Ellen Dilissen

IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?

BackgroundThe role of Th2 cells (producing interleukin (IL-)4, IL-5 and IL-13) in allergic asthma is well-defined. A distinct proinflammatory T cell lineage has recently been identified, called Th17 cells, producing IL-17A, a cytokine that induces CXCL8 (IL-8) and recruits neutrophils. Neutrophilic infiltration in the airways is prominent in severe asthma exacerbations and may contribute to airway gland hypersecretion, bronchial hyper-reactivity and airway wall remodelling in asthma.Aimto study the production of IL-17 in asthmatic airways at the mRNA level, and to correlate this with IL-8 mRNA, neutrophilic inflammation and asthma severity.MethodsWe obtained airway cells by sputum induction from healthy individuals (n = 15) and from asthmatic patients (n = 39). Neutrophils were counted on cytospins and IL-17A and IL-8 mRNA expression was quantified by real-time RT-PCR (n = 11 controls and 33 asthmatics).ResultsSputum IL-17A and IL-8 mRNA levels are significantly elevated in asthma patients compared to healthy controls. IL-17 mRNA levels are significantly correlated with CD3γ mRNA levels in asthmatic patients and mRNA levels of IL-17A and IL-8 correlated with each other and with sputum neutrophil counts. High sputum IL-8 and IL-17A mRNA levels were also found in moderate-to-severe (persistent) asthmatics on inhaled steroid treatment.ConclusionThe data suggest that Th17 cell infiltration in asthmatic airways links T cell activity with neutrophilic inflammation in asthma.

Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients

Background: Asthma is a chronic inflammatory disorder of the airways driven by T cell activation. Th2 cells and their cytokines are thought to play a role in the pathophysiology of allergic as well as non-allergic asthma. Methods: Airway cells were obtained by sputum induction from 15 healthy and 39 asthmatic individuals and the airway T cell cytokine profiles (interleukin (IL)-4, IL-5, IL-13, IL-10 and interferon (IFN)-γ) at the mRNA level were studied by real time RT-PCR. Results: Asthma patients had increased expression of IL-5 (p = 0.001) and IL-13 (p = 0.03) mRNA in sputum compared with non-asthmatic controls. IL-4 mRNA and IFN-γ mRNA were detectable in the sputum of 44% and 21% of patients, respectively, but not in controls. Sputum IL-10 mRNA levels did not differ significantly between patients and controls. Sputum mRNA expression levels of IL-4, IL-5, and IL-13 were significantly correlated with the percentage of eosinophils and were higher in subjects with allergic asthma than in those with non-allergic asthma (p = 0.03, p = 0.02 and p = 0.0002, respectively); they did not differ between mild asthmatic subjects and those with moderate to severe asthma. In contrast, IFN-γ mRNA expression was higher in non-allergic than in allergic patients (p = 0.04) and higher in patients with moderate to severe asthma than in those with mild asthma (p<0.01). Sputum IL-5 mRNA levels (but not the other cytokine mRNA levels) were also correlated with exhaled nitric oxide (eNO) and with bronchial hyperreactivity expressed as the histamine concentration resulting in a 20% decrease in forced expiratory volume in 1 second. Conclusion: Real time RT-PCR analysis of mRNA in induced sputum confirms a predominance of Th2 cytokines in both allergic and non-allergic asthma. IL-5 levels reflect eosinophil infiltration as well as eNO levels and hyperreactivity, and levels of the Th1 cytokine IFN-γ indicate asthma severity. The technique is a promising tool for use in further studies of asthma severity and disease activity.

Effects of high altitude and cold air exposure on airway inflammation in patients with asthma

Aims Eighteen patients with asthma were evaluated during preparation to climb to extreme altitude in order to study the effects of low fractional inspired oxygen (FiO2), prolonged exposure to cold air and high altitude on lung function, asthma control and airway inflammation. Methods Spirometry and airway inflammation (fractional exhaled nitric oxide (FeNO) and induced sputum) were studied under different test conditions: hypoxic (FiO2=11%) exercise test, 24-hour cold exposure (−5°C) and before, during and after an expedition that involved climbing the Aconcagua mountain (6965 m). Results Forced expiratory volume in 1 s (FEV1) and FeNO values were slightly lower (p<0.04) after 1 h of normobaric hypoxia. FEV1 decreased (p=0.009) after 24-hour cold exposure, accompanied by increased sputum neutrophilia (p<0.01). During the expedition FEV1 and forced vital capacity decreased (maximum FEV1 decrease of 12.3% at 4300 m) and asthma symptoms slightly increased. After the expedition the Asthma Control Test score and prebronchodilator FEV1 were reduced (p<0.02), sputum neutrophil count was increased (p=0.04) and sputum myeloperoxidase levels, sputum interleukin 17 mRNA, serum and sputum vascular endothelial growth factor A levels were significantly higher compared with baseline. Patients with asthma with the lowest oxygen saturation during the hypoxic exercise test were more prone to develop acute mountain sickness. Conclusions Exposure to environmental conditions at high altitude (hypoxia, exercise, cold) was associated with a moderate loss of asthma control, increased airway obstruction and neutrophilic airway inflammation. The cold temperature is probably the most important contributing factor as 24-hour cold exposure by itself induced similar effects.

Piliation of Lactobacillus rhamnosus GG Promotes Adhesion, Phagocytosis, and Cytokine Modulation in Macrophages

ABSTRACT Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.

Development of siRNA-loaded chitosan nanoparticles targeting Galectin-1 for the treatment of glioblastoma multiforme via intranasal administration

Galectin-1 (Gal-1) is a naturally occurring galactose-binding lectin, which is overexpressed in glioblastoma multiforme (GBM). Gal-1 is associated with tumor progression, and is a potent immune suppressor in the tumor micro-environment. To inhibit Gal-1 in GBM, an effective therapy is required that reaches the central nervous system tumor, with limited systemic effects. In this study, we report for the first time that concentrated chitosan nanoparticle suspensions can deliver small interfering RNA (siRNA) into the central nervous system tumor within hours after intranasal administration. These nanoparticles are able to complex siRNA targeting Gal-1 to a high percentage, and protect them from RNAse degradation. Moreover, a successful intracellular delivery of anti-Gal-1 siRNA resulted in a decreased expression of Gal-1 in both murine and human GBM cells. Sequence specific RNAinterference, resulted in more than 50% Gal-1 reduction in tumor bearing mice. This study indicates that the intranasal pathway is an underexplored transport route for delivering siRNA-based therapies targeting Gal-1 in the treatment of GBM.

Obese Individuals with Asthma Preferentially Have a High IL-5/IL-17A/IL-25 Sputum Inflammatory Pattern

1. Desai D, Newby C, Symon FA, Haldar P, Shah S, Gupta S, Bafadhel M, Singapuri A, Siddiqui S, Woods J, et al. Elevated sputum interleukin-5 and submucosal eosinophilia in obese individuals with severe asthma. Am J Respir Crit Care Med 2013;188: 657–663. 2. Haldar P, Pavord ID, Shaw DE, Berry MA, Thomas M, Brightling CE, Wardlaw AJ, Green RH. Cluster analysis and clinical asthma phenotypes. Am J Respir Crit Care Med 2008;178:218–224. 3. Broekema M, Timens W, Vonk JM, Volbeda F, Lodewijk ME, Hylkema MN, Ten Hacken NH, Postma DS. Persisting remodeling and less airway wall eosinophil activation in complete remission of asthma. Am J Respir Crit Care Med 2011;183:310–316. 4. Volbeda F, Broekema M, Lodewijk ME, Hylkema MN, Reddel HK, Timens W, Postma DS, ten Hacken NH. Clinical control of asthma associates with measures of airway inflammation. Thorax 2013;68: 19–24. 5. Broekema M, ten Hacken NH, Volbeda F, Lodewijk ME, Hylkema MN, Postma DS, Timens W. Airway epithelial changes in smokers but not in ex-smokers with asthma. Am J Respir Crit Care Med 2009;180: 1170–1178. 6. Global Initiative for Asthma. Guidelines [updated 2012 Dec]. Available from: www.ginasthma.org 7. van Veen IH, Ten Brinke A, Sterk PJ, Rabe KF, Bel EH. Airway inflammation in obese and nonobese patients with difficult-to-treat asthma. Allergy 2008;63:570–574.

Airway inflammation in patients with chronic non-asthmatic cough

Introduction Chronic non-asthmatic cough (CC) is a clinical challenge and underlying pathophysiological mechanisms remain still not completely understood. One of the most common comorbidities in CC is gastro-oesophageal reflux disease (GORD). Airway epithelium damage can contribute to airway inflammation in CC. Aims We studied airway inflammation in patients with CC compared to healthy controls. Patients with GORD were treated with proton pump inhibitors (PPI) and cough response to PPI was evaluated. Patients and methods Sputum was induced in 41 adults with CC and 20 healthy non-smokers who were age and sex matched. We compared sputum differential cell count by cytospin and cytokine and chemokine production at the mRNA and/or protein levels by real-time (RT)-PCR and cytokine bead array (CBA), between patients with CC and healthy subjects. Furthermore we studied airway inflammation in patients with different comorbidities. Results No differences in sputum differential cell counts were observed between patients with CC and healthy subjects. Sputum monocyte chemoattractant protein-1 (MCP-1) protein levels were significantly higher in patients when compared to controls. Thymic stromal lymphopoietin (TSLP) mRNA was significantly more often expressed in sputum of patients with CC than from healthy controls. Sputum transforming growth factor (TGF)-β levels did not differ between patients and controls, but were significantly lower in the PPI responders compared to the non-responders; p=0.047. There is no evidence for impaired T helper cell (Th)1/Th2/Th17 balance in CC. Patients with reflux oesophagitis (RO) have significantly more sputum eosinophils than patients without RO. Conclusions CC is a condition presenting with different disease phenotypes. High sputum MCP-1 levels are present in a large group of patients with CC and a majority of these patients with CC have increased sputum TSLP levels, most likely produced by damaged airway epithelial cells.

Feasibility to apply eucapnic voluntary hyperventilation in young elite athletes.

INTRODUCTION Exercise-induced bronchoconstriction (EIB) is more common in athletes compared to the general population. The eucapnic voluntary hyperventilation test is used to detect EIB in adult athletes. It is however unclear whether this technique is also applicable to young athletes. METHODS Young athletes (basketball (n = 13), football (n = 19), swimming (n = 12)) were recruited at the start of their elite sports career (12-14 years). Eight age-matched controls were also recruited. Eucapnic voluntary hyperventilation test was performed according to ATS guidelines in all subjects. A second (after 1 year, n = 32) and third (after 2 years, n = 39) measurement was performed in a subgroup of athletes and controls. RESULTS At time of first evaluation, 3/13 basketball players, 4/19 football players, 5/11 swimmers and 1/8 controls met criteria for EIB (fall in FEV1≥10% after EVH). A ventilation rate of >85% of the maximal voluntary ventilation (MVV) is recommended by current guidelines (for adults) but was only achieved by a low number of individuals (first occasion: 27%, third occasion: 45%) However, MVV in young athletes corresponds to 30 times FEV1, which is equivalent to 85% of MVV in adults. A threshold of 70% of MVV (21 times FEV1) is feasible in the majority of young athletes. CONCLUSION EIB is present in a substantial number of individuals at the age of 12-14 years, especially in swimmers. This underscores the importance of screening for EIB at this age. EVH is feasible in young elite athletes, however target ventilation needs to be adjusted accordingly.

Low cord blood Foxp3/CD3γ mRNA ratios: a marker of increased risk for allergy development

Data from birth cohort studies suggest that increased cord blood total IgE and reduced cord blood regulatory T cells increase the risk of developing allergic sensitization and atopic dermatitis.

Neonatal IL-10 production and risk of allergy development

We read with interest the recent report of Belderbos and coworkers in this journal showing that low neonatal TLR-4-mediated IL-10 production is associated with subsequent development of atopic dermatitis [1]. The authors stimulated whole blood of 1-month-old neonates with ultrapure lipopolysaccharide (LPS) and Interferon (IFN)-γ, and measured IL-10 in the culture supernatants. Neonates who developed physician-diagnosed atopic dermatitis at age 1 year had 1.8-fold lower TLR-4-mediated IL-10 production at the age of 1 month [1]. In a birth cohort ‘Follow-up of Newborns Immune development in relation to Allergic outcome’ (FONIA) study, we obtained data on the association between allergen-induced IL-10 production by cord blood cells and prospective risk of allergy development. The diagnosis of ‘clinical allergy’ was based on the presence of symptoms (skin, gastrointestinal or respiratory) in association with relevant IgE sensitization [2]. Cord blood mononuclear cells (CBMC) (n = 67) isolated immediately after uncomplicated vaginal delivery were stimulated by either ovalbumine (OVA) or by recombinant(r) Der p 2 (a kind gift from JM-Saint Rémy, KULeuven, Belgium). r IL-4 was added because we have previously found that rIL-4 increases in vitro allergen-induced IL-10 production by T cells from non-allergic subjects, but not by those from allergic subjects [3]. As shown in Fig. 1, IL-10 was significantly lower in OVA/IL-4 stimulated CBMC cultures from children who had developed clinical allergy at age 6 (n = 19) compared with the others (n = 45). IL-10 above 0.5 pg/mL was present in OVA/IL-4 stimulated cultures from 19 of 45 non-allergics and predicted a non-allergic outcome at age 6 (Relative Risk (RR) (in fact ‘relative benefit’) 1.49 (95% CI 1.1–2.0), Chi-square df 6.1; P = 0.007). Nine children were egg-allergic at 12 months and in only one of these nine children, OVA/IL-4-induced