results TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL −/− mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL −/− mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. Conclusion TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

Background Asthma exacerbations represent a significant disease burden and are commonly caused by rhinovirus (RV), which is sensed by Toll-like receptors (TLR) such as TLR7. Some asthmatics have impaired interferon (IFN) responses to RV, but the underlying mechanisms of this clinically relevant observation are poorly understood. Objectives To investigate the importance of intact TLR7 signalling in vivo during RV exacerbation using mouse models of house dust mite (HDM)-induced allergic airways disease exacerbated by a superimposed RV infection. Methods Wild-type and TLR7-deficient ( Tlr7 −/− ) BALB/c mice were intranasally sensitised and challenged with HDM prior to infection with RV1B. In some experiments, mice were administered recombinant IFN or adoptively transferred with plasmacytoid dendritic cells (pDC). Results Allergic Tlr7 −/− mice displayed impaired IFN release upon RV1B infection, increased virus replication and exaggerated eosinophilic inflammation and airways hyper reactivity. Treatment with exogenous IFN or adoptive transfer of TLR7-competent pDCs blocked these exaggerated inflammatory responses and boosted IFNγ release in the absence of host TLR7 signalling. TLR7 expression in the lungs was suppressed by allergic inflammation and by interleukin (IL)-5-induced eosinophilia in the absence of allergy. Subjects with moderate-to-severe asthma and eosinophilic but not neutrophilic airways inflammation, despite inhaled steroids, showed reduced TLR7 and IFNλ2/3 expression in endobronchial biopsies. Furthermore, TLR7 expression inversely correlated with percentage of sputum eosinophils. Conclusions This implicates IL-5-induced airways eosinophilia as a negative regulator of TLR7 expression and antiviral responses, which provides a molecular mechanism underpinning the effect of eosinophil-targeting treatments for the prevention of asthma exacerbations.

Rhinovirus (RV) infections are common and have the potential to exacerbate asthma. We have determined the lung transcriptome in RV strain 1B–infected naive BALB/c mice (nonallergic) and identified CCL7 and IFN regulatory factor (IRF)-7 among the most upregulated mRNA transcripts in the lung. To investigate their roles we employed anti-CCL7 Abs and an IRF-7–targeting small interfering RNA in vivo. Neutralizing CCL7 or inhibiting IRF-7 limited neutrophil and macrophage influx and IFN responses in nonallergic mice. Neutralizing CCL7 also reduced activation of NF-κB p65 and p50 subunits, as well as airway hyperreactivity (AHR) in nonallergic mice. However, neither NF-κB subunit activation nor AHR was abolished with infection of allergic mice after neutralizing CCL7, despite a reduction in the number of neutrophils, macrophages, and eosinophils. IRF-7 small interfering RNA primarily suppressed IFN-α and IFN-β levels during infection of allergic mice. Our data highlight a pivotal role of CCL7 and IRF-7 in RV-induced inflammation and IFN responses and link NF-κB signaling to the development of AHR.

Allergic asthma is a complex disease characterized by acute inflammation of the airways that over time leads to the development of significant structural changes termed remodeling. TNF-related apoptosis-inducing ligand (TRAIL) has an important regulatory role in acute allergic airways inflammation through up-regulation of the E3 ubiquitin ligase Midline-1 (MID-1), which limits protein phosphatase 2A (PP2A) activity and downstream dephosphorylation of proinflammatory signaling molecules. The relevance of TRAIL in the development of airways remodeling has yet to be determined. In this study, the lungs of wild-type (WT) BALB/c and Tnfsf10 knockout (TRAIL-/-) mice were chronically exposed to ovalbumin (OVA) for 12 weeks to induce hallmark features of chronic allergic airways disease, including airways hyperreactivity (AHR), subepithelial collagen deposition, goblet cell hyperplasia, and smooth muscle hypertrophy. TRAIL-/- mice were largely protected from the development of AHR and peribronchial eosinophilia and had reduced levels of mast cells in the airways. This correlated with lower levels of cytokines, including IL-4, -5, -10, and -13, and with lower levels of proinflammatory chemokines from cultured cells isolated from the draining lymph nodes. TRAIL-/- mice were also protected from the characteristic features of airways remodeling, including peribronchial fibrosis, smooth muscle hypertrophy, and mucus hypersecretion, which correlated with reduced TGF-β1 levels in the lungs. MID-1 expression was reduced in TRAIL-/- mice and up-regulated in allergic WT mice. Raising PP2A activity using 2-amino-4-(4-heptyloyphenol)-2-methylbutan-1-ol in allergic WT mice reduced eosinophilia, TGF-β1, and peribronchial fibrosis. This study shows that TRAIL promotes airways remodeling in an OVA-induced model of chronic allergic airways disease. Targeting TRAIL and its downstream proinflammatory signaling pathway involving PP2A may be of therapeutic benefit in reducing the hallmark features of airways remodeling observed in chronic allergic airways inflammation.

Background β-Agonists are used for relief and control of asthma symptoms by reversing bronchoconstriction. They might also have anti-inflammatory properties, but the underpinning mechanisms remain poorly understood. Recently, a direct interaction between formoterol and protein phosphatase 2A (PP2A) has been described in vitro . Objective We sought to elucidate the molecular mechanisms by which β-agonists exert anti-inflammatory effects in allergen-driven and rhinovirus 1B–exacerbated allergic airways disease (AAD). Methods Mice were sensitized and then challenged with house dust mite to induce AAD while receiving treatment with salmeterol, formoterol, or salbutamol. Mice were also infected with rhinovirus 1B to exacerbate lung inflammation and therapeutically administered salmeterol, dexamethasone, or the PP2A-activating drug (S)-2-amino-4-(4-[heptyloxy]phenyl)-2-methylbutan-1-ol (AAL[S]). Results Systemic or intranasal administration of salmeterol protected against the development of allergen- and rhinovirus-induced airway hyperreactivity and decreased eosinophil recruitment to the lungs as effectively as dexamethasone. Formoterol and salbutamol also showed anti-inflammatory properties. Salmeterol, but not dexamethasone, increased PP2A activity, which reduced CCL11, CCL20, and CXCL2 expression and reduced levels of phosphorylated extracellular signal-regulated kinase 1 and active nuclear factor κB subunits in the lungs. The anti-inflammatory effect of salmeterol was blocked by targeting the catalytic subunit of PP2A with small RNA interference. Conversely, increasing PP2A activity with AAL(S) abolished rhinovirus-induced airway hyperreactivity, eosinophil influx, and CCL11, CCL20, and CXCL2 expression. Salmeterol also directly activated immunoprecipitated PP2A in vitro isolated from human airway epithelial cells. Conclusions Salmeterol exerts anti-inflammatory effects by increasing PP2A activity in AAD and rhinovirus-induced lung inflammation, which might potentially account for some of its clinical benefits.

Respiratory infections in early life can lead to chronic respiratory disease. Chlamydia infections are common causes of respiratory disease, particularly pneumonia in neonates, and are linked to permanent reductions in pulmonary function and the induction of asthma. However, the immune responses that protect against early-life infection and the mechanisms that lead to chronic lung disease are incompletely understood. Here we identify novel roles for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in promoting Chlamydia respiratory infection-induced pathology in early life, and subsequent chronic lung disease. By infecting TRAIL-deficient neonatal mice and using neutralizing antibodies against this factor and its receptors in wild-type mice, we demonstrate that TRAIL is critical in promoting infection-induced histopathology, inflammation, and mucus hypersecretion, as well as subsequent alveolar enlargement and impaired lung function. This suggests that therapeutic agents that target TRAIL or its receptors may be effective treatments for early-life respiratory infections and associated chronic lung disease.

Background Retrospective studies of childhood peanut allergy demonstrate serum-specific IgE (IgE) levels against the peanut allergen Ara h2 may help predict a clinical reaction at food challenge. Fraction of exhaled nitric oxide (FeNO) is a non-invasive tool correlating to allergic airways inflammation and has been independently associated with increased food-specific IgE. Objective To assess the validity of serum-specific Ara h2 IgE measured prospectively to diagnose peanut allergy and explore the utility of FeNO as a non-invasive screening tool for childhood food challenge. Methods We recruited 53 participants from a cohort of consecutive children scheduled for an open-labelled peanut food challenge (OFC) by their paediatric allergist. Participants underwent skin prick test (SPT) measurement for sensitization to whole peanut extract, and serum was collected for Ara h2-specific IgE. FeNO was also measured in all cooperative children before the challenge. OFC and assessment of reaction were undertaken by clinicians blinded to test results. Results Ara h2-specific IgE and FeNO each showed improved diagnostic accuracy when compared to SPT. Receiver operator characteristic curve analysis gave an area under the curve (AUC) for Ara h2 sIgE of 0.84 (95% CI, 0.72-0.96). The AUC for FeNO, 0.83 (95% CI, 0.71-0.95), was equivalent to that of Ara h2. Combined AUC for SPT, sIgE to Ara h2 and FeNO was 0.96 (95% CI 0.90-1.00). There was no correlation between FeNO and serum nitrite levels (rs = -0.13, P = 0.6, n = 18). Conclusion and clinical relevance Prospectively measured Ara h2-specific IgE improves diagnostic accuracy and reduces unsuccessful challenge to peanut. FeNO levels may provide improved diagnostic accuracy in a paediatric population undergoing OFC. The proposed FeNO-based diagnostic algorithm requires further validation studies.

Background There are few studies investigating the relationship between respiratory viral infection in pregnancy and asthma in the offspring, and none among mothers with asthma. Infants of mothers with asthma are more likely to wheeze and have a higher risk of developing asthma than infants of non-asthmatic mothers. Methods A prospective cohort study of viral infection in pregnancy was conducted between 2007 and 2009, and a subgroup of infants of mothers with asthma was followed up at 6 and 12 months of age. During common colds, nasal and throat swabs were collected from mothers and respiratory viruses detected by polymerase chain reaction. Respiratory health of infants was assessed by parent-completed questionnaire. Results Twelve-month-old infants whose mothers had confirmed viral infections in pregnancy (n = 26) reported more frequent wheeze (40% had 4–12 wheeze attacks compared with 0%), sleep disturbed by wheeze (1 night per week or more in 60% vs. 11%), beta agonist treatment for wheeze (27% vs. 0%), prolonged colds (2 wk or longer 31% vs. 0%), more eczema (40% vs. 6.3%), and parent-perceived asthma (32% vs. 0%), compared with infants whose mothers had common colds without laboratory-confirmed viral infection (n = 16). Conclusions This study demonstrates a relationship between maternal respiratory viral infection in pregnancy and wheezing illness in infants of mothers with asthma. Viral infections are the most common cause of asthma exacerbations in pregnancy, and infants of asthmatic mothers are at increased risk of asthma themselves. Further research is needed to elucidate the mechanisms involved.

Background The single-center double-blind, randomized controlled Managing Asthma in Pregnancy (MAP) trial in Newcastle, Australia, compared a treatment algorithm using the fraction of exhaled nitric oxide (FENO) in combination with asthma symptoms (FENO group) against a treatment algorithm using clinical symptoms only (clinical group) in pregnant asthmatic women (Australian New Zealand Clinical Trials Registry, no. 12607000561482). The primary outcome was a 50% reduction in asthma exacerbations during pregnancy in the FENO group. However, the effect of FENO-guided management on the development of asthma in the offspring is unknown. Objective We sought to investigate the effect of FENO-guided asthma management during pregnancy on asthma incidence in childhood. Methods A total of 179 mothers consented to participate in the Growing into Asthma (GIA) double-blind follow-up study with the primary aim to determine the effect of FENO-guided asthma management on childhood asthma incidence. Results A total of 140 children (78%) were followed up at 4 to 6 years of age. FENO-guided as compared to symptoms-only approach significantly reduced doctor-diagnosed asthma (25.9% vs 43.2%; odds ratio [OR], 0.46, 95% CI, 0.22-0.96; P = .04). Furthermore, frequent wheeze (OR, 0.27; 95% CI, 0.09-0.87; P = .03), use of short-acting β-agonists (OR, 0.49; 95% CI, 0.25-0.97; P = .04), and emergency department visits for asthma (OR, 0.17; 95% CI, 0.04-0.76; P = .02) in the past 12 months were less common in children born to mothers from the FENO group. Doctor-diagnosed asthma was associated with common risk alleles for early onset asthma at gene locus 17q21 ( P = .01 for rs8069176; P = .03 for rs8076131), and higher airways resistance ( P = .02) and FENO levels ( P = .03). A causal mediation analysis suggested natural indirect effects of FENO-guided asthma management on childhood asthma through "any use" and "time to first change in dose" of inhaled corticosteroids during the MAP trial (OR: 0.83; 95% CI: 0.59-0.99, and OR: 0.90; 95% CI: 0.70-1.03, respectively). Conclusions FENO-guided asthma management during pregnancy prevented doctor-diagnosed asthma in the offspring at preschool age, in part mediated through changes in use and dosing of inhaled corticosteroids during the MAP trial.

Summary Background Asthma is a complex disease that involves both genetic factors and environmental exposures. Aberrant epigenetic modifications, such as DNA methylation, may be important in asthma development. Fetal exposure to maternal asthma during critical periods of in utero development may lead to epigenetic alterations that predispose infants to a greater risk of developing asthma themselves. We investigated alterations in the DNA methylation profile of peripheral blood from infants exposed to maternal asthma during pregnancy. Methods Peripheral blood was collected from 12-month-old infants born to women with (n = 25) and without (n = 15) doctor diagnosed asthma during pregnancy. Genomic DNA was extracted, bisulfite converted, and hybridized to Infinium Methylation 27 arrays (Illumina), containing over27,000 CpGs from 14,495 genes. CpG loci in only autosomal genes were classified as differentially methylated at the 99% level (P 22 and delta beta >0.06). Results There were 70 CpG loci, corresponding to 67 genes that were significantly differentially methylated. Twelve CpG loci (11 genes) showed greater than 10% comparative difference in DNA methylation, including hyper-methylated loci of FAM181A, MRI1, PIWIL1, CHFR, DEFA1, MRPL28, AURKA, and hypo-methylated loci of NALP1L5, MAP8KIP3, ACAT2, and PM20D1 in maternal asthma. Methylation of MAPK8IP3 was significantly negatively correlated with maternal blood eosinophils (r = −0.38; P = 0.022), maternal eNO (r = −0.44; P = 0.005), and maternal serum total IgE (r = −0.39, P = 0.015). Methylation of AURKA negatively correlated with maternal hemoglobin (r = −0.43; P = 0.008), infants height (r = −0.51; P

Summary Chronic inflammatory diseases (e.g. asthma and chronic obstructive pulmonary disease) are leading causes of morbidity and mortality world-wide and effective treatments are limited. These disorders can often be attributed to abnormal immune responses to environmental stimuli and infections. Mechanisms leading to inflammation are complex, resulting from interactions of structural cells and activation of both the adaptive and innate arms of the immune system. The activation of structural and immune cells involves both temporary and permanent changes in gene expression in these cells, which underpin chronic inflammation and tissue dysfunction. miRNAs are small non-coding RNAs increasingly being recognized to play important roles in the post-transcriptional regulation of gene expression in mammalian cells by regulating translation. Individual miRNAs can exert their effects by directly inhibiting the translation or stability of multiple mRNAs simultaneously. Thus, the expression or blockade of function of a single miRNA (miR) can result in pronounced alterations in protein expression within a given cell. Dysregulation of miRNA expression may subsequently alter cellular function, and in certain situations predispose to disease. Our current understanding of the role of miRNA in the regulation of inflammatory disease (e.g. allergic diseases) remains limited. In this review, we provide an overview of the current understanding of miRNA biogenesis and function, the roles miRNA play in the regulation of immune cell function and their potential contribution to inflammatory diseases. We also highlight strategies to alter miRNA function for experimental or therapeutic gain, and discuss the potential utility and limitations of targeting these molecules as anti-inflammatory strategies.

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13(-/-) mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13(-/-) mice induced persistent AHR. Infection and associated pathology were restored in infected Il13(-/-) mice by reconstitution with IL-13. Stat6(-/-) mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.

Allergic airway inflammation is associated with activation of innate immune pathways by allergens. Acute exacerbations of asthma are commonly associated with rhinovirus infection. Here we show that, after exposure to house dust mite (HDM) or rhinovirus infection, the E3 ubiquitin ligase midline 1 (MID1) is upregulated in mouse bronchial epithelium. HDM regulates MID1 expression in a Toll-like receptor 4 (TLR4)- and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent manner. MID1 decreases protein phosphatase 2A (PP2A) activity through association with its catalytic subunit PP2Ac. siRNA-mediated knockdown of MID1 or pharmacological activation of PP2A using a nonphosphorylatable FTY720 analog in mice exposed to HDM reduces airway hyperreactivity and inflammation, including the expression of interleukin-25 (IL-25), IL-33 and CCL20, IL-5 and IL-13 release, nuclear factor (NF)κB activity, p38 mitogen-activated protein kinase (MAPK) phosphorylation, accumulation of eosinophils, T lymphocytes and myeloid dendritic cells, and the number of mucus-producing cells. MID1 inhibition also limited rhinovirus-induced exacerbation of allergic airway disease. We found that MID1 was upregulated in primary human bronchial epithelial cells upon HDM or rhinovirus exposure, and this correlated with TRAIL and CCL20 expression. Together, these findings identify a key role of MID1 in allergic airway inflammation and links innate immune pathway activation to the development and exacerbation of asthma.

Introduction: Steroid-insensitive (SI) asthma is of considerable clinical and economic significance and improved therapies are urgently required, however, the development of therapies has been hampered by a lack of understanding of the processes that underpin disease. Aim: To develop experimental models of SI asthma that represent different subtypes of clinical disease and use these models to elucidate universal drivers of SI asthma. Methods: Mouse models of Chlamydia , Haemophilus , influenza and respiratory syncytial virus (RSV) lung infections were superimposed with a model of steroid-sensitive ovalbumin (Ova)-induced allergic airways disease (AAD). The effects of infection on AAD and response to dexamethasone (DEX) treatment were examined. Results: We show that Chlamydia and Haemophilus infections drive neutrophil-dominated inflammation, while influenza and RSV infections drive eosinophil-dominated inflammation, in AAD. DEX does not suppress inflammation or airways hyper-responsiveness in all four models. These models of SIAAD represent neutrophil and eosinophil-enriched subtypes of SI asthma. Using gene expression and microRNA (miR) array analyses of these models we identified several factors that are universally dysregulated in SIAAD. Significantly, we show that suppression of miR-21 is effective for restoring steroid sensitivity in all four models. Conclusion: We have developed unique models of SI asthma and used these models to identify factors that are universally dysregulated in SI disease. These factors may represent novel targets for therapies that are broadly effective for the treatment of different subtypes of SI asthma in humans.

Background Ara h2 sIgE serum levels improve the diagnostic accuracy for predicting peanut allergy, but the use of Ara h2 purified protein as a skin prick test (SPT), has not been substantially evaluated. The fraction of exhaled nitric oxide (FeNO) shows promise as a novel biomarker of peanut allergy. Reproducibility of these measures has not been determined. The aim was to assess the accuracy and reproducibility (over a time-period of at least 12 months) of SPT to Ara h2 in comparison with four predictors of clinical peanut allergy (Peanut SPT, Ara h2 specific Immunoglobulin E (sIgE), Peanut sIgE and FeNO). Methods Twenty-seven children were recruited in a follow-up of a prospective cohort of fifty-six children at least 12 months after an open-labelled peanut food challenge. Their repeat assessment involved a questionnaire, SPT to peanut and Ara h2 purified protein, FeNO and sIgE to peanut and Ara h2 measurements. Results Ara h2 SPT was no worse in accuracy when compared with peanut SPT, FeNO, Ara h2 sIgE and peanut sIgE (AUC 0.908 compared with 0.887, 0.889, 0.935 and 0.804 respectively) for predicting allergic reaction at previous food challenge. SPT for peanut and Ara h2 demonstrated limited reproducibility (ICC = 0.51 and 0.44); while FeNO demonstrated good reproducibility (ICC = 0.73) and sIgE for peanut and Ara h2 were highly reproducible (ICC = 0.81 and 0.85). Conclusions In this population, Ara h2 SPT was no worse in accuracy when compared with current testing for the evaluation of clinical peanut allergy, but had—like peanut SPT—poor reproducibility. FeNO, peanut sIgE and Ara h2 sIgE were consistently reproducible despite an interval of at least 12 months between the repeated measurements. Electronic supplementary material The online version of this article (doi:10.1186/s13223-016-0143-z) contains supplementary material, which is available to authorized users.

© 2016 American Academy of Allergy, Asthma & Immunology Background Severe steroid-insensitive asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms of disease pathogenesis. Steroid-insensitive asthma is associated with respiratory tract infections and noneosinophilic endotypes, including neutrophilic forms of disease. However, steroid-insensitive patients with eosinophil-enriched inflammation have also been described. The mechanisms that underpin infection-induced, severe steroid-insensitive asthma can be elucidated by using mouse models of disease. Objective We sought to develop representative mouse models of severe, steroid-insensitive asthma and to use them to identify pathogenic mechanisms and investigate new treatment approaches. Methods Novel mouse models of Chlamydia, Haemophilus influenzae, influenza, and respiratory syncytial virus respiratory tract infections and ovalbumin-induced, severe, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed and interrogated. Results Infection induced increases in the levels of microRNA (miRNA)-21 (miR-21) expression in the lung during SSIAAD, whereas expression of the miR-21 target phosphatase and tensin homolog was reduced. This was associated with an increase in levels of phosphorylated Akt, an indicator of phosphoinositide 3-kinase (PI3K) activity, and decreased nuclear histone deacetylase (HDAC)2 levels. Treatment with an miR-21–specific antagomir (Ant-21) increased phosphatase and tensin homolog levels. Treatment with Ant-21, or the pan-PI3K inhibitor LY294002, reduced PI3K activity and restored HDAC2 levels. This led to suppression of airway hyperresponsiveness and restored steroid sensitivity to allergic airway disease. These observations were replicated with SSIAAD associated with 4 different pathogens. Conclusion We identify a previously unrecognized role for an miR-21/PI3K/HDAC2 axis in SSIAAD. Our data highlight miR-21 as a novel therapeutic target for the treatment of this form of asthma.

Introduction:Some asthmatics display impaired type 1 IFN responses and exaggerated inflammatory responses upon Rhinovirus (RV) infection, however, the molecular basis for these observations remain obscure despite intensive research. Recently, impaired production of antiviral molecules and IFN-induced cytokines have been described in asthmatics when peripheral bloodmononuclear cells (PBMC) were stimulated with the TLR7 agonist Imiquimod but not in response to the TLR3 agonist poly I : C (Roponen et al. ERJ 2009). Interestingly, some asthmatics display variations in their TLR7 genotype that may be related to impaired TLR7 function. Aim:To identify the molecular basis for the aberrant response to RV observed in asthmatics. Method:Bronchial biopsies were taken from well characterized asthmatics and healthy controls. Expression of key pathogen recognition receptors including TLR3 and 7 were enumerated using qPCR. WT and TLR7-/- BALB/c mouse models of RV1B induced exacerbation of house dust mite driven allergic airways disease were employed to determine the relevance of TLR7 signallingin antiviral responses and inflammation. Results:Expression of TLR7 was found to be significantly reduced in bronchial biopsies from eosinophilic but not neutrophilic asthmatics. TLR7-/- mice were found to have a deficient response to RV1B infection with reduced levels of type I and III interferons and elevated viral titre at 24 hr post infection when compared to wild type controls. Treatment with type I or type III interferons limited RV1B replication as well as impaired both neutrophilic and eosinophilic inflammation.Conclusion:Our study suggests deficient TLR7 function as a crucial mechanism underpinning impaired IFN responses to RV1B in allergic airways inflammation. Restoration of IFN not only reduced viral replication but also impaired RV1B-induced inflammation. These studies may indicate that augmentation of TLR7-regulated effector functions could be exploited as a novel therapeutic approach for the treatment of virus-induced asthma exacerbation in asthmatics with deficient IFN responses and/or eosinophilic inflammation.

To cite this article: Weckmann M, Kopp MV, Heinzmann A, Mattes J. Haplotypes covering the TNFSF10 gene are associated with bronchial asthma. Pediatric Allergy Immunology 2011: 22: 25–30. Abstract Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is elevated in the airways of subjects with asthma and has been linked to the development of allergic airway disease by promoting STAT6-dependent T helper 2 cell (TH2) effector functions. To determine whether single nucleotide polymorphisms (SNPs) in the TNFSF10 gene are associated with bronchial asthma we genotyped 498 Caucasian subjects living in Southern Germany for eight SNPs in the TNFSF10 by restriction fragment length polymorphism analysis. In contrast to single SNPs, haplotypes constructed from eight SNPs were robustly associated with asthma (p = 0.00012). A small haplotype approach returned four alleles consisting of two (rs3136586/ rs3136598), three (rs12488654/rs3136586/rs3136598 and rs3136586/rs3136598/rs3136604), and four SNPs (rs12488654/ rs3136586/ rs3136598/ rs3136604) that were highly associated with asthma (p = 0.00005, p = 0.00008, p = 0.00017 and p = 0.00038). Combinations of SNPs in the TNFSF10 allele were strongly associated with asthma supporting the concept that TRAIL is important in the development of hallmark features of this disease.

Allergic asthma is an inflammatory disease of the lung characterized by abnormal T helper-2 (T H 2) lymphocyte responses to inhaled antigens. The molecular mechanisms leading to the generation of T H 2 responses remain unclear, although toll-like receptors (TLRs) present on innate immune cells play a pivotal role in sensing molecular patterns and in programming adaptive T cell responses. Here we show that in vivo activation of TLR4 by house dust mite antigens leads to the induction of allergic disease, a process that is associated with expression of a unique subset of small, noncoding microRNAs. Selective blockade of microRNA (miR)-126 suppressed the asthmatic phenotype, resulting in diminished T H 2 responses, inflammation, airways hyperresponsiveness, eosinophil recruitment, and mucus hypersecretion. miR-126 blockade resulted in augmented expression of POU domain class 2 associating factor 1, which activates the transcription factor PU.1 that alters T H 2 cell function via negative regulation of GATA3 expression. In summary, this study presents a functional connection between miRNA expression and asthma pathogenesis, and our data suggest that targeting miRNA in the airways may lead to anti-inflammatory treatments for allergic asthma.

Summary 1. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cells while leaving most non-transformed cells unharmed. Binding of TRAIL to its death receptors (DR4 and DR5) activates the extrinsic apoptotic pathway by recruiting procaspase 8 into the death-inducing silencing complex. Cleavage of the BH-3 only peptide Bid by caspase 8 links the apoptotic TRAIL signal to the mitochondrial pathway and the subsequent release of cytochrome c. 2. In addition, TRAIL binds to neutralizing decoy receptors (DcR1 and DcR2). Signalling through DcR2, DR4 and DR5 can activate pro-inflammatory intracellular molecules such as mitogen-activated protein kinase, protein kinase B and nuclear factor-κB. 3. Recent studies have identified an important role for TRAIL in regulating immune responses to viruses, self-antigen and allergens. Increased concentrations of TRAIL are found in virus infections of the lung and TRAIL affects the antiviral response and resolution of infection. In addition, TRAIL is upregulated in the airways of asthmatics and inhibition results in reduced inflammation, T helper 2 cytokine and CCL20 release, as well as abolishing the development of airway hyperreactivity in experimental models. 4. Characterization of the specific receptor systems activated and the pro-inflammatory factors regulated by TRAIL in vivo may lead to the development of novel therapeutic strategies for diseases as diverse as infection, autoimmunity and asthma.

Rationale: One of the immunopathological features of allergic inflammation is the infiltration of helper T type 2 (Th2) cells to the site of disease. Activation of innate pattern recognition receptors such as Toll-like receptors (TLRs) plays a critical role in helper T type 1 cell differentiation, yet their contribution to the generation of Th2 responses to clinically relevant aeroallergens remains poorly defined.Objectives: To determine the requirement for TLR2, TLR4, and the Toll/IL-1 receptor domain adaptor protein MyD88 in a murine model of allergic asthma.Methods: Wild-type and factor-deficient (−/−) mice were sensitized intranasally to the common allergen house dust mite (HDM) and challenged 2 weeks later on four consecutive days. Measurements of allergic airway inflammation, T-cell cytokine production, and airway hyperreactivity were performed 24 hours later.Measurements and Main Results: Mice deficient in MyD88 were protected from the cardinal features of allergic asthma, including granulocytic in...

Chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory respiratory disorder, often induced by cigarette smoke (CS) exposure. The development of effective therapies is impaired by a lack of understanding of the underlining mechanisms. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with inflammatory and apoptotic properties. We interrogated a mouse model of CS-induced experimental COPD and human tissues to identify a novel role for TRAIL in COPD pathogenesis. CS exposure of wild-type mice increased TRAIL and its receptor messenger RNA (mRNA) expression and protein levels, as well as the number of TRAIL(+)CD11b(+) monocytes in the lung. TRAIL and its receptor mRNA were also increased in human COPD. CS-exposed TRAIL-deficient mice had decreased pulmonary inflammation, pro-inflammatory mediators, emphysema-like alveolar enlargement, and improved lung function. TRAIL-deficient mice also developed spontaneous small airway changes with increased epithelial cell thickness and collagen deposition, independent of CS exposure. Importantly, therapeutic neutralization of TRAIL, after the establishment of early-stage experimental COPD, reduced pulmonary inflammation, emphysema-like alveolar enlargement, and small airway changes. These data provide further evidence for TRAIL being a pivotal inflammatory factor in respiratory diseases, and the first preclinical evidence to suggest that therapeutic agents that target TRAIL may be effective in COPD therapy.

Pathogenic bacterial infections of the lung are life threatening and underpin chronic lung diseases. Current treatments are often ineffective potentially due to increasing antibiotic resistance and impairment of innate immunity by disease processes and steroid therapy. Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses. Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity. Moreover, inhibition of miR-328 in respiratory models of infection, steroid-induced immunosuppression, and smoke-induced emphysema enhances bacterial clearance. Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases.

© Schilter et al. Background and purpose: The persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A. Methods: Mice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A. Results: Selective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity. Conclusions and implications: This study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.

Asthma is prevalent in Western countries, and recent explanations have evoked the actions of the gut microbiota. Here we show that feeding mice a high-fibre diet yields a distinctive gut microbiota, which increases the levels of the short-chain fatty acid, acetate. High-fibre or acetate-feeding led to marked suppression of allergic airways disease (AAD, a model for human asthma), by enhancing T-regulatory cell numbers and function. Acetate increases acetylation at the Foxp3 promoter, likely through HDAC9 inhibition. Epigenetic effects of fibre/acetate in adult mice led us to examine the influence of maternal intake of fibre/acetate. High-fibre/acetate feeding of pregnant mice imparts on their adult offspring an inability to develop robust AAD. High fibre/acetate suppresses expression of certain genes in the mouse fetal lung linked to both human asthma and mouse AAD. Thus, diet acting on the gut microbiota profoundly influences airway responses, and may represent an approach to prevent asthma, including during pregnancy.

Asthma affects 10% of the population in Westernized countries, being most common in children. It is a heterogeneous condition characterized by chronic allergic airway inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR) to normally innocuous antigens. Combination therapies with inhaled corticosteroids and bronchodilators effectively manage mild to moderate asthma, but there are no cures, and patients with severe asthma do not respond to these treatments. The inception of asthma is linked to respiratory viral (respiratory syncytial virus, rhinovirus) and bacterial (Chlamydia, Mycoplasma) infections. The examination of mouse models of early-life infections and allergic airway disease (AAD) provides valuable insights into the mechanisms of disease inception that may lead to the development of more effective therapeutics. For example, early-life, but not adult, Chlamydia respiratory infections in mice permanently modify immunity and lung physiology. This increases the severity of AAD by promoting IL-13 expression, mucus hypersecretion, and AHR. We have identified novel roles for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and IL-13 in promoting infection-induced pathology in early life and subsequent chronic lung disease. Genetic deletion of TRAIL or IL-13 variously protected against neonatal infection-induced inflammation, mucus hypersecretion, altered lung structure, AHR, and impaired lung function. Therapeutic neutralization of these factors prevented infection-induced severe AAD. Other novel mechanisms and avenues for intervention are also being explored. Such studies indicate the immunological mechanisms that may underpin the association between early-life respiratory infections and the development of more severe asthma and may facilitate the development of tailored preventions and treatments.

Eosinophil degranulation is thought to play a pivotal role in the pathogenesis of allergic disorders. Although mouse models of allergic disorders have been used extensively to identify the contribution of eosinophils to disease, ultrastructural evidence of active granule disassembly has not been reported. In this investigation, we characterized the degree of eosinophil activation in the bone marrow, blood, lung tissue, and airways lumen [bronchoalveolar lavage fluid (BALF)] of ovalbumin-sensitized and aero-challenged wild-type and interleukin-5 transgenic mice. Degranulation was most prominent in and primarily compartmentalized to the airways lumen. Eosinophils released granule proteins by the process of piecemeal degranulation (PMD). Accordingly, recruitment and activation of eosinophils in the lung correlated with the detection of cell-free eosinophil peroxidase in BALF and with the induction of airways hyper-reactivity. As in previous studies with human eosinophils, degranulation of isolated mouse cells did not occur until after adherence to extracellular matrix. However, higher concentrations of exogenous stimuli appear to be required to trigger adherence and degranulation (piecemeal) of mouse eosinophils when compared with values reported for studies of human eosinophils. Thus, mouse eosinophils undergo PMD during allergic inflammation, and in turn, this process may contribute to pathogenesis. However, the degranulation process in the allergic lung of mice is primarily compartmentalized to the airway lumen. Understanding the mechanism of eosinophil degranulation in the airway lumen may provide important insights into how this process occurs in human respiratory diseases.

Background IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-γ and IL-13 release of CBMC of healthy neonates. Methods CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-γ, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 μg/ml) and an allergen-specific stimulus (BLG 25 μg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. Results IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-γ and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-γ, 6154; IL-13, 4357; BLG: IFN-γ, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-γ release (PHA: 6154; PHA + IL-18: 13474, p =0.0001; BLG: 801; BLG + IL-18: 1077, p =0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p =0.16) or BLG + IL-18 (124, p =0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. Conclusions IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-γ release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.

Summary Objective The aim of our study was to conduct a prospective investigation into the potential association of cord blood proliferative response and cytokine production in response to various stimuli on the development of atopic dermatitis (AD) at the age of 3 years. Methods Cord blood mononuclear cells (CBMC) from 40 healthy term neonates were isolated. The proliferative response of CBMC stimulated with IL-2, betalactoglobulin (BLG) and house dust mite allergen (Der p 1) was assessed by liquid scintillation counting and the stimulation index (SI) was calculated. The cytokines interleukin (IL-)13, interferon (IFN-)γ, IL-10 and IL-18 in the cell culture supernatants in response to phytohaemagglutinin (PHA), Der p 1 and BLG were measured using the ELISA technique. After 3 years, symptoms of AD were obtained with a questionnaire completed by the parents. Results We observed significantly higher IL-13 levels in response to PHA in children who subsequently developed symptoms of AD (S: median, 291 pg/mL) compared with asymptomatic children (No-S: 149 pg/mL; P=0.021, Wilcoxon test). Similarly, in response to Der p 1 significantly higher IL-13 levels were observed in symptomatic children (S: 168.6; No-S: 61.6 pg/mL; P=0.0084). In response to BLG, IL-13 levels were 287.2 (S) and 123.6 pg/mL (No-S; P=0.19). No significant differences were found when comparing the IFN-γ levels in CBMC cultures stimulated with PHA (S: 10.2; No-S: 17.6 IU/L; P=0.78), Der p 1 (S: 307.6; No-S: 616.2 IU/L; P=0.2) or BLG (S: 18; No-S: 28.5 IU/L; P=0.83; Fig. 2). The IL-18 and IL-10 levels and the stimulation index in response to IL-2, BLG and Der p 1 showed no significant difference between children who subsequently developed symptoms of AD and asymptomatic children. Figure 2. Release of interferon-gamma (IFN-γ) in response to phytohaemagglutinin (PHA), house dust mite allergen (Der p 1) and betalactoglobulin (BLG) in children who developed symptoms of atopic dermatitis at the age of 3 years (‘symptomatic’) and asymptomatic children. The median value and the 5th and 95th percentile are presented. Differences between the groups were calculated with the Wilcoxon test and P-values are reported. Conclusion Our data suggest that enhanced IL-13 levels at birth are associated with the subsequent development of atopic symptoms at the age of 3 years.

Currently most attempts at cancer immunotherapy involve the generation of CD8+ cytotoxic T lymphocytes (CTLs) against tumor-associated antigens. Many tumors, however, have been immunoselected to evade recognition by CTLs and thus alternative approaches to cancer immunotherapy are urgently needed. Here we demonstrate that CD4+ T cells that recognize a secreted tumor-specific antigen and exhibit a cytokine secretion profile characteristic of Th2 cells, are capable of clearing established lung and visceral metastases of a CTL-resistant melanoma. Clearance of lung metastases by the Th2 cells was found to be totally dependent on the eosinophil chemokine, eotaxin, and partially dependent on the transcription activator signal transducer and activator of transcription 6 (STAT6), with degranulating eosinophils within the tumors inducing tumor regression. In contrast, tumor-specific CD4+ Th1 cells, that recruited macrophages into the tumors, had no effect on tumor growth. This work provides the basis for a new approach to adoptive T cell immunotherapy of cancer.

Background: Atopy has been linked to chromosome 11q22, a region that harbors the IL18 gene. IL-18 enhances IL-4/IL-13 production and induces IgE production that is directly associated with the pathogenesis of atopic disorders. Objective: We sought to investigate whether genetic abnormalities in the regulatory regions of the IL18 gene predispose, in part, to susceptibility to atopy. Methods: Among a white population of 105 families, the oldest child was examined with regard to atopic phenotypes and single-nucleotide polymorphisms (SNPs) within the IL18 gene. Results: We have identified 5 novel SNPs in the IL18 gene (–920[t/c], –133[c/g], and –132[a/g] in promoter 2 [upstream of exon 2]; +179[c/a; Ser35Ser] in exon 4; and +486[c/t; Phe137Phe] in exon 6). Three SNPs are located in promoter 2, and one (–133[c/g]; nuclear factor 1 site) was significantly associated with high serum IgE levels ( P = .001; odds ratio, 3.96) and specific sensitization to common allergens ( P = .005; OR, 4.12). In addition, previously identified SNPs in exon 1 (+113[t/g] and +127[c/t]) and in promoter 1 (–137[g/c], GATA3 site) of the IL18 gene were significantly associated with high IgE levels ( P ≤ .005; OR, 3.27-3.90) and specific sensitization ( P = .02 to .008; OR, 3.27-3.83). The SNP +127(g/t) in exon 1 was also a susceptibility locus for seasonal allergic rhinitis ( P = .008; OR, 3.22). Conclusion: IL18 might be responsible for the linkage effects seen in the chromosomal region 11q22, which has been found previously with the phenotype "sensitization to mite allergen." Thus a suspected direct role of IL18 in the pathogenesis of atopy has been strengthened by the presence of 8 common SNPs in the promoter regions of IL18 . (J Allergy Clin Immunol 2003;111:117-22.)

Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease.