Cytokine profiles in asthma families depend on age and phenotype

Background

Circulating cytokine patterns may be relevant for the diagnosis of asthma, for the discrimination of certain phenotypes, and prognostic factors for exacerbation of disease.

Methodology/Principal Findings

In this study we investigated serum samples from 944 individuals of 218 asthma-affected families by a multiplex, microsphere based system detecting at high sensitivity eleven asthma associated mediators: eotaxin (CCL11), granulocyte macrophage stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-4 (IL-4), IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17 and tumor necrosis factor alpha (TNFα). Single cytokine levels were largely similar between asthmatic and healthy individuals when analysing asthma as single disease entity. Regulatory differences between parental and pediatric asthma were reflected by six of the eleven mediators analyzed (eotaxin, IL-4, IL-5, IL-10, IL-12, TNFα). IL-12 (p40) and IL-5 were the best predictor for extrinsic asthma in children with an increased odds ratio of 2.85 and 1.96 per log pg/ml increase (IL-12 (p40): 1.2–6.8, p = 0.019, and IL-5: 1.2–2.5, p = 0.025). Frequent asthma attacks in children are associated with elevated IL-5 serum levels (p = 0.013). Cytokine patterns seem to be individually balanced in both, healthy and diseased adults and children, with various cytokines correlating among each other (IL-17 and IFNγ (r_s = 0.67), IL-4 and IL-5 (r_s = 0.55), IFNγ and GM-CSF (r_s = 0.54)).

Conclusion/Significance

Our data support mainly an age- but also an asthma phenotype-dependent systemic immune regulation.     

Differential Dermal Expression of CCL17 and CCL18 in Tuberculoid and Lepromatous Leprosy

Background

Leprosy is characterized by polar clinical, histologic and immunological presentations. Previous immunologic studies of leprosy polarity were limited by the repertoire of cytokines known at the time.

Methodology

We used a candidate gene approach to measure mRNA levels in skin biopsies from leprosy lesions. mRNA from 24 chemokines and cytokines, and 6 immune cell type markers were measured from 85 Nepalese leprosy subjects. Selected findings were confirmed with immunohistochemistry.

Principal Results

Expression of three soluble mediators (CCL18, CCL17 and IL-10) and one macrophage cell type marker (CD14) was significantly elevated in lepromatous (CCL18, IL-10 and CD14) or tuberculoid (CCL17) lesions. Higher CCL18 protein expression by immunohistochemistry and a trend in increased serum CCL18 in lepromatous lesions was observed. No cytokines were associated with erythema nodosum leprosum or Type I reversal reaction following multiple comparison correction. Hierarchical clustering suggested that CCL18 was correlated with cell markers CD209 and CD14, while neither CCL17 nor CCL18 were highly correlated with classical TH1 and TH2 cytokines.

Conclusions

Our findings suggest that CCL17 and CCL18 dermal expression is associated with leprosy polarity.     

Differential effects of simvastatin on IL-13-induced cytokine gene expression in primary mouse tracheal epithelial cells

Background

Asthma causes significant morbidity worldwide in adults and children alike, and incurs large healthcare costs. The statin drugs, which treat hyperlipidemia and cardiovascular diseases, have pleiotropic effects beyond lowering cholesterol, including immunomodulatory, anti-inflammatory, and anti-fibrotic properties which may benefit lung health. Using an allergic mouse model of asthma, we previously demonstrated a benefit of statins in reducing peribronchiolar eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia, and lung IL-4 and IL-13 production.

Objectives

In this study, we evaluated whether simvastatin inhibits IL-13-induced pro-inflammatory gene expression of asthma-related cytokines in well-differentiated primary mouse tracheal epithelial (MTE) cell cultures. We hypothesized that simvastatin reduces the expression of IL-13-inducible genes in MTE cells.

Methods

We harvested tracheal epithelial cells from naïve BALB/c mice, grew them under air-liquid interface (ALI) cell culture conditions, then assessed IL-13-induced gene expression in MTE cells using a quantitative real-time PCR mouse gene array kit.

Results

We found that simvastatin had differential effects on IL-13-mediated gene expression (inhibited eotaxin-1; MCP-1,-2,-3; and osteopontin (SPP1), while it induced caspase-1 and CCL20 (MIP-3α)) in MTE cells. For other asthma-relevant genes such as TNF, IL-4, IL-10, CCL12 (MCP-5), CCL5 (RANTES), and CCR3, there were no significant IL-13-inducible or statin effects on gene expression.

Conclusions

Simvastatin modulates the gene expression of selected IL-13-inducible pro-inflammatory cytokines and chemokines in primary mouse tracheal epithelial cells. The airway epithelium may be a viable target tissue for the statin drugs. Further research is needed to assess the mechanisms of how statins modulate epithelial gene expression.     

Smoking in Asthma Is Associated with Elevated Levels of Corticosteroid Resistant Sputum Cytokines—An Exploratory Study

Background

Current cigarette smoking is associated with reduced acute responses to corticosteroids and worse clinical outcomes in stable chronic asthma. The mechanism by which current smoking promotes this altered behavior is currently unclear. Whilst cytokines can induce corticosteroid insensitivity in-vitro, how current and former smoking affects airway cytokine concentrations and their responses to oral corticosteroids in stable chronic asthma is unclear.

Objectives

To examine blood and sputum cytokine concentrations in never, ex and current smokers with asthma before and after oral corticosteroids.

Methods

Exploratory study utilizing two weeks of oral dexamethasone (equivalent to 40 mg/day prednisolone) in 22 current, 21 never and 10 ex-smokers with asthma. Induced sputum supernatant and plasma was obtained before and after oral dexamethasone. 25 cytokines were measured by multiplex microbead system (Invitrogen, UK) on a Luminex platform.

Results

Smokers with asthma had elevated sputum cytokine interleukin (IL) -6, -7, and -12 concentrations compared to never smokers with asthma. Few sputum cytokine concentrations changed in response to dexamethasone IL-17 and IFNα increased in smokers, CCL4 increased in never smokers and CCL5 and CXCL10 reduced in ex-smokers with asthma. Ex-smokers with asthma appeared to have evidence of an ongoing corticosteroid resistant elevation of cytokines despite smoking cessation. Several plasma cytokines were lower in smokers with asthma compared to never smokers with asthma.

Conclusion

Cigarette smoking in asthma is associated with a corticosteroid insensitive increase in multiple airway cytokines. Distinct airway cytokine profiles are present in current smokers and never smokers with asthma and could provide an explanatory mechanism for the altered clinical behavior observed in smokers with asthma.     

Hemolytic Streptococcus May Exacerbate Kidney Damage in IgA Nephropathy through CCL20 Response to the Effect of Th17 Cells

Background

The exacerbation of IgA nephropathy (IgAN) is related to respiratory tract infection with hemolytic streptococcus (HS), but the mechanism is unknown. In this study we investigated the role of chemokine ligand 20 (CCL20) in response to the effect of T helper 17 (Th17) cells in the pathogenesis of IgAN associated with HS.

Methods

Thirty mice were randomly divided into five groups: control mice (control), IgAN mice (IgAN), HS-infected IgAN mice (HS-IgAN), CCL20-treated IgAN mice (CCL20-IgAN), and CCL20-treated HS infected IgAN mice (CCL20-HS-IgAN). IgAN mice were induced with lipopolysaccharide, carbon tetrachloride and bovine serum albumin. Then the mice were sensitized with CCL20 antibody and infected with alpha-hemolytic streptococcus (α-HS) isolated from tonsils in sequence. Urine Albumin-Creatinine ratio and sediments were measured. The pathological changes in kidney and lung tissues were observed under microscopy. Th17 cells and regulatory T cells (Tregs) in kidneys were tested by flow cytometry. CCL20, IL-17A, IL-6 and IL-21 in the kidneys were detected by ELISA.

Results

The IgAN mice had albuminuria and microscopic hematuria, renal mesangial proliferation, IgA deposition, high electron dense deposition in glomerular mesangial region, decreased frequency of Tregs, increased frequency of Th17 and Th17-Treg ratio. Furthermore, Th17-related cytokines CCL20, IL-17A, IL-6 and IL-21 were all increased in the kidneys of IgAN mice. Compared with IgAN mice, the manifestations in HS-IgAN mice were more severe, but alleviated in CCL20-treated groups.

Conclusion

α-HS may exacerbate kidney damage in IgAN through CCL20 response to the effect of Th17 cells.     

Anti-inflammatory deficiencies in neutrophilic asthma: reduced galectin-3 and IL-1RA/IL-1β

Background

Galectin-3 (gal-3), a member of the β-galactoside-binding animal lectins, is involved in the recruitment, activation and removal of neutrophils. Neutrophilic asthma is characterized by a persistent elevation of airway neutrophils and impaired efferocytosis. We hypothesized that sputum gal-3 would be reduced in neutrophilic asthma and the expression of gal-3 would be associated with other markers of neutrophilic inflammation.

Methods

Adults with asthma (n = 80) underwent a sputum induction following clinical assessment and blood collection. Sputum was dispersed for a differential cell count and ELISA assessment of gal-3, gal-3 binding protein (BP), interleukin (IL)-1β, IL-1 receptor antagonist (RA), IL-8 and IL-6. Gal-3 and gal-3BP immunoreactivity were assessed in mixed sputum cells.

Results

Sputum gal-3 (median, (q1,q3)) was significantly reduced in neutrophilic asthma (183 ng/mL (91,287)) compared with eosinophilic (293 ng/mL (188,471), p = 0.021) and paucigranulocytic asthma (399 ng/mL (213,514), p = 0.004). The gal-3/gal-3BP ratio and IL-1RA/IL-1β ratio were significantly reduced, while gal-3BP and IL-1β were significantly elevated in neutrophilic asthma compared with eosinophilic and paucigranulocytic asthma.

Conclusion

Patients with neutrophilic asthma have impairment in anti-inflammatory ratio of gal-3/gal-3BP and IL-1RA/IL-1β which provides a further framework for exploration into pathologic mechanisms of asthma phenotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0163-5) contains supplementary material, which is available to authorized users.     

IL-9 Induces CCL11 Expression via STAT3 Signalling in Human Airway Smooth Muscle Cells

Background

Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood.

Methodology/Principal Findings

In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3β over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression.

Conclusion/Significance

Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.     

Nasal Epithelial Cells Can Act as a Physiological Surrogate for Paediatric Asthma Studies

Introduction

Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.

Methods

Paired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis.

Results

Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13.

Conclusions

We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.     

Differences in candidate gene association between European ancestry and African American asthmatic children

Background

Candidate gene case-control studies have identified several single nucleotide polymorphisms (SNPs) that are associated with asthma susceptibility. Most of these studies have been restricted to evaluations of specific SNPs within a single gene and within populations from European ancestry. Recently, there is increasing interest in understanding racial differences in genetic risk associated with childhood asthma. Our aim was to compare association patterns of asthma candidate genes between children of European and African ancestry.

Methodology/Principal Findings

Using a custom-designed Illumina SNP array, we genotyped 1,485 children within the Greater Cincinnati Pediatric Clinic Repository and Cincinnati Genomic Control Cohort for 259 SNPs in 28 genes and evaluated their associations with asthma. We identified 14 SNPs located in 6 genes that were significantly associated (p-values <0.05) with childhood asthma in African Americans. Among Caucasians, 13 SNPs in 5 genes were associated with childhood asthma. Two SNPs in IL4 were associated with asthma in both races (p-values <0.05). Gene-gene interaction studies identified race specific sets of genes that best discriminate between asthmatic children and non-allergic controls.

Conclusions/Significance

We identified IL4 as having a role in asthma susceptibility in both African American and Caucasian children. However, while IL4 SNPs were associated with asthma in asthmatic children with European and African ancestry, the relative contributions of the most replicated asthma-associated SNPs varied by ancestry. These data provides valuable insights into the pathways that may predispose to asthma in individuals with European vs. African ancestry.     

Sleep-disordered breathing and asthma: evidence from a large multicentric epidemiological study in China

Background

Previous studies have postulated that sleep-disordered breathing (SDB) may be associated with the occurrence and exacerbation of asthma. However, there was limited quantitative evidence on the topic. This study aimed at investigating the prevalence and predisposing factors of asthma, and quantifying the association between SDB and asthma among school-aged children in China. In addition, a comprehensive meta-analysis of the published evidences and our findings were further conducted.

Methods

To test the hypothesis, we conducted a multicentric cross-sectional study involving 22,478 children aged 5–12 years recruited from eight cities in China. Furthermore, a meta-analysis based on both previously published studies and our cross-sectional study was performed.

Results

The prevalence rate of SDB and asthma was 12.0% and 3.5% among our cross-sectional study sample. It was demonstrated that symptoms of SDB, such as habitual snoring (OR = 1.28, 95%CI: 1.01-1.62), and obstructive sleep apnea (OSA) (OR = 1.92, 95%CI: 1.34-2.76), were significantly associated with asthma, after adjusting for potential confounding factors. In the meta-analysis, SDB was correlated with the prevalence of asthma in both children (OR = 1.58, 95%CI: 1.35-1.80) and adults (OR = 1.55, 95%CI: 1.42-1.67).

Conclusions

Our results provide further evidence for the independent association between SDB and asthma. The clinical significance of our findings lies in the emphasis that children undergoing examination or treatment for asthma should be routinely screened for sleep problems. Further systematic study is required to illuminate the underlying mechanism.     

Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model

Background

Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.

Aim

We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice.

Methods

BALB/c mice were intranasally sensitized with 1 μg HDM on day 0 followed by daily intranasal challenge with PBS or 10 μg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 μg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected.

Results

Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue.

Conclusion

Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0171-0) contains supplementary material, which is available to authorized users.     

Food allergy enhances allergic asthma in mice

Background

Atopic march refers to the typical transition from a food allergy in early childhood to allergic asthma in older children and adults. However the precise interplay of events involving gut, skin and pulmonary inflammation in this process is not completely understood.

Objectives

To develop a mouse model of mixed food and respiratory allergy mimicking the atopic march and better understand the impact of food allergies on asthma.

Methods

Food allergy to ovalbumin (OVA) was induced through intra-peritoneal sensitization and intra-gastric challenge, and/or a respiratory allergy to house dust mite (HDM) was obtained through percutaneous sensitization and intra-nasal challenges with dermatophagoides farinae (Der f) extract. Digestive, respiratory and systemic parameters were analyzed.

Results

OVA-mediated gut allergy was associated with an increase in jejunum permeability, and a worsening of Der f-induced asthma with stronger airway hyperresponsiveness and pulmonary cell infiltration, notably eosinophils. There was overproduction of the pro-eosinophil chemokine RANTES in broncho-alveolar lavages associated with an enhanced Th2 cytokine secretion and increased total and Der f-specific IgE when the two allergies were present. Both AHR and lung inflammation increased after a second pulmonary challenge.

Conclusion

Gut sensitization to OVA amplifies Der f-induced asthma in mice.     

Role of Abl in airway hyperresponsiveness and airway remodeling

Background

Asthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.

Methods

To evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.

Results

The expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.

Conclusions

These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.     

Involvement of CCR6/CCL20/IL-17 axis in NSCLC disease progression

Objectives

Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.

Methods

A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.

Results

CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.

Conclusion

Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.     

Poverty,dirt, infections and non-atopic wheezing in children from a Brazilian urban center

Background

The causation of asthma is poorly understood. Risk factors for atopic and non-atopic asthma may be different. This study aimed to analyze the associations between markers of poverty, dirt and infections and wheezing in atopic and non-atopic children.

Methods

1445 children were recruited from a population-based cohort in Salvador, Brazil. Wheezing was assessed using the ISAAC questionnaire and atopy defined as allergen-specific IgE ≥0.70 kU/L. Relevant social factors, environmental exposures and serological markers for childhood infections were investigated as risk factors using multivariate multinomial logistic regression.

Results

Common risk factors for wheezing in atopic and non-atopic children, respectively, were parental asthma and respiratory infection in early childhood. No other factor was associated with wheezing in atopic children. Factors associated with wheezing in non-atopics were low maternal educational level (OR 1.49, 95% CI 0.98-2.38), low frequency of room cleaning (OR 2.49, 95% CI 1.27-4.90), presence of rodents in the house (OR 1.48, 95% CI 1.06-2.09), and day care attendance (OR 1.52, 95% CI 1.01-2.29).

Conclusions

Non-atopic wheezing was associated with risk factors indicative of poverty, dirt and infections. Further research is required to more precisely define the mediating exposures and the mechanisms by which they may cause non-atopic wheeze.     

Pooled genome-wide analysis to identify novel risk loci for pediatric allergic asthma

Background

Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach.

Methodology/Principal Findings

We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models.

Conclusion/Significance

Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population.     

Pathways activated during human asthma exacerbation as revealed by gene expression patterns in blood

Background

Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations.

Methodology/Principal Findings

Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations.

Conclusions/Significance

This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.     

Risk Factors for Asthma-Related Healthcare Use: Longitudinal Analysis Using the NHI Claims Database in a Korean Asthma Cohort

Background

Though insurance claims data are useful for researching asthma, they have important limitations, such as a diagnostic inaccuracy and a lack of clinical information. To overcome these drawbacks, we used the novel method by merging the clinical data from our asthma cohort with the National Health Insurance (NHI) claims data.

Methods and Results

Longitudinal analysis of asthma-related healthcare use from the NHI claims database, merged with data of 736 patients registered in a Korean asthma cohort, was conducted for three consecutive years from registration of the cohort. Asthma-related asthma healthcare referred to outpatient and emergency department visits, hospitalizations, and the use of systemic corticosteroids. Univariate and multivariate logistic regression analysis was used to evaluate risk factors for asthma-related healthcare. Over three years after enrollment, many patients changed from tertiary to primary/secondary hospitals with a lack of maintenance of inhaled corticosteroid-based controllers. An independent risk factor for emergency visits was a previous history of asthma exacerbation. In hospitalizations, old age and Asthma Control Test (ACT) score variability were independent risk factors. An independent risk factor for per person cumulative duration of systemic corticosteroids was the FEV₁ (Forced expiratory volume in one second)%. The use of systemic corticosteroids was independently associated with being female, the FEV₁%, and ACT score variability.

Conclusion

We found that old age, being female, long-standing asthma, a low FEV₁%, asthma brittleness, asthma drug compliance, and a history of asthma exacerbation were independent risk factors for increased asthma-related healthcare use in Korea.     

Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Background

Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective

To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods

Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results

OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions

Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.