Timothy Oates

Pediatric severe asthma is characterized by eosinophilia and remodeling without TH2 cytokines

BACKGROUND The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood. OBJECTIVES We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the T(H)2 cytokines IL-4, IL-5, and IL-13. METHODS Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen T(H)2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation. RESULTS Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5(+) and IL-13(+) cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid T(H)2 cytokines had significantly lower lung function than those with undetectable BAL fluid T(H)2 cytokines. CONCLUSIONS STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the T(H)2 mediators that are thought to drive allergic asthma were mostly absent.

IL-33 promotes airway remodeling in pediatric patients with severe steroid-resistant asthma.

BACKGROUND TH2 cytokines are not responsible for the ongoing symptoms and pathology in children with severe therapy-resistant asthma (STRA). IL-33 induces airway hyperresponsiveness, but its role in airway remodeling and steroid resistance is unknown. OBJECTIVE We sought to investigate the relationship between IL-33 and airway remodeling in pediatric patients with STRA. METHODS IL-33 levels were quantified in neonatal mice given inhaled house dust mite (HDM), and the effect of blocking IL-13 on remodeling and IL-33 levels was assessed. HDM-induced allergic airways disease (AAD) in neonatal ST2(-/-) mice lacking the IL-33 receptor was assessed, together with collagen production after IL-33 administration. The effect of steroid therapy on IL-33 levels in patients with neonatal AAD was explored. IL-33 expression was quantified in endobronchial biopsy (EB) specimens from children with STRA and related to remodeling, and collagen production by airway fibroblasts from pediatric patients stimulated with IL-33 and budesonide was quantified. RESULTS Blocking IL-13 after AAD was established in neonatal mice and did not reduce remodeling or IL-33 levels; airway hyperresponsiveness was only partially reduced. IL-33 promoted collagen synthesis both from asthmatic fibroblasts from pediatric patients and after intranasal administration in mice. Increased cellular expression of IL-33, but not IL-13, was associated with increased reticular basement membrane thickness in EB specimens from children with STRA, whereas remodeling was absent in HDM-exposed ST2(-/-) mice. IL-33 levels were maintained, whereas IL-13 levels were abrogated by steroid treatment in neonatal HDM-exposed mice and in EB specimens from children with STRA. CONCLUSION IL-33 is a relatively steroid-resistant mediator that promotes airway remodeling in patients with STRA and is an important therapeutic target.

Increased airway smooth muscle in preschool wheezers who have asthma at school age

BACKGROUND Increased airway smooth muscle (ASM) is a feature of established asthma in schoolchildren, but nothing is known about ASM in preschool wheezers. OBJECTIVE We sought to determine endobronchial biopsy specimen ASM area fraction in preschool wheezers and its association with asthma at school age. METHODS ASM area, reticular basement membrane thickness, and mucosal eosinophil and ASM mast cell values were quantified in endobronchial biopsy specimens previously obtained from preschool children undergoing clinically indicated bronchoscopy: severe recurrent wheezers (n=47; median age, 26 months) and nonwheezing control subjects (n=21; median age, 15 months). Children were followed up, and asthma status was established at age 6 to 11 years. Preschool airway pathology was examined in relation to asthma at school age. RESULTS Forty-two (62%) of 68 children had 1 or more evaluable biopsy specimens for ASM. At school age, 51 of 68 children were followed up, and 15 (40%) of 37 preschool wheezers had asthma. Children who had asthma and an evaluable biopsy specimen had increased preschool ASM area fraction (n=8; median age, 8.2 years [range, 6-10.4 years]; median ASM, 0.12 [range, 0.08-0.16]) compared with that seen in children without asthma (n=24; median age, 7.3 years [range, 5.9-11 years]; median ASM, 0.07 [range, 0.02-0.23]; P=.007). However, preschool reticular basement membrane thickness and mucosal eosinophil or ASM mast cell values were not different between those who did or did not have asthma at school age. CONCLUSION Increased preschool ASM is associated with those children who have asthma at school age. Thus a focus on early changes in ASM might be important in understanding the subsequent development of childhood asthma.

Expression of GATA family of transcription factors in T-cells, monocytes and bronchial biopsies

GATA-binding proteins are a subfamily of zinc finger transcription factors with six members (GATA-1-6) that interact with the GATA deoxyribonucleic acid (DNA) sequence. This sequence is found in the regulatory regions of many genes including those encoding T-helper 2 (Th2)-like cytokines, receptors, adhesion molecules and enzymes, which may be important in the pathogenesis of bronchial asthma. The expression of GATA-3, 4 and -6 was investigated in peripheral blood T-lymphocytes and monocytes and bronchial biopsies from 11 normal subjects and 10 steroid-naive asthmatic patients. Using Western blot analysis, T-cells from asthmatic subjects expressed 5 times the level of GATA-3 compared to that in normals. Confocal microscopy indicated that GATA-3 expression was both nuclear and cytoplasmic. GATA DNA binding complex containing GATA-3 was elevated in Th2 cells as determined by electrophorectic mobility shift assay. In contrast, monocytes from normal and asthmatic subjects expressed GATA-4 and -6 in equal amounts, but no GATA-3 was found. Using immunohistochemistry in bronchial biopsies, epithelial cells expressed high levels of GATA-3, GATA-4 and GATA-6 proteins. Comparison of Western blots of bronchial biopsies showed no significant differences between normal and asthmatic subjects. In conclusion, the increased expression of GATA-3 in asthmatic T-cells may underlie augmented T-helper 2-like cytokines in this disease. However, the unaltered GATA-3 expression in epithelial cells suggests a distinct role for GATA-3 in these cells unrelated to T-helper 2-like cytokine release. Finally, no evidence was found for an increased expression of GATA-4 and GATA-6 in asthma.

Effect of Transforming Growth Factor-beta Receptor I Kinase Inhibitor 2,4-Disubstituted Pteridine (SD-208) in Chronic Allergic Airway Inflammation and Remodeling

Transforming growth factor (TGF)-β is a multifunctional regulator of cell growth and differentiation with both pro- and anti-inflammatory properties. We used an inhibitor of TGF-β receptor I (TGF-βRI) kinase, SD-208 (2,4-disubstituted pteridine, a ATP-competitive inhibitor of TGF-βRI kinase), to determine the role of TGF-β in airway allergic inflammation and remodeling. Brown-Norway rats sensitized and repeatedly exposed to ovalbumin (OVA) aerosol challenge were orally administered SD-208 twice daily, before each of six OVA exposures to determine the preventive effects, or only before each of the last three of six OVA exposures to investigate its reversal effects. SD-208 (60 mg/kg) reversed bronchial hyperresponsiveness (BHR) induced by repeated allergen exposure, but it did not prevent it. SD-208 prevented changes in serum total and OVA-specific IgE, but it did not reverse them. SD-208 had both a preventive and reversal effect on airway inflammation as measured by major basic protein-positive eosinophils and CD2+ T-cell counts in mucosal airways, cell proliferation measured by 5-bromo-2′-deoxyuridine expression in airway smooth muscle (ASM) cells and epithelial cells, and goblet cell hyperplasia induced by repeated allergen challenges. There was a significant decrease in intracellular Smad2/3 expression. SD-208 did not significantly decrease the increased ASM thickness induced by allergen exposure. These findings support a proinflammatory and proremodeling role for TGF-β in allergic airway inflammation. Inhibition of TGF-βRI kinase activities by SD-208 may be a useful approach to the reversal of BHR and to the prevention and reversal of inflammatory and remodeling features of chronic asthma.

STAT6 expression in T cells, alveolar macrophages and bronchial biopsies of normal and asthmatic subjects

BackgroundAsthma is characterised by increased numbers of Th2-like cells in the airways and IgE secretion. Generation of Th2 cells requires interleukin (IL)-4 and IL-13 acting through their specific receptors and activating the transcription factor, signal transducer and activator of transcription 6 (STAT6). STAT6 knockout mice fail to produce IgE, airway hyperresponsiveness and bronchoalveolar lavage eosinophilia after allergen sensitisation, suggesting a critical role for STAT6 in allergic responses.MethodsWe have investigated the expression of STAT6 in peripheral blood T-lymphocytes, alveolar macrophages and bronchial biopsies from 17 normal subjects and 18 mild-moderate steroid-naïve stable asthmatic patients.ResultsSTAT6 expression was variable and was detected in T-lymphocytes, macrophages and bronchial epithelial cells from all subjects with no difference between normal and stable asthmatic subjects.ConclusionsSTAT6 expression in different cells suggests that it may be important in regulating the expression of not only Th2-like cytokines in T cells of man, but may also regulate STAT-inducible genes in alveolar macrophages and airway epithelial cells.

S33 Vitamin D and airway remodelling in paediatric severe therapy resistant asthma

Background Serum vitamin D levels have been related to asthma control and medication use in children with mild to moderate disease, but little is known about the relationship between serum vitamin D levels and airway remodelling and mucosal inflammation in asthma. We hypothesised that lower serum vitamin D levels would be associated with increased airway inflammation and remodelling and lower lung function in children with severe therapy resistant asthma (STRA). Methods Nineteen children aged between 6 and 16 years with STRA underwent spirometry, fiberoptic bronchoscopy, endobronchial biopsy, and measurement of serum vitamin D (25(OH)D3 nmol/l). Endobronchial biopsies stained with H&E were used to quantify airway remodelling (reticular basement membrane thickness, smooth muscle mass and epithelial shedding). Immunohistochemistry was used to quantify smooth muscle cell proliferation using proliferating cell nuclear antigen, and inflammatory cells (eosinophils, neutrophils and mast cells). Results Seventeen of 19 children with STRA were vitamin D insufficient (<50 nmol/l), median (range) serum 25(OH)D3 29 (21–39) nmol/l. There was no relationship between serum 25(OH)D3 and submucosal eosinophils, neutrophils or mast cells. Airway smooth muscle mass was inversely related to serum 25(OH)D3 (r=−0.6, p=0.007) (Abstract S33 figure 1), but there was no relationship between vitamin D levels and reticular basement membrane thickness or epithelial shedding. Lung function was not related to serum vitamin D levels, however bronchodilator reversibility was inversely related to serum 25(OH)D3 levels (r=−0.53, p=0.02).Abstract S33 Figure 1 Correlation between volume fraction of airway smooth muscle and serum 25(OH)D3 in paediatric STRA. Conclusions Vitamin D insufficiency is common in children with STRA. Lower vitamin D levels in children with STRA were associated with increased airway smooth muscle and increased bronchodilator reversibility. Randomised controlled trials of vitamin D supplementation are warranted in paediatric STRA.

S88 Mast cell myositis is associated with persistent airflow limitation (PAL) in childhood severe asthma (SA)

Background Studies of airway inflammation and remodelling may help us to understand the pathophysiology of SA. Adult studies have shown mast cell inflammation within smooth muscle is specific to asthma and is associated with airway hyperresponsiveness (AHR). However, this has not been studied in childhood disease. Hypothesis Children with SA have increased submucosal eosinophils and mast cells within smooth muscle compared to age-matched mild asthmatics and non-asthmatic controls. Methods 75 children, mean age 11.8 (5.6–17.3) years, 53 with SA, 7 with mild/moderate asthma (MA) and 15 non-asthmatic controls (bronchoscoped for upper airway symptoms) were included. All underwent spirometry and bronchodilator reversibility, fractional exhaled nitric oxide (FeNO) measurement, fibreoptic bronchoscopy with bronchoalveolar lavage (BAL) and endobronchial biopsy (EB). EB were stained for: eosinophils (congo red), neutrophils (neutrophil elastase), mast cells (mast cell tryptase); and reticular basement membrane (RBM) thickness, epithelial shedding and volume fraction (Vv) of smooth muscle. Results See Abstract S88 Table 1. Children with SA had significantly increased BAL and submucosal eosinophils compared to controls. There were no significant group differences in submucosal mast cells, but the presence of mast cells within smooth muscle exhibited a non-significant trend to be increased in SA and MA. Children with mast cells within smooth muscle were more likely to have PAL (post bronchodilator, post steroid trial FEV1<80% predicted) (p<0.05). The Vv of subepithelial tissue occupied by airway smooth muscle (ASM) was only increased in SA.Abstract S88 Table 1 Airway inflammation and remodelling in severe, mild/moderate asthma and non asthmatic control subjects Severe asthma (n=53) Mild/moderate asthma (n=7) Control (n=15) p BAL eosinophils % 2.7 (1–51) 0.7 (0–27.7) 0 (0–5.7) <0.001 BAL neutrophils % 3.3 (0.3–73.7) 1.7 (0–7.3) 2.7 (0.6–14) NS Mucosal eosinophils (/mm2) 11.2 (0–209.3) 3.7 (0–14.5) 0 (0–25.1) 0.01 Mucosal neutrophils (/mm2) 9.8 (0–125.6) 11.4 (0–22.2) 1.2 (0–58.3) NS Mucosal mast cells (/mm2) 45.7 (0–185) 63.1 (9.2–79.7) 60.5 (0–165.6) NS Muscle mast cells (/mm2) 12.3 (0–299) 18.3 (0–72.8) 0 (0–50) NS Vv (sm/subepithelium) 0.20 (0–0.65) 0.06 (0–0.3) 0.09 (0–0.16) 0.002 Values are median (range). All highlighted p values denote difference between severe asthma and controls. BAL, bronchoalveolarlavage; RBM, reticular basement membrane; sm, smooth muscle; Vv, volume fraction of airway smooth muscle indexed to subepithelium. Conclusions Children with SA have increased luminal and submucosal eosinophilia. However, in contrast to reports in adults of AHR being associated with mast cell myositis, we have found severe asthmatic children with mast cell myositis were more likely to have PAL. Mast cell myositis may be a feature of severe asthma in children.

S87 Relationship between bronchial reticular basement membrane thickness (RBM) and smooth muscle mass in childhood severe asthma (SA)

Background In asthma there is a direct relationship between tissue eosinophilic inflammation and RBM thickness. However, relationships between components of remodelling have not been explored, but may be important especially in determining disease severity. There are no reports of an association between smooth muscle mass and asthma severity in children. We hypothesised that increases in both RBM thickness and smooth muscle mass is present in children with SA, only increased RBM thickness is present in mild asthma and neither feature is present in controls. Methods 75 children, mean age 11.8 (5.6–17.3) years, 53 with SA, 7 with mild/moderate asthma and 15 non-asthmatic controls (bronchoscoped for upper airway symptoms) were included. All underwent fibreoptic bronchoscopy with endobronchial biopsies (EB). EB were processed to paraffin, and 5 μm sections were cut and stained with haematoxylin and eosin and used to quantify RBM thickness, epithelial shedding and volume fraction (Vv) of subepithelial smooth muscle indexed to submucosa. Results Epithelial shedding was increased in atopic but not asthmatic subjects, (p=0.02 and p=0.37, respectively), and in children with asthma was correlated with exhaled nitric oxide (r=0.4, p=0.005). RBM thickness was increased in severe asthmatics compared to controls (p<0.0001), but a trend only to increased thickness was seen in mild asthmatics compared to controls (median (range) values: 6 (4.4–8.4) and 4 (3.1–7.5) μm, respectively; p=0.06). The Vv of subepithelial airway smooth muscle was only increased in severe asthmatics compared to controls (0.20 (0–0.65) and 0.09 (0–0.16), respectively; p=0.002). Interestingly, there was a positive relationship between RBM thickness and smooth muscle Vv fraction in asthmatics, but not in controls (r=0.31, p=0.02 and r=0.5, p=0.07, respectively) (Abstract S87 Figure 1).Abstract S87 Figure 1 Correlation between RBM and volume fraction of airway smooth muscle in asthma. Discussion We report for the first time a direct relationship between RBM thickness and airway smooth muscle mass in paediatric asthma. It is unknown if the relationship is causal, or both are driven by a common underlying process. Combinations of components of airway remodelling, rather than single factors, may prove to be more informative when phenotyping children with severe asthma.