TLR7/8 regulates type I and type III interferon signalling in rhinovirus 1b-induced allergic asthma.

INTRODUCTION: Interferon (IFN) responses have been reported to be defective in rhinovirus (RV)-induced asthma. The heterodimeric receptor of type I IFN (IFN-α/ß) is composed of IFN-αR1 and IFN-αR2. Ligand binding to the IFN-α/ß receptor complex activates signal transducer and activator of transcription (STAT) proteins STAT1 and STAT2 intracellularly. Although type III IFN (IFN-λ) binds to a different receptor containing IFN-λR1 and interleukin-10R2, its triggering leads to activation of the same downstream transcription factors. Here, we analysed the effects of RV on IFN type I and III receptors, and asked about possible Toll-like receptor 7/8 (TLR7/8) agonist R848-mediated IFN-αR1 and IFN-λR1 regulation. METHODS: We measured IFN-α, IFN-ß and IFN-λ and their receptor levels in peripheral blood mononuclear cell (PBMC) supernatants and cell pellets stimulated with RV1b and R848 in two cohorts of children with and without asthma recruited at pre-school age (PreDicta) and at primary school age (AGENDAS) as well as in cell supernatants from total lung cells isolated from mice. RESULTS: We observed that R848 induced IFN-λR mRNA expression in PBMCs of healthy and asthmatic children, but suppressed IFN-αR mRNA levels. In murine lung cells, RV1b alone and together with R848 suppressed IFN-αR protein in T-cells compared with controls and in total lung IFN-λR mRNA compared with RV1b infection alone. CONCLUSIONS: In PBMCs from pre-school age children, IFN-αR mRNA was reduced and IFN-λR1 mRNA was induced upon treatment with the TLR7/8 agonist R848, thus suggesting new avenues for induction of antiviral immune responses in paediatric asthma.

Clinical correlates of rhinovirus infection in preschool asthma.

BACKGROUND: Investigation of preschool asthma is important since not all children outgrow their illness during this age. Data are scarce on the role of rhinovirus (RV) infections in this patient group. OBJECTIVES: To investigate the role of RV infections in preschool asthma: (i) susceptibility factors, (ii) clinical course, and (iii) medium-term outcome. METHODS: A total of 130 asthmatic children aged 4-6 years from the multinational PreDicta cohort were prospectively followed for a 12-month period. Allergy tests and a standard health questionnaire were carried out at study entry. Respiratory virus presence in nasopharyngeal washes was studied at illness visits and at 3 scheduled visits. RESULTS: At study entry, mean age of the children was 5.3 years. Of 571 visits, 54% were positive for any virus and 39% for RV. Patient characteristics were only assessed with RV infection due to low number of other viruses. The use of supplementary vitamin D was inversely associated with RV infection (P < .05). RV infection was associated with more severe course of acute illness in terms of more severe nighttime coughing, more sleep disturbances, and more days with runny nose (all P < .05). RV infection was also associated with more severe disease course during the 12-month follow-up in terms of more nights with awakenings and more days of exercise-related symptoms (both P < .05). CONCLUSIONS: Vitamin D supplementation may have an anti-rhinovirus effect. Both short- and medium-term outcomes suggest RV infection to be an important clinical marker of instable preschool asthma.

Risk Factors for Complicated Lymphadenitis Caused by Nontuberculous Mycobacteria in Children.

Nontuberculous mycobacteria (NTM) are an emerging cause of infections, including chronic lymphadenitis in children. To identify risk factors for NTM lymphadenitis, particularly complicated disease, we collected epidemiologic, clinical, and microbiological data on 138 cases of NTM lymphadenitis in children across 13 centers in Germany and Austria. We assessed lifestyle factors but did not identify specific risk behaviors. We noted that more cases of NTM lymphadenitis occurred during cold months than during warm months. Moreover, we noted female sex and age <5.5 years as potential risk factors. Complete extirpation of the affected lymph node appeared to be the best therapeutic measure. We integrated the study data to develop a simple risk score to predict unfavorable clinical outcomes for NTM lymphadenitis.

Rhinovirus Species-Specific Antibodies Differentially Reflect Clinical Outcomes in Health and Asthma.

Rationale: Rhinoviruses (RVs) are major triggers of common cold and acute asthma exacerbations. RV species A, B, and C may have distinct clinical impact; however, little is known regarding RV species-specific antibody responses in health and asthma.Objectives: To describe and compare total and RV species-specific antibody levels in healthy children and children with asthma, away from an acute event.Methods: Serum samples from 163 preschool children with mild to moderate asthma and 72 healthy control subjects from the multinational Predicta cohort were analyzed using the recently developed PreDicta RV antibody chip.Measurements and Main Results: RV antibody levels varied, with RV-C and RV-A being higher than RV-B in both groups. Compared with control subjects, asthma was characterized by significantly higher levels of antibodies to RV-A and RV-C, but not RV-B. RV antibody levels positively correlated with the number of common colds over the previous year in healthy children, and wheeze episodes in children with asthma. Antibody levels also positively correlated with asthma severity but not with current asthma control.Conclusions: The variable humoral response to RV species in both groups suggests a differential infectivity pattern between RV species. In healthy preschoolers, RV antibodies accumulate with colds. In asthma, RV-A and RV-C antibodies are much higher and further increase with disease severity and wheeze episodes. Higher antibody levels in asthma may be caused by a compromised innate immune response, leading to increased exposure of the adaptive immune response to the virus. Importantly, there is no apparent protection with increasing levels of antibodies.

Contribution of repeated infections in asthma persistence from preschool to school age: Design and characteristics of the PreDicta cohort.

BACKGROUND: The PreDicta cohort was designed to prospectively evaluate wheeze/asthma persistence in preschoolers in association with viral/microbial exposures and immunological responses. We present the cohort design and demographic/disease characteristics and evaluate unsupervised and predefined phenotypic subgroups at inclusion. METHODS: PreDicta is a 2-year prospective study conducted in five European regions, including children 4-6 years with a diagnosis of asthma as cases and healthy age-matched controls. At baseline, detailed information on demographics, asthma and allergy-related disease activity, exposures, and lifestyle were recorded. Lung function, airway inflammation, and immune responses were also assessed. Power analysis confirmed that the cohort is adequate to answer the initial hypothesis. RESULTS: A total of 167 asthmatic children (102 males) and 66 healthy controls (30 males) were included. Groups were homogeneous in respect to most baseline characteristics, with the exception of male gender in cases (61%) and exposure to tobacco smoke. Comorbidities and number and duration of infections were significantly higher in asthmatics than controls. 55.7% of asthmatic children had at least one positive skin prick test to aeroallergens (controls: 33.3%, P = .002). Spirometric and exhaled nitric oxide values were within normal limits; only baseline FEV0.5 and FEV1 reversibility values were significantly different between groups. Viral infections were the most common triggers (89.2%) independent of severity, control, or atopy; however, overlapping phenotypes were also common. Severity and control clustered together in an unsupervised analysis, separating moderate from mild disease. CONCLUSIONS: The PreDicta cohort presented no differences in non-asthma related measures; however, it is well balanced regarding key phenotypic characteristics representative of "preschool asthma".

IFN-α/IFN-λ responses to respiratory viruses in paediatric asthma.

We analysed the influence of rhinovirus (RV) in nasopharyngeal fluid (NPF) on type I and III interferon (IFN) responses (e.g. IFN-α and IFN -: λ) and their signal transduction, at baseline and during disease exacerbation, in cohorts of pre-school children with and without asthma.At the time of recruitment into the Europe-wide study PreDicta, and during symptoms, NPF was collected and the local RV colonisation was analysed. Peripheral blood mononuclear cells (PBMCs) were challenged in vitro with RV or not. RNA was analysed by quantitative real-time PCR and gene arrays. Serum was analysed with ELISA for IFNs and C-reactive protein.We found that PBMCs from asthmatic children infected in vitro with the RV1b serotype upregulated MYD88, IRF1, STAT1 and STAT2 mRNA, whereas MYD88, IRF1, STAT1 and IRF9 were predominantly induced in control children. Moreover, during symptomatic visits because of disease exacerbation associated with RV detection in NPF, IFN-α production was found increased, while IFN-λ secretion was already induced by RV in asthmatic children at baseline.During asthma exacerbations associated with RV, asthmatic children can induce IFN-α secretion, indicating a hyperactive immune response to repeated respiratory virus infection.

The activating protein 1 transcription factor basic leucine zipper transcription factor, ATF-like (BATF), regulates lymphocyte- and mast cell-driven immune responses in the setting of allergic asthma.

BACKGROUND: Mice without the basic leucine zipper transcription factor, ATF-like (BATF) gene (Batf(-/-)) lack TH17 and follicular helper T cells, which demonstrates that Batf is a transcription factor important for T- and B-cell differentiation. OBJECTIVE: In this study we examined whether BATF expression would influence allergic asthma. METHODS: In a cohort of preschool control children and children with asthma, we analyzed BATF mRNA expression using real-time PCR in PBMCs. In a murine model of allergic asthma, we analyzed differences in this allergic disease between wild-type, Batf transgenic, and Batf(-/-) mice. RESULTS: In the absence of corticosteroid treatment, children with recurrent asthma have a significant increase in BATF mRNA expression in their PBMCs. Batf(-/-) mice display a significant reduction in the pathophysiologic responses seen in asthmatic wild-type littermates. Moreover, we discovered a decrease in IL-3 production and IL-3-dependent mast cell development in Batf(-/-) mice. By contrast, IFN-γ was induced in lung CD4(+) and CD8(+) T cells. Intranasal delivery of anti-IFN-γ antibodies induced airway hyperresponsiveness and inflammation in wild-type but not in Batf(-/-) mice. Transgenic overexpression of Batf under the control of the CD2 promoter/enhancer augmented lung inflammation and IgE levels in the setting of experimental asthma. CONCLUSION: BATF is increased in non-steroid-treated asthmatic children. Targeting BATF expression resulted in amelioration of the pathophysiologic responses seen in children with allergic asthma, and BATF has emerged as a novel target for antiasthma interventions.

Early respiratory and ocular involvement in X-linked hypohidrotic ectodermal dysplasia.

UNLABELLED: X-linked hypohidrotic ectodermal dysplasia (XLHED; ectodysplasin deficiency) has been classically described as affecting hair, sweat glands, and dentition. What may be underappreciated is the effect ectodysplasin deficiency has on glands surrounding the airways and eyes and the resulting chronic health issues. In this study, 12 male children (age range 6-13 years) and 14 male adults with XLHED (18-58 years of age) were investigated by pulmonary function tests, measurement of fractional exhaled nitric oxide, and by ophthalmologic assessments. Twelve healthy individuals (six children, six adults) served as controls. Signs of airway constriction and inflammation were detected in eight children with XLHED, including the youngest subject, and in ten adult XLHED patients. Increased tear osmolarity, reduced tear film break-up time, and other ocular abnormalities were also present at an early age. Five of 12 XLHED subjects not reporting a history of asthma and 7 of the 12 patients not reporting a history of dry eye issues showed at least two abnormal test results in the respective organ system. The presence of residual sweat ducts, suggestive of partial ectodysplasin gene expression, correlated with milder disease in two XLHED subjects with mutations affecting the collagen-like domain of ectodysplasin. CONCLUSION: The high prevalence of asthma-like symptoms in XLHED patients as young as 6 years and a similar prevalence of dry eye problems indicate that screening evaluation, regular monitoring, and consideration of therapeutic intervention should begin in early childhood.

Vitamin D3 resolved human and experimental asthma via B lymphocyte-induced maturation protein 1 in T cells and innate lymphoid cells.

Background: Vitamin D3 (VitD3) is known to have immunomodulatory functions, and VitD3 deficiency is associated with more severe asthma. Objective: We aimed to assess the immunoregulatory effects of VitD3 food supplementation on asthma manifestation, with particular focus on T cells and type 2 innate lymphoid cells. Methods: Preschool children and adult asthmatic cohorts were analyzed in the context of VitD3 supplementation and serum levels. In a murine model of ovalbumin-induced asthma, effects of diet VitD3 sufficiency and deficiency on T cells and type 2 innate lymphoid cells immune mechanisms were investigated. Results: We found less severe and better-controlled asthma phenotypes along with reduced need for steroid medication in preschool children and asthmatic adults with VitD3 supplementation. VitD3 serum levels correlated with B lymphocyte-induced maturation protein 1 (Blimp-1) expression in blood peripheral mononuclear cells. VitD3-supplement-fed mice showed decreased asthmatic traits, with a decrease in IgE serum levels, reduced airway mucus, and increased IL-10 production by lung cells. Furthermore, we discovered an upregulation of effector T cells and Blimp-1+ lung tissue-resident memory T cells as well as induction of anti-inflammatory Blimp-1+ lung innate lymphoid cells producing IL-10. Conclusion: Supplementing VitD3 resulted in amelioration of clinical asthma manifestations in human studies as well as in experimental allergic asthma, indicating that VitD3 shifts proinflammatory immune responses to anti-inflammatory immune responses via upregulating Blimp-1 in lung innate lymphoid cells and tissue-resident memory cells.

An Immunoregulatory Role of Interleukin-3 in Allergic Asthma.

Background: Allergic asthma is a chronic airway inflammatory disease associated with airway mucus hyper-production. ILC2 cells, which express the Th2 transcription factor GATA3, have been associated with allergic asthma. The cytokine IL-3 is known to support eosinophil, basophil and mucosal mast cell differentiation and survival; however, its role on T regulatory cells as well as on lung ILC2 and in pediatric asthma needs further investigation. Objectives: To investigate the role of IL-3 in preschool children and to explore its therapeutic role in experimental asthma. Methods: In a cohort of preschool children with and without asthma, we analyzed the secretion of IL-3 in nasopharyngeal fluid (NPF) and IL-3 receptor (R) alpha chain mRNA expression in peripheral blood mononuclear cells (PBMCs). In a murine model of allergic asthma, we analyzed the phenotype of wild-type untreated and rIL-3 intranasally treated asthmatic mice. Results: IL-3 was found downregulated in the nasopharyngeal fluid of children with partially controlled asthma, as compared to control children. Moreover, IL-3 was found induced in phytohemagglutinin (PHA)-stimulated PBMCs from children with asthma and treated with steroids. Finally, IL-3 in NPF directly correlated with the anti-inflammatory molecule sST2 in steroid-treated asthmatic children. Intranasal rIL-3 delivery in vivo during the challenge phase decreased airway mucus production and inflammatory eosinophils. Moreover, rIL-3 given during the challenge phase, reduced lung ST2intGATA3+ILC2, accompanied by an induction of T regulatory cells in the airways. Conclusions: IL-3 was found associated with steroid-resolved asthma. Moreover, treatment with rIL-3 resulted in amelioration of airway eosinophilia and mucus production, two main pathophysiological conditions associated with asthma in a murine model of allergic asthma. Thus, rIL-3 opens new strategies for immunotherapy of this disease.

Targeted deletion of Interleukin-3 results in asthma exacerbations.

The cytokine interleukin-3 (IL-3) acts on early hematopoietic precursor cells. In humans, Treg cells secrete IL-3 and repress inflammatory cells except for basophils. The present study aims to elucidate the contribution of IL-3 in the development and the course of allergic asthma. We therefore analyzed the secretion of IL-3 in PBMCs and total blood cells in two cohorts of pre-school children with and without asthma. In a murine model of allergic asthma, we analyzed the phenotype of IL-3-/- mice compared to wild-type mice. PBMCs from asthmatic children showed increased IL-3 secretion, which directly correlated with improved lung function. IL-3-/- asthmatic mice showed increased asthmatic traits. Moreover, IL-3-deficient mice had a defect in T regulatory cells in the lung. In conclusion, IL-3 downregulation was found associated with more severe allergic asthma in pre-school children. Consistently, targeting IL-3 resulted in an induced pathophysiological response in a murine model of allergic asthma.

Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) drives the resolution of allergic asthma.

RANTES is implicated in allergic asthma and in T cell-dependent clearance of infection. RANTES receptor family comprises CCR1, CCR3, and CCR5, which are G-protein-coupled receptors consisting of seven transmembrane helices. Infections with respiratory viruses like Rhinovirus cause induction of RANTES production by epithelial cells. Here, we studied the role of RANTES in the peripheral blood mononuclear cells in cohorts of children with and without asthma and validated and extended this study to the airways of adults with and without asthma. We further translated these studies to a murine model of asthma induced by house dust mite allergen in wild-type RANTES and CCR5-deficient mice. Here we show an unpredicted therapeutic role of RANTES in the resolution of allergen-induced asthma by orchestrating the transition of effector GATA-3+CD4+ T cells into immune-regulatory-type T cells and inflammatory eosinophils into resident eosinophils as well as increased IL-10 production in the lung.

Respiratory infections regulated blood cells IFN-ß-PD-L1 pathway in pediatric asthma.

BACKGROUND: Respiratory infections, in general, and rhinovirus infection specifically are the main reason for asthma exacerbation in children and programmed cell death protein 1 ligand (PD-L1) expression inhibits T cell responses. OBJECTIVE: Could the interferon (IFN) type I expression in peripheral blood mononuclear cells (PBMCs) improve disease exacerbation in pediatric asthma? RESULTS: Here we found increased level of PD-L1 messenger RNA (mRNA) in total blood cells isolated from preschool children with virus-induced asthma, with lower percentage of forced expiratory volume in 1 second and with high serum levels of the C-reactive-protein. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that, in the presence of infection in the airways of preschool children, worse asthma is associated with induced PD-L1 mRNA expression in blood cells. Further, type I IFN, IFN-ß, a cytokine that is involved in the clearance of infections, was found to be associated with a better lung function in asthmatic children. These data suggest that improving peripheral blood IFN type I expression in PBMCs in pediatric asthma could improve disease exacerbation due to suppressing PD-L1 expression in blood cells.

Role of nuclear factor of activated T cells 2 (NFATc2) in allergic asthma.

BACKGROUND: We recently described increased NFATc1, IRF4, and NIP45 messenger RNA (mRNA) expression in peripheral blood mononuclear cells (PBMCs) of asthmatic children and adults with multiple allergies. OBJECTIVE: NFATc2 has been described to associate with IRF4 to induce interleukin-4, and to be inhibited by T-bet. Here, we analyzed the role of NFATc2 in asthmatic children and adults. METHODS: PBMCs were isolated from the blood of control of asthmatics subjects. Some PBMCs were analyzed untreated and some cultured with and without phytohemagglutinin. Then, RNA was extracted from the cells and cytokines were measured in the supernatants via enzyme-linked immunosorbent assay or multiplex analysis. RNA was then reverse-transcribed and NFATc1, NFATC2, IRF4, and T-bet mRNA were analyzed by real-time polymerase chain reaction. In addition, in peripheral blood cells, NFATc2 expression was analyzed, in a population of asthmatic children and adults from the Asthma BRIDGE study. RESULTS: In addition to NFATc1 and NIP45, also NFATc2 was found upregulated in PBMCs and peripheral blood cells from asthmatic children and adults with allergic asthma. Moreover, NFATc1 directly correlated with lymphocytes number whereas NFATc2 correlated with peripheral eosinophilia in asthma. CONCLUSIONS: In addition to NFATc1 and NIP45, NFATc2 was found upregulated in asthma. Moreover, NFATc1 mRNA correlated with lymphocytes both in control and asthma, and NFATC1 and NFATc2 mRNA showed a direct correlation with eosinophils in controls but not in asthma, indicating that NFATc1 is associated with lymphocytes and not eosinophils in asthma. CLINICAL SIGNIFICANCE: Targeting NFATc2 in T lymphocytes might ameliorate the allergic phenotype in asthmatic subjects.

Evolution of Airway Inflammation in Preschoolers with Asthma-Results of a Two-Year Longitudinal Study.

Fractional exhaled nitric oxide (FeNO) is a non-invasive marker for eosinophilic airway inflammation and has been used for monitoring asthma. Here, we assess the characteristics of FeNO from preschool to school age, in parallel with asthma activity. A total of 167 asthmatic children and 66 healthy, age-matched controls were included in the 2-year prospective PreDicta study evaluating wheeze/asthma persistence in preschool-aged children. Information on asthma/rhinitis activity, infections and atopy was recorded at baseline. Follow-up visits were performed at 6-month intervals, as well as upon exacerbation/cold and 4-6 weeks later in the asthmatic group. We obtained 539 FeNO measurements from asthmatics and 42 from controls. At baseline, FeNO values did not differ between the two groups (median: 3.0 ppb vs. 2.0 ppb, respectively). FeNO values at 6, 12, 18 and 24 months (4.0, CI: 0.0-8.6; 6.0, CI: 2.8-12.0; 8.0, CI: 4.0-14.0; 8.5, CI: 4.4-14.5 ppb, respectively) increased with age (correlation p ≤ 0.001) and atopy (p = 0.03). FeNO was non-significantly increased from baseline to the symptomatic visit, while it decreased after convalescence (p = 0.007). Markers of disease activity, such as wheezing episodes and days with asthma were associated with increased FeNO values during the study (p < 0.05 for all). Age, atopy and disease activity were found to be important FeNO determinants in preschool children. Longitudinal and individualized FeNO assessment may be valuable in monitoring asthmatic children with recurrent wheezing or mild asthma.

Tracheostomy in young patients: indications and long-term outcome.

Diagnostic and treatment modalities have changed substantially over the past years in the field of pediatrics and neonatal medicine. As a result, the indications and outcome after tracheostomy in young patients have evolved. The aim of this study is to present our experience with pediatric tracheostomies and provide an up-to-date review of the literature with special focus on current trends. The complete medical records of 85 children and adolescents (up to age 18) which underwent tracheostomy from January 1990 until March 2008 were reviewed. Telephone interviews were conducted to evaluate the childrens further clinical course. The indications for tracheostomy were upper airway obstruction (27%), craniofacial syndromes (3.5%), long-term mechanical ventilation (22.3%), neurological deficit (25.9%), trauma and sequelae (16.5%) and bilateral vocal cord paralysis (4.7%). The average age of patients at the time of tracheostomy was 4.7 years (range, 2 days-18 years) but there were significant differences between the six indication groups. Children under the age of 7 years comprised 72.9% of all patients. The mean cannulation time was 21.6 months; 50.6% of the patients could be successfully decannulated. Life-threatening complications occurred in 6 patients (7%). The total mortality rate was 18.8%; the tracheostomy related mortality rate was 0%. In the past 30 years, short-term tracheostomy was commonly performed for infectious causes such as epiglottitis. Nowadays, the majority of patients are very young children with severe and chronic diseases. This fact accounts for the relatively low decannulation rates, long cannulation times and high mortality. The tracheostomy related mortality on the other hand, is comparatively low.