Fahy, John V.

AbstractIntroductionPrevious bronchoalveolar lavage fluid (BALF) proteomic analysis has evaluated limited numbers of subjects for only a few proteins of interest, which may differ between asthma and normal controls. Our objective was to examine a more comprehensive inflammatory biomarker panel in quantitative proteomic analysis for a large asthma cohort to identify molecular phenotypes distinguishing severe from nonsevere asthma.MethodsBronchoalveolar lavage fluid from 48 severe and 77 nonsevere adult asthma subjects were assessed for 75 inflammatory proteins, normalized to BALF total protein concentration. Validation of BALF differences was sought through equivalent protein analysis of autologous sputum. Subjects' data, stratified by asthma severity, were analysed by standard statistical tests, principal component analysis and 5 machine learning algorithms.ResultsThe severe group had lower lung function and greater health care utilization. Significantly increased BALF proteins for severe asthma compared to nonsevere asthma were fibroblast growth factor 2 (FGF2), TGFα, IL1Ra, IL2, IL4, CCL8, CCL13 and CXCL7 and significantly decreased were platelet‐derived growth factor a‐a dimer (PDGFaa), vascular endothelial growth factor (VEGF), interleukin 5 (IL5), CCL17, CCL22, CXCL9 and CXCL10. Four protein differences were replicated in sputum. FGF2, PDGFaa and CXCL7 were independently identified by 5 machine learning algorithms as the most important variables for discriminating severe and nonsevere asthma. Increased and decreased proteins identified for the severe cluster showed significant protein–protein interactions for chemokine and cytokine signalling, growth factor activity, and eosinophil and neutrophil chemotaxis differing between subjects with severe and nonsevere asthma.ConclusionThese inflammatory protein results confirm altered airway remodelling and cytokine/chemokine activity recruiting leukocytes into the airways of severe compared to nonsevere asthma as important processes even in stable status.

AbstractBackgroundHeterozygote carriers of potentially pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have increased asthma risk. However, the frequency and impact of CFTR variation among individuals with asthma is unknown.ObjectiveTo determine whether potentially pathogenic CFTR variants associate with disease severity and whether individuals with two potentially pathogenic variants exist in a severe asthma‐enriched cohort.MethodsWe analyzed sequencing data spanning a 190.5Kb region of CFTR in participants from the Severe Asthma Research Program (SARP1‐3). Potentially pathogenic, rare CFTR variants (frequency < 0.05) were classified as CF‐causing or of varying clinical consequences (VVCC) (CFTR2. org). Regression‐based models tested for association between CFTR genotypes (0‐2 potentially pathogenic variants) and severity outcomes.ResultsOf 1401 participants, 9.5% (134) had one potentially pathogenic variant, occurring more frequently in non‐Hispanic white (NHW, 10.1% [84 of 831]) compared to African American individuals (AA, 5.2% [22 of 426]). We found ≥2 potentially pathogenic CFTR variants in 1.4% (19); 0.5% (4) of NHW and 2.8% (12) of AA. Potentially pathogenic CFTR variant genotypes (≥1 or ≥2 variants) were not cumulatively associated with lung function or exacerbations. In NHW, we found three F508del compound heterozygotes with F508del and a VVCC (two 5 T; TG12[c.1210‐11 T > G] and one Arg1070Trp) and a homozygote for the VVCC, 5 T; TG12.ConclusionsWe found potentially pathogenic CFTR variants within a severe asthma‐enriched cohort, including three compound heterozygote genotypes variably associated with CF in NHW individuals. These findings provide the rationale for CFTR sequencing and phenotyping of CF‐related traits in individuals with severe asthma.

Background: Heterozygote carriers of potentially pathogenic variants in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene have increased asthma risk. However, the frequency and impact of CFTR variation among individuals with asthma is unknown. Objective: To determine whether potentially pathogenic CFTR variants associate with disease severity and whether individuals with two potentially pathogenic variants exist in a severe asthma-enriched cohort . Methods : We analyzed sequencing data spanning a 190.5Kb region of CFTR in participants from the Severe Asthma Research Program (SARP1-3). Potentially pathogenic, rare CFTR variants (frequency<0.05) were classified as CF-causing or of varying clinical consequences (VVCC) (CFTR2.org). Regression-based models tested for association between CFTR genotypes (0-2 potentially pathogenic variants) and severity outcomes. Results: Of 1401 participants, 9.5% (134) had one potentially pathogenic variant, occurring more frequently in non-Hispanic white (NHW, 10.1% [84 of 831]) compared to African American individuals (AA, 5.2% [22 of 426]). We found ≥2 potentially pathogenic CFTR variants in 1.4% (19); 0.5% (4) of NHW and 2.8% (12) of AA. Potentially pathogenic CFTR variant genotypes (≥1 or ≥2 variants) were not cumulatively associated with lung function or exacerbations. In NHW, we found three F508del compound heterozygotes with F508del and a VVCC (two 5T;TG12[c.1210-11T>G] and one Arg1070Trp) and a homozygote for the VVCC, 5T;TG12. Conclusions: We found potentially pathogenic CFTR variants within a severe asthma-enriched cohort , including three compound heterozygote genotypes variably associated with CF in NHW individuals. These findings provide the rationale for CFTR sequencing and phenotyping of CF-related traits in individuals with severe asthma.

Abstract In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Rα expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Rα is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Rα mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Rα, which in turn contributes to the presence of sIL-5Rα. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Rα was accompanied by an increase in sIL-5Rα in the supernatant. IL-5 had no ligand-specific effect on mIL-5Rα or sIL-5Rα mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Rα, suggesting that sIL-5Rα may be produced by proteolytic cleavage of mIL-5Rα. IL-5 transiently reduced surface expression of β-chain, but had no effect on the expression of GM-CSFRα. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Rα rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Rα and release sIL-5Rα in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.

Abstract IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Rα on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Rα in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Rα on airway eosinophils compared with circulating cells. Furthermore, sIL-5Rα concentrations were elevated in BAL fluid, but steady state levels of sIL-5Rα mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Rα on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Rα, the presence of sIL-5Rα, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.

Abstract Airway inflammation is increasingly recognized as a pivotal component of asthma. Because allergens provoke bronchial constriction and inflammation in allergic subjects, bronchial antigen challenge has emerged as a powerful technique for evaluating mechanisms involved in this process. In this study, we compare whole lung antigen challenge (WLAC) with segmentai bronchoprovocation (SBP) in eight allergic, non-asthmatic, non-smoking subjects, and evaluated the response by bronchoalveolar lavage (BAL) prior to, and 48 h after antigen challenge. Both challenge techniques evoked airway inflammation, manifest as an increase in total cells and eosinophils recovered by BAL, an increase in total protein concentration, and enhanced production of superoxide anion by airspace cells. The degree to which these changes occurred was significantly greater with SBP than WLAC, and only SBP evoked persistent measurable change in alveolar macrophage density and eosinophil granule protein concentrations. Moreover, although both techniques were associated with a comparable immediate fall in FEV1, only WLAC resulted in statistically significant persistent physiologic changes 48 h afterwards. We conclude that, as anticipated, SBP produces more intense airway inflammation in allergic subjects, does not result in late airway obstruction, and offers specific advantages in studying allergen-driven airway inflammation.

Abstract Human blood eosinophils exposed ex vivo to hematopoietic cytokines (e.g., IL-5 or GM-CSF) subsequently display enhanced responsiveness to numerous chemoattractants, such as chemokines, platelet-activating factor, or FMLP, through a process known as priming. Airway eosinophils, obtained by bronchoalveolar lavage after segmental Ag challenge, also exhibit enhanced responsiveness to selected chemoattractants, suggesting that they are primed during cell trafficking from the blood to the airway. Earlier work has shown that chemoattractants stimulate greater activation of ERK1 and ERK2 following IL-5 priming in vitro, thus revealing that ERK1/ERK2 activity can be a molecular readout of priming under these circumstances. Because few studies have examined the intracellular mechanisms regulating priming as it relates to human airway eosinophils, we evaluated the responsiveness of blood and airway eosinophils to chemoattractants (FMLP, platelet-activating factor, CCL11, CCL5, CXCL8) with respect to degranulation, adherence to fibronectin, or Ras–ERK signaling cascade activation. When compared with blood eosinophils, airway eosinophils exhibited greater FMLP-stimulated eosinophil-derived neurotoxin release as well as augmented FMLP- and CCL11-stimulated adherence to fibronectin. In airway eosinophils, FMLP, CCL11, and CCL5 stimulated greater activation of Ras or ERK1/ERK2 when compared with baseline. Ras activation by FMLP in blood eosinophils was also enhanced following IL-5 priming. These studies are consistent with a model of in vivo priming of eosinophils by IL-5 or related cytokines following allergen challenge, and further demonstrate the key role of priming in the chemoattractant-stimulated responses of eosinophils. These data also demonstrate the importance of the Ras–ERK signaling pathway in the regulation of eosinophil responses to chemoattractants in the airway. Human airway eosinophils respond to several chemoattractants with increased activation of the Ras–ERK cascade, eosinophil-derived neurotoxin release, and adherence to fibronectin relative to blood eosinophils.