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Rationale: The role of obesity-associated insulin resistance (IR) in airflow limitation in asthma is uncertain. Objectives: Using data in the Severe Asthma Research Program 3 (SARP-3), we evaluated relationships between homeostatic measure of IR (HOMA-IR), lung function (cross-sectional and longitudinal analyses), and treatment responses to bronchodilators and corticosteroids. Methods: HOMA-IR values were categorized as without (5.0). Lung function included FEV1 and FVC measured before and after treatment with inhaled albuterol and intramuscular triamcinolone acetonide and yearly for 5 years. Measurements and Main Results: Among 307 participants in SARP-3, 170 (55%) were obese and 140 (46%) had IR. Compared with patients without IR, those with IR had significantly lower values for FEV1 and FVC, and these lower values were not attributable to obesity effects. Compared with patients without IR, those with IR had lower FEV1 responses to β-adrenergic agonists and systemic corticosteroids. The annualized decline in FEV1 was significantly greater in patients with moderate IR (-41 ml/year) and severe IR (-32 ml/year,) than in patients without IR (-13 ml/year, P

Rationale: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 (angiotensin-converting enzyme 2), and TMPRSS2 (transmembrane protease serine 2) mediate viral infection of host cells. We reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells among patients with asthma may identify subgroups at risk for COVID-19 morbidity.Objectives: To determine the relationship between demographic features and sputum ACE2 and TMPRSS2 gene expression in asthma.Methods: We analyzed gene expression for ACE2 and TMPRSS2, and for ICAM-1 (intercellular adhesion molecule 1) (rhinovirus receptor as a comparator) in sputum cells from 330 participants in SARP-3 (Severe Asthma Research Program-3) and 79 healthy control subjects.Measurements and Main Results: Gene expression of ACE2 was lower than TMPRSS2, and expression levels of both genes were similar in asthma and health. Among patients with asthma, male sex, African American race, and history of diabetes mellitus were associated with higher expression of ACE2 and TMPRSS2. Use of inhaled corticosteroids (ICS) was associated with lower expression of ACE2 and TMPRSS2, but treatment with triamcinolone acetonide did not decrease expression of either gene. These findings differed from those for ICAM-1, where gene expression was increased in asthma and less consistent differences were observed related to sex, race, and use of ICS.Conclusions: Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values

BackgroundAirway type 2 inflammation is usually corticosteroid sensitive, but the role of type 2 inflammation as a mechanism of asthma in patients receiving high-dose inhaled corticosteroids (ICSs) is uncertain.ObjectiveWe sought to determine whether airway type 2 inflammation persists in patients treated with ICSs and to evaluate the clinical features of patients with steroid-resistant airway type 2 inflammation.MethodsWe used quantitative PCR to generate a composite metric of type 2 cytokine gene expression (type 2 gene mean [T2GM]) in induced sputum cells from healthy control subjects, patients with severe asthma receiving ICSs (n = 174), and patients with nonsevere asthma receiving ICSs (n = 85). We explored relationships between asthma outcomes and T2GM values and the utility of noninvasive biomarkers of airway T2GM.ResultsSputum cell T2GM values in asthmatic patients were significantly increased and remained high after treatment with intramuscular triamcinolone. We used the median T2GM value as a cutoff to classify steroid-treated type 2-low and steroid-resistant type 2-high (srT2-high) subgroups. Compared with patients with steroid-treated type 2-low asthma, those with srT2-high asthma were older and had more severe asthma. Blood eosinophil cell counts predicted srT2-high asthma when body mass index was less than 40 kg/m2 but not when it was 40 kg/m2 or greater, whereas blood IgE levels strongly predicted srT2-high asthma when age was less than 34 years but not when it was 34 years or greater.ConclusionDespite ICS therapy, many asthmatic patients have persistent airway type 2 inflammation (srT2-high asthma), and these patients are older and have more severe disease. Body weight and age modify the performance of blood-based biomarkers of airway type 2 inflammation.

Background: Eosinophils have numerous roles in type 2 inflammation depending on their activation states in the blood and airway or after encounter with inflammatory mediators. Airway epithelial cells have a sentinel role in the lung and, by instructing eosinophils, likely have a foundational role in asthma pathogenesis. Summary: In this review, we discuss various topics related to eosinophil-epithelial cell interactions in asthma, including the influence of eosinophils and eosinophil products, e.g., granule proteins, on epithelial cell function, expression, secretion, and plasticity; the effects of epithelial released factors, including oxylipins, cytokines, and other mediators on eosinophils, e.g., on their activation, expression, and survival; possible mechanisms of eosinophil-epithelial cell adhesion; and the role of intra-epithelial eosinophils in asthma. Key Messages: We suggest that eosinophils and their products can have both injurious and beneficial effects on airway epithelial cells in asthma and that there are bidirectional interactions and signaling between eosinophils and airway epithelial cells in asthma. [ABSTRACT FROM AUTHOR]

In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov NCT01606826)FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology.

Severe asthma is typically characterized by chronic airway inflammation that is refractory to corticosteroids and associated with excess morbidity. Patients were recruited into the National Heart, Lung, and Blood Institute-sponsored Severe Asthma Research Program and comprehensively phenotyped by bronchoscopy. Bronchoalveolar lavage (BAL) cells were analyzed by flow cytometry. Compared with healthy individuals (n = 21), patients with asthma (n = 53) had fewer BAL natural killer (NK) cells. Patients with severe asthma (n = 29) had a marked increase in the ratios of CD4+ T cells to NK cells and neutrophils to NK cells. BAL NK cells in severe asthma were skewed toward the cytotoxic CD56dim subset, with significantly increased BAL fluid levels of the cytotoxic mediator granzyme A. The numbers of BAL CD56dim NK cells and CCR6-CCR4- T helper 1-enriched CD4+ T cells correlated inversely with lung function [forced expiratory volume in 1 s (FEV1) % predicted] in asthma. Relative to cells from healthy controls, peripheral blood NK cells from asthmatic patients had impaired killing of K562 myeloid target cells despite releasing more cytotoxic mediators. Ex vivo exposure to dexamethasone markedly decreased blood NK cell lysis of target cells and cytotoxic mediator release. NK cells expressed airway lipoxin A4/formyl peptide receptor 2 receptors, and in contrast to dexamethasone, lipoxin A4-exposed NK cells had preserved functional responses. Together, our findings indicate that the immunology of the severe asthma airway is characterized by decreased NK cell cytotoxicity with increased numbers of target leukocytes, which is exacerbated by corticosteroids that further disable NK cell function. These failed resolution mechanisms likely contribute to persistent airway inflammation in severe asthma.

Rare eosinophil-associated disorders (EADs), including hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, and eosinophilic gastrointestinal disorders, are a heterogeneous group of conditions characterized by blood and/or tissue hypereosinophilia and eosinophil-related clinical manifestations. Although the recent availability of biologic therapies that directly and indirectly target eosinophils has the potential to dramatically improve treatment options for all EADs, clinical trials addressing their safety and efficacy in rare EADs have been relatively few. Consequently, patient access to therapy is limited for many biologics, and the establishment of evidence-based treatment guidelines has been extremely difficult. In this regard, multicenter retrospective collaborative studies focusing on disease manifestations and treatment responses in rare EADs have provided invaluable data for physicians managing patients with these conditions and helped identify important questions for future translational research. During the Clinical Pre-Meeting Workshop held in association with the July 2023 biennial meeting of the International Eosinophil Society in Hamilton, Ontario, Canada, the successes and limitations of pivotal multicenter retrospective studies in EADs were summarized and unmet needs regarding the establishment of guidelines for use of biologics in rare EADs were discussed. Key topics of interest included (1) clinical outcome measures, (2) minimally invasive biomarkers of disease activity, (3) predictors of response to biologic agents, and (4) long-term safety of eosinophil depletion. Herein, we report a summary of these discussions, presenting a state-of-the-art overview of data currently available for each of these topics, the limitations of the data, and avenues for future data generation through implementation of multidisciplinary and multicenter studies. [ABSTRACT FROM AUTHOR]

BackgroundSevere asthma is a complex heterogeneous disease associated with older age and obesity. The presence of eosinophilic (type 2) inflammation in some but not all patients with severe asthma predicts responsiveness to current treatments, but new treatment approaches will require a better understanding of non-type 2 mechanisms of severe asthma. We considered the possibility that systemic inflammation, which arises in subgroups of obese and older patients, increases the severity of asthma. Interleukin-6 (IL-6) is a biomarker of systemic inflammation and metabolic dysfunction, and we aimed to explore the association between IL-6 concentrations, metabolic dysfunction, and asthma severity.MethodsIn this cross-sectional analysis, patients were recruited from two cohorts: mainly non-severe asthmatics from the University of California San Francisco (UCSF) and mainly severe asthmatics from the Severe Asthma Research Program (SARP). We generated a reference range for plasma IL-6 in a cohort of healthy control patients. We compared the clinical characteristics of asthmatics with plasma IL-6 concentrations above (IL-6 high) and below (IL-6 low) the upper 95% centile value for plasma IL-6 concentration in the healthy cohort. We also compared how pulmonary function, frequency of asthma exacerbations, and frequency of severe asthma differed between IL-6 low and IL-6 high asthma populations in the two asthma cohorts.FindingsBetween Jan 1, 2005, and Dec 31, 2014, we recruited 249 patients from UCSF and between Nov 1, 2012, and Oct 1, 2014, we recruited 387 patients from SARP. The upper 95th centile value for plasma IL-6 concentration in the healthy cohort (n=93) was 3·1 pg/mL, and 14% (36/249) of UCSF cohort and 26% (102/387) of the SARP cohort had plasma IL-6 concentrations above this upper limit. The IL-6 high patients in both asthma cohorts had a significantly higher average BMI (p

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n = 439) and from healthy controls (n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation. NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways. [ABSTRACT FROM AUTHOR]

Use of antibodies against tyrosine phosphorylated PYbeta1 peptide reveals the localization of the PYbeta1 subunit to podosomes of RSV-transformed NIH 3T3 cells, in a manner different from that of phosphotyrosine and beta1 integrins. One SH2 domain of the p85 phosphatidylinositol-3-kinase subunit binds only to the PYbeta1 integrin subunit and not to the non-phosphorylated peptide. Tyrosine phosphorylation of cytoplasmic domain of beta1 subunit influences cellular signaling pathways.

Background: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell‐free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. Objective: The present study explores the mechanism of CD32‐ and αMß2 integrin‐dependent eosinophil cytolysis of IL3‐primed blood eosinophils seeded on heat‐aggregated immunoglobulin G (HA‐IgG). Methods: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. Results: Cytolysis of IL3‐primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p‐38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule‐associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. Conclusion and Clinical Relevance: In this CD32‐ and αMß2 integrin‐dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p‐38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil‐driven tissue damage. [ABSTRACT FROM AUTHOR]