Background: The nasal allergen challenge (NAC) is a tool for evaluating upper airway allergic responses. In the CRITICAL study, NAC with cockroach allergen was used to confirm clinical reactivity in sensitized individuals from urban environments before enrollment onto a subcutaneous immunotherapy trial. Objective: Immunologic and transcriptomic predictors of NAC responsiveness were identified. Methods: NAC was performed in 103 participants. Clinical responses were assessed by the Total Nasal Symptom Score (TNSS) and sneeze score (TSNEEZ), which scale positively with symptom severity. Baseline immunologic markers—including skin prick test wheal size, cockroach-specific IgE, IgG, and IgG4, and T-cell responses were evaluated. Nasal lavage samples were analyzed for gene expression modules. Clinical trial registration: ClinicalTrials.gov NCT03541187. Results: Larger skin prick test wheal sizes were significantly associated with positive NAC outcomes (2.2 mm larger than negative NAC) and lower reactive doses (hazard ratio = 1.10). Cockroach (i6 extract)-specific IgE levels were inversely correlated with TNSS, while levels of IgE, IgG, and IgG4 specific to the extract used for therapy showed no association. Higher IL-10 T-cell responses were observed in those without reaction, while Bla g 5 and Bla g 9 dominance correlated negatively with TNSS and positively with TSNEEZ, respectively. Transcriptomic analysis revealed that higher expression of eosinophil- and neutrophil-associated modules and lower expression of type 1 interferon, macrophage, and epithelial barrier modules were linked to positive NAC responses. Conclusion: NAC responsiveness to cockroach extract is influenced by skin test reactivity, T- and B-cell regulation, and nasal gene expression. These findings highlight the role of both adaptive and innate immunity in allergic airway responses and suggest potential biomarkers for clinical reactivity.

Importance:While biologic therapies targeting type 2 (T2) inflammation reduce acute exacerbation rates in children with asthma and T2 inflammation, exacerbations still occur, and the underlying molecular mechanisms are poorly defined. Objective:To identify multiple distinct molecular mechanisms implicated in asthma exacerbations by characterizing respiratory illnesses among urban children with eosinophilic asthma enrolled in a clinical trial comparing treatment with mepolizumab vs placebo. Design, Setting, and Participants:This is a secondary analysis of the Mechanisms Underlying Asthma Exacerbations Prevented and Persistent With Immune-Based Therapy: A Systems Approach Phase 2 (MUPPITS-2) double-blind, placebo-controlled, parallel-group, randomized clinical trial comparing treatment with mepolizumab vs placebo among children with exacerbation-prone asthma in low-income urban centers in 9 US cities. Data analysis was performed from September 2022 to April 2025. Intervention:Participants were randomized to receive either mepolizumab (aged 6-11 years: 40 mg; aged 12-17 years: 100 mg) or matching placebo by subcutaneous injection once every 4 weeks for 52 weeks. Main Outcomes and Measures:The primary measurement was a transcriptomic modular analysis by RNA sequencing of nasal samples obtained during acute respiratory illnesses. Associations among upper airway transcriptional signatures, the clinical outcome of respiratory illnesses, and pulmonary functions were investigated. Results:Of the 290 participants enrolled in the MUPPITS-2 trial, 108 participants (median [IQR] age, 10.0 [9.0-13.0] years; 48 [44%] female) were sampled during 176 acute respiratory illness events. During illness events resulting in asthma exacerbations, children receiving mepolizumab demonstrated decreased expression of an eosinophil-associated module associated with T2 inflammation (log2 fold change [FC] estimate, -0.60; false discovery rate [FDR] < .05) but increased expression of gene modules associated with epithelial and macrophage inflammatory pathways relative to children receiving placebo (log2 FC estimates, 0.22-0.85; FDR < .05). Both groups showed higher expression of mucus secretion and cellular stress response pathways during exacerbations relative to nonexacerbation illnesses. The mepolizumab group demonstrated upregulation of epithelial inflammatory pathways in exacerbations irrespective of a respiratory virus, while macrophage pathways contributed specifically to viral exacerbations. Three distinct, semiorthogonal inflammatory axes were shown to underlie the majority of the heterogeneity among exacerbations in the 2 groups. Conclusions and Relevance:The study's findings implicate multiple alternative inflammatory pathways associated with the epithelium and macrophages, as well as mucus hypersecretion, as mechanisms of residual acute exacerbations in children receiving mepolizumab. Further, they indicate that multiple distinct inflammatory axes can independently contribute to asthma exacerbations. Trial Registration:ClinicalTrials.gov Identifier: NCT03292588.

Mepolizumab (anti-IL5 therapy) reduces asthma exacerbations in urban children with exacerbation-prone eosinophilic asthma. We previously utilized nasal transcriptomics to identify inflammatory pathways (gene co-expression modules) associated with asthma exacerbations despite this therapy. In this study, we applied differential gene correlation analysis on these targeted gene co-expression modules to gain better insight into the treatment effects on correlation structure within gene networks. Mepolizumab treatment resulted in loss of correlation amongst eosinophil-specific genes but conservation and even strengthening of correlation amongst mast cell-specific genes, T2 cytokines, and airway epithelial inflammatory genes. Notably, mepolizumab induced significant gain in correlation of genes associated with multiple aspects of airway epithelial inflammation including those related to extracellular matrix production and nitric oxide synthesis, and this change was associated with a poor clinical response to mepolizumab. These findings highlight that using differential gene correlation analysis offers insight into the molecular regulatory effects of treatment on gene interactions and may lead to better understanding of disease mechanisms and therapeutic responses. ClinicalTrials.gov ID: NCT03292588.

While biologic therapies targeting type 2 (T2) inflammation reduce acute exacerbation rates in children with asthma and T2 inflammation, exacerbations still occur, and the underlying molecular mechanisms are poorly defined. To identify multiple distinct molecular mechanisms implicated in asthma exacerbations by characterizing respiratory illnesses among urban children with eosinophilic asthma enrolled in a clinical trial comparing treatment with mepolizumab vs placebo. This is a secondary analysis of the Mechanisms Underlying Asthma Exacerbations Prevented and Persistent With Immune-Based Therapy: A Systems Approach Phase 2 (MUPPITS-2) double-blind, placebo-controlled, parallel-group, randomized clinical trial comparing treatment with mepolizumab vs placebo among children with exacerbation-prone asthma in low-income urban centers in 9 US cities. Data analysis was performed from September 2022 to April 2025. Participants were randomized to receive either mepolizumab (aged 6-11 years: 40 mg; aged 12-17 years: 100 mg) or matching placebo by subcutaneous injection once every 4 weeks for 52 weeks. The primary measurement was a transcriptomic modular analysis by RNA sequencing of nasal samples obtained during acute respiratory illnesses. Associations among upper airway transcriptional signatures, the clinical outcome of respiratory illnesses, and pulmonary functions were investigated. Of the 290 participants enrolled in the MUPPITS-2 trial, 108 participants (median [IQR] age, 10.0 [9.0-13.0] years; 48 [44%] female) were sampled during 176 acute respiratory illness events. During illness events resulting in asthma exacerbations, children receiving mepolizumab demonstrated decreased expression of an eosinophil-associated module associated with T2 inflammation (log2 fold change [FC] estimate, −0.60; false discovery rate [FDR] < .05) but increased expression of gene modules associated with epithelial and macrophage inflammatory pathways relative to children receiving placebo (log2 FC estimates, 0.22-0.85; FDR < .05). Both groups showed higher expression of mucus secretion and cellular stress response pathways during exacerbations relative to nonexacerbation illnesses. The mepolizumab group demonstrated upregulation of epithelial inflammatory pathways in exacerbations irrespective of a respiratory virus, while macrophage pathways contributed specifically to viral exacerbations. Three distinct, semiorthogonal inflammatory axes were shown to underlie the majority of the heterogeneity among exacerbations in the 2 groups. The study’s findings implicate multiple alternative inflammatory pathways associated with the epithelium and macrophages, as well as mucus hypersecretion, as mechanisms of residual acute exacerbations in children receiving mepolizumab. Further, they indicate that multiple distinct inflammatory axes can independently contribute to asthma exacerbations. ClinicalTrials.gov Identifier: NCT03292588

Background: Determining why some upper respiratory illnesses provoke asthma exacerbations remains an unmet need. Objective: We sought to identify transcriptome-wide gene expression changes associated with colds that progress to exacerbation. Methods: Two hundred eight urban children (6-17 years) with exacerbation-prone asthma were prospectively monitored for up to 2 cold illnesses. Exacerbation illnesses (Ex(+)), defined as colds leading to asthma exacerbations requiring systemic corticosteroids within 10 days, were compared to colds that resolved without exacerbation (Ex(-)). Peripheral blood and nasal lavage samples were collected at baseline and during colds for RNA sequencing. Interferon gene expression was compared between Ex(+) and Ex(-) illnesses. Generalized additive modeling revealed interferon response kinetics. Multiple linear regression models compared interferon expression to clinical variables. Results: One hundred six participants were evaluated during 154 colds. There was greater upregulation of differentially expressed interferon genes during Ex(+) illnesses compared to Ex(-). Ex(+) illnesses had greater average and steeper rise in interferon expression. Within 3 days of illness, interferon expression was positively associated with nasal rhinovirus quantity (nasal: adjusted R-2 = 0.48, P = .015; blood: adjusted R-2 = 0.22, P = .013), and interferon expression was negatively associated with percentage predicted forced expiratory volume in 1 second (nasal: beta = -0.010, P = .048; blood: beta = -0.008, P = .023). Participants with lower baseline interferon expression had shorter time to exacerbation, higher risk for exacerbation with viral illnesses, and greater increase in interferon expression during viral colds (nasal: beta = -0.80, P < .0001; blood: beta = -0.75, P < .0001). Conclusion: Dysregulated interferon responses are important contributors to asthma exacerbation risk in children. Low baseline interferon expression followed by greater upregulation of interferon pathways in airway and blood during respiratory illnesses increased exacerbation risk. Targeting this pathway in at-risk individuals holds promise for the personalized prevention of asthma exacerbations.

Background: While biologic therapies targeting type 2 (T2) inflammation reduce acute exacerbation rates in children with asthma and T2 inflammation, exacerbations still occur, and the underlying molecular mechanisms are poorly defined. We aimed to identify the multiple distinct molecular mechanisms implicated in asthma exacerbations in children by contrasting exacerbations among urban children with eosinophilic asthma enrolled in a clinical trial comparing treatment with mepolizumab versus placebo.Methods: We performed transcriptomic modular analysis of nasal samples obtained during acute respiratory illnesses from urban children with exacerbation-prone eosinophilic asthma enrolled in the MUPPITS-2 clinical trial, which compared treatment with mepolizumab versus placebo. We investigated associations among upper airway transcriptional signatures, respiratory illnesses that resulted in exacerbations, pulmonary function, and biomarkers of T2 inflammation.Findings: Of the 290 participants enrolled in the MUPPITS-2 trial, 108 participants were sampled during 176 acute respiratory illness events. During illness events resulting in asthma exacerbations, children receiving mepolizumab demonstrated decreased expression of an eosinophil-associated module related to T2 inflammation but increased expression of gene modules associated with epithelial and macrophage inflammatory pathways relative to children receiving placebo. Both groups showed higher expression of mucus secretion and cellular stress response pathways during exacerbations relative to non-exacerbation illnesses. The mepolizumab group demonstrated upregulation of epithelial inflammatory pathways in exacerbations irrespective of a respiratory virus while macrophage pathways occurred specifically in viral exacerbations.Interpretation: Our findings implicate multiple alternative inflammatory pathways related to the epithelium and macrophages, as well as mucus hypersecretion, as probable mechanisms responsible for residual acute exacerbations in children receiving mepolizumab. Further, they indicate that multiple distinct inflammatory axes contribute to asthma exacerbations.Funding: The research reported in this publication was supported by NIH-NIAID award numbers 5UM1AI114271, UM1AI160040, and UM2AI117870, with additional support provided through award numbers UL1TRG01422, UL1RR025741, UL1TR000150, UL1TR001422, UL1 TR002535, UL1TR001876, and 5UL1TR001425–03. Additional support was also provided through an unrestricted grant from GlaxoSmithKline.Declaration of Interest: All authors except A. Togias and P. Becker report grants from NIH/NIAID during the conduct of study. W. Busse reports consulting fees from Novartis, GlaxoSmithKline, Genentech, Sanofi, AstraZeneca and Regeneron, royalties from Elsevier outside the submitted work. M. Gill reports an honorarium for and support for travel to the 2017 AAAAI meeting during the conduct of study and monetary compensation from the American Academy of Pediatrics for her work teaching the biannual Pediatrics board review course, PREP The Course. K. Hershey reports grants from Adare, during the conduct of the study. D. Jackson reports personal fees from Novartis, Pfizer, Regeneron, AstraZeneca, Sanofi and Vifor Pharma, grants and personal fees from GlaxoSmithKline and grants from NIH/NHLBI, outside the submitted work. M. Kattan reports personal fees from Regeneron, outside the submitted work. R. Gruchalla reports government employment from Center for Biologics Evaluation and Research as well as personal fees from Consulting Massachusetts Medical Society, outside the submitted work. A. Liu reports personal fees from Phadia ThermoFisher as consulting honoraria, grants and non-financial support from ResMed/Propeller Health, non-financial support from Revenio, grants and personal fees from Avillion and personal fees from Labcorp, outside the submitted work. L. Bacharier reports book royalties from Elsevier, consulting fees from Sanofi, Regeneron, Genentech, GlaxoSmithKline, DBV technologies, Teva, Medscape, Kinaset, OM Pharma and AstraZeneca, honoraria from Sanofi, Regeneron and GlaxoSmithKline, participation in advisory board for DBV Technologies, AstraZeneca and Vertex, leadership role in American Academy of Allergy Asthma & Immunology and American Board of Allergy and Immunology and medical writing services for Sanofi/Regeneron. J. Gern reports consulting fees from AstraZeneca and Meissa Vaccines, two patents related to the methods to enhance the production of rhinoviruses and stock options with Meissa Vaccines. S. Teach reports grants from NIH–NHLBI, payment from Medscape and personal fees from Uptodate. S. Lynch reports personal fees from Siolta Therapeutics, personal fees from Sanofi, a patent Reductive prodrug cancer chemothera (Stan449-PRV) issued, a patent Combination antibiotic and antibody therapy for the treatment of Pseudomonas aeruginosa infection; WO 2010091189 A1 with royalties paid to KaloBios Inc., a patent Therapeutic microbial consortium for induction of immune tolerance licensed to Siolta Therapeutics, a patent Systems and methods for detecting antibiotic resistance (WO 2012027302 A3) issued, a patent Nitroreductase enzymes. US 7687474 B2 issued, a patent Sinusitis diagnostics and treatments WO 2013155370 A1 issued, a patent Methods and systems for phylogenetic analysis US 20120264637 A1 issued, and a patent methods and compositions relating to epoxide hydrolase genes issued to Siolta Therapeutics and reports that she is a co-founder of Siolta Therapeutics, a start up company that is developing a mixed-species microbial oral therapeutic for induction of immune tolerance. All other authors declare no competing interests.Ethical Approval: The protocol was approved by a central IRB. The legal guardians of all study participants provided written informed consent and children provided assent.

BACKGROUND: Characterization of allergic responses to cockroach (CR), a common aeroallergen associated with asthma, has focused mainly on IgE reactivity, but little is known about T cell responses, particularly in children. We conducted a functional evaluation of CR allergen-specific T cell reactivity in a cohort of CR allergic children with asthma. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 71 children, with mild-to-moderate asthma who were enrolled in a CR immunotherapy (IT) clinical trial, prior to treatment initiation. PBMC were stimulated with peptide pools derived from 11 CR allergens, and CD4+ T cell responses assessed by intracellular cytokine staining. RESULTS: Highly heterogeneous responses in T cell reactivity were observed among participants, both in terms of the magnitude of cytokine response and allergen immunodominance. Reactivity against Bla g 9 and Bla g 5 was most frequent. The phenotype of the T cell response was dominated by IL-4 production and a Th2 polarized profile in 54.9% of participants, but IFN production and Th1 polarization was observed in 25.3% of the participants. The numbers of regulatory CD4+ T cells were also highly variable and the magnitude of effector responses and Th2 polarization were positively correlated with serum IgE levels specific to a clinical CR extract. CONCLUSIONS: Our results demonstrate that in children with mild-to-moderate asthma, CR-specific T cell responses display a wide range of magnitude, allergen dominance, and polarization. These results will enable examination of whether any of the variables measured are affected by IT and/or are predictive of clinical outcomes.

The chronic inflammatory component of asthma is propagated by granulocytes, including neutrophils and eosinophils, in the peripheral circulation and airway. Previous studies have suggested that these cells have an altered expression of adhesion-related molecules and a propensity for the release of granule contents that may contribute to tissue damage and enhance inflammatory complications in patients with status asthmaticus. The goal of this prospective cohort study at a tertiary care pediatric hospital with a large population of asthma patients was to assess the role of granulocyte-based inflammation in the development of asthma exacerbation. Subjects were enrolled from two patient populations: those with mild-to-moderate asthma exacerbations seen in the emergency department and those with severe asthma admitted to the intensive care unit (PICU). Clinical data were collected, and blood was drawn. Granulocytes were immediately purified, and the phenotype was assessed, including the expression of cell surface markers, elastase release, and cytokine production. Severe asthmatics admitted to the PICU displayed a significantly higher total neutrophil count when compared with healthy donors. Moreover, little to no eosinophils were found in granulocyte preparations from severe asthmatics. Circulating neutrophils from severe asthmatics admitted to the PICU displayed significantly increased elastase release ex vivo when compared with the PMN from healthy donors. These data suggest that the neutrophil-based activation and release of inflammatory products displayed by severe asthmatics may contribute to the propagation of asthma exacerbations.

Background: The discriminatory and racist policy of historical redlining in the United States during the 1930s played a role in perpetuating contemporary environmental health disparities. Objective: Our objectives were to determine associations between home and school pollutant exposure (fine particulate matter [PM2.5], NO2) and respiratory outcomes (Composite Asthma Severity Index, lung function) among school-aged children with asthma and examine whether associations differed between children who resided and/or attended school in historically redlined compared to non-redlined neighborhoods. Methods: Children ages 6 to 17 with moderate-to-severe asthma (N = 240) from 9 US cities were included. Combined home and school exposure to PM2.5 and NO2 was calculated based on geospatially assessed monthly averaged outdoor pollutant concentrations. Repeated measures of Composite Asthma Severity Index and lung function were collected. Results: Overall, 37.5% of children resided and/or attended schools in historically redlined neighborhoods. Children in historically redlined neighborhoods had greater exposure to NO2 (median: 15.4 vs 12.1 parts per billion) and closer distance to a highway (median: 0.86 vs 1.23 km), compared to those in non-redlined neighborhoods (P < .01). Overall, PM2.5 was not associated with asthma severity or lung function. However, among children in redlined neighborhoods, higher PM2.5 was associated with worse asthma severity (P < .005). No association was observed between pollutants and lung function or asthma severity among children in non-redlined neighborhoods (P > .005). Conclusions: Our findings highlight the significance of historical redlining and current environmental health disparities among school-aged children with asthma, specifically, the environmental injustice of PM2.5 exposure and its associations with respiratory health.

BACKGROUND:MUPPITS-2 was a randomized, placebo-controlled clinical trial that demonstrated mepolizumab (anti-IL-5) reduced exacerbations and blood and airway eosinophils in urban children with severe eosinophilic asthma. Despite this reduction in eosinophilia, exacerbation risk persisted in certain patients treated with mepolizumab. This raises the possibility that subpopulations of airway eosinophils exist that contribute to breakthrough exacerbations. OBJECTIVE:We aimed to determine the effect of mepolizumab on airway eosinophils in childhood asthma. METHODS:Sputum samples were obtained from 53 MUPPITS-2 participants. Airway eosinophils were characterized using mass cytometry and grouped into subpopulations using unsupervised clustering analyses of 38 surface and intracellular markers. Differences in frequency and immunophenotype of sputum eosinophil subpopulations were assessed based on treatment arm and frequency of exacerbations. RESULTS:Median sputum eosinophils were significantly lower among participants treated with mepolizumab compared with placebo (58% lower, 0.35% difference [95% CI 0.01, 0.74], P = .04). Clustering analysis identified 3 subpopulations of sputum eosinophils with varied expression of CD62L. CD62Lint and CD62Lhi eosinophils exhibited significantly elevated activation marker and eosinophil peroxidase expression, respectively. In mepolizumab-treated participants, CD62Lint and CD62Lhi eosinophils were more abundant in participants who experienced exacerbations than in those who did not (100% higher for CD62Lint, 0.04% difference [95% CI 0.0, 0.13], P = .04; 93% higher for CD62Lhi, 0.21% difference [95% CI 0.0, 0.77], P = .04). CONCLUSIONS:Children with eosinophilic asthma treated with mepolizumab had significantly lower sputum eosinophils. However, CD62Lint and CD62Lhi eosinophils were significantly elevated in children on mepolizumab who had exacerbations, suggesting that eosinophil subpopulations exist that contribute to exacerbations despite anti-IL-5 treatment.

BackgroundCockroach allergy contributes to morbidity among urban children with asthma. Few trials address the effect of subcutaneous immunotherapy (SCIT) with cockroach allergen among these at-risk children.ObjectiveTo determine if nasal allergen challenge (NAC) responses to cockroach allergen would improve following one year of SCIT.MethodsUrban children with asthma, that were cockroach-sensitized and reactive on NAC, participated in a yearlong randomized double-blind placebo-controlled SCIT trial using German cockroach extract. The primary endpoint was the change in mean total nasal symptoms scores (TNSS) during NAC after 12 months of SCIT. Changes in nasal transcriptomic responses during NAC, skin prick test (SPT) wheal size, serum allergen-specific antibody production and T-cell responses to cockroach allergen were assessed.ResultsChanges in mean NAC TNSS did not differ between SCIT-assigned (n=28) versus placebo-assigned (n=29) participants (p=0.63). Nasal transcriptomic responses correlated with TNSS, but a treatment effect was not observed. Cockroach serum specific IgE (sIgE) decreased to a similar extent in both groups, while decreased cockroach SPT wheal size was greater among SCIT participants (p=0.04). A 200-fold increase in cockroach sIgG4 was observed among subjects receiving SCIT (p<0.001) but was unchanged in the placebo group. T-cell interleukin-4 responses following cockroach allergen stimulation decreased to a greater extent among SCIT versus placebo (p=0.002), while no effect was observed for interleukin-10 or interferon-gamma.ConclusionA year of SCIT failed to alter NAC TNSS and nasal transcriptome responses to cockroach allergen challenge despite systemic effects on allergen-specific skin tests, induction of serum sIgG4 production and down-modulation of allergen stimulated T-cell responses.

Human Dendritic Cell (DC) responses are linked to type 2 (T2) inflammation and asthma pathogenesis. Biologic therapies targeting allergic mediators IgE and IL-5 improve DC function (PMID 28870461, 35413355). IL-4Ra blockade improves clinical outcomes in T2 asthma but underlying mechanisms remain partially undefined (PMID 34879449). We aimed to investigate the impact of IL-4 on human blood mDC functions.

Background: Most genetic studies of asthma and allergy have focused on common variation in individuals primarily of European ancestry. Studying the role of rare variation in quantitative phenotypes and in asthma phenotypes in populations of diverse ancestries can provide additional, important insights into the development of these traits. Objective: We sought to examine the contribution of rare variants to different asthma- or allergy-associated quantitative traits in children with diverse ancestries and explore their role in asthma phenotypes. Methods: We examined whole-genome sequencing data from children participants in longitudinal studies of asthma (n = 1035; parent-identified as 67% Black and 25% Hispanic) to identify rare variants (minor allele frequency < 0.01). We assigned variants to genes and tested for associations using an omnibus variant -set test between each of 24,902 genes and 8 asthma-associated quantitative traits. On combining our results with external data on predicted gene expression in humans and mouse knockout studies, we identified 3 candidate genes. A burden of rare variants in each gene and in a combined 3 -gene score was tested for its associations with clinical phenotypes of asthma. Finally, published single-cell gene expression data in lower airway mucosal cells after allergen challenge were used to assess transcriptional responses to allergen. Results: Rare variants in USF1 were significantly associated with blood neutrophil count (P = 2.18 x 10(-7)); rare variants in TNFRSF21 with total IgE (P = 6.47 x 10(-6)) and PIK3R6 with eosinophil count (P = 4.10 x 10(-5)) reached suggestive significance. These 3 findings were supported by independent data from human and mouse studies. A burden of rare variants in TNFRSF21 and in a 3 -gene score was associated with allergyrelated phenotypes in cohorts of children with mild and severe asthma. Furthermore, TNFRSF21 was significantly upregulated in bronchial basal epithelial cells from adults with allergic asthma but not in adults with allergies (but not asthma) after allergen challenge. Conclusions: We report novel associations between rare variants in genes and allergic and inflammatory phenotypes in children with diverse ancestries, highlighting TNFRSF21 as contributing to the development of allergic asthma.

Neutrophils and eosinophils share common hematopoietic precursors and usually diverge into distinct lineages with unique markers before being released from their hematopoietic site, which is the bone marrow (BM). However, previous studies identified an immature Ly6g(+) Il-5Rα(+) neutrophil population in mouse BM, expressing both neutrophil and eosinophil markers suggesting hematopoietic flexibility. Moreover, others have reported neutrophil populations expressing eosinophil-specific cell surface markers in tissues and altered disease states, confusing the field regarding eosinophil origins, function, and classification. Despite these reports, it is still unclear whether hematopoietic flexibility exists in human granulocytes. To answer this, we utilized single-cell RNA sequencing (scRNA-seq) and CITE-seq to profile human BM and circulating neutrophils and eosinophils at different stages of differentiation and determine whether neutrophil plasticity plays role in asthmatic inflammation. We show that immature metamyelocyte neutrophils in humans expand during severe asthmatic inflammation and express both neutrophil and eosinophil markers. We also show an increase in tri-lobed eosinophils with mixed neutrophil and eosinophil markers in allergic asthma and that IL-5 promotes differentiation of immature blood neutrophils into tri-lobed eosinophilic phenotypes suggesting a mechanism of emergency granulopoiesis to promote myeloid inflammatory or remodeling response in patients with chronic asthma. By providing insights into unexpectedly flexible granulocyte biology and demonstrating emergency hematopoiesis in asthma, our results highlight the importance of granulocyte plasticity in eosinophil development and allergic diseases.

Background Asthma prevalence and severity have markedly increased with urbanisation, and children in low-income urban centres have among the greatest asthma morbidity. Outdoor air pollution has been associated with adverse respiratory effects in children with asthma. However, the mechanisms by which air pollution exposure exacerbates asthma, and how these mechanisms compare with exacerbations induced by respiratory viruses, are poorly understood. We aimed to investigate the associations between regional air pollutant concentrations, respiratory illnesses, lung function, and upper airway transcriptional signatures in children with asthma, with particular focus on asthma exacerbations occurring in the absence of respiratory virus. Methods We performed a retrospective analysis of data from the MUPPITS1 cohort and validated our findings in the ICATA cohort. The MUPPITS1 cohort recruited 208 children aged 6-17 years living in urban areas across nine US cities with exacerbation-prone asthma between Oct 7, 2015, and Oct 18, 2016, and monitored them during reported respiratory illnesses. The last MUPPITS1 study visit occurred on Jan 6, 2017. The ICATA cohort recruited 419 participants aged 6-20 years with persistent allergic asthma living in urban sites across eight US cities between Oct 23, 2006, and March 25, 2008, and the last study visit occurred on Dec 30, 2009. We included participants from the MUPPITS1 cohort who reported a respiratory illness at some point during the follow-up and participants from the ICATA cohort who had nasal samples collected during respiratory illness or at a scheduled visit. We used air quality index values and air pollutant concentrations for PM (2 center dot 5), PM10, O-3, NO2, SO2, CO, and Pb from the US Environmental Protection Agency spanning the years of both cohorts, and matched values and concentrations to each illness for each participant. We investigated the associations between regional air pollutant concentrations and respiratory illnesses and asthma exacerbations, pulmonary function, and upper airway transcriptional signatures by use of a combination of generalised additive models, case crossover analyses, and generalised linear mixed-effects models. Findings Of the 208 participants from the MUPPITS1 cohort and 419 participants from the ICATA cohort, 168 participants in the MUPPITS1 cohort (98 male participants and 70 female participants) and 189 participants in the ICATA cohort (115 male participants and 74 female participants) were included in our analysis. We identified that increased air quality index values, driven predominantly by increased PM2 center dot 5 and O-3 concentrations, were significantly associated with asthma exacerbations and decreases in pulmonary function that occurred in the absence of a provoking viral infection. Moreover, individual pollutants were significantly associated with altered gene expression in coordinated inflammatory pathways, including PM2 center dot 5 with increased epithelial induction of tissue kallikreins, mucus hypersecretion, and barrier functions and O-3 with increased type-2 inflammation. Interpretation Our findings suggest that air pollution is an important independent risk factor for asthma exacerbations in children living in urban areas and is potentially linked to exacerbations through specific inflammatory pathways in the airway. Further investigation of these potential mechanistic pathways could inform asthma prevention and management approaches.

Impaired lung function in early life is associated with the subsequent development of chronic respiratory disease. Most genetic associations with lung function have been identified in adults of European descent and therefore may not represent those most relevant to pediatric populations and populations of different ancestries. In this study, we performed genome-wide association analyses of lung function in a multiethnic cohort of children (n = 1,035) living in low-income urban neighborhoods. We identified one novel locus at the TDRD9 gene in chromosome 14q32.33 associated with percent predicted forced expiratory volume in one second (FEV 1 ) (p = 2.4x10 -9 ; β z = -0.31, 95% CI = -0.41- -0.21). Mendelian randomization and mediation analyses revealed that this genetic effect on FEV 1 was partially mediated by DNA methylation levels at this locus in airway epithelial cells, which were also associated with environmental tobacco smoke exposure (p = 0.015). Promoter-enhancer interactions in airway epithelial cells revealed chromatin interaction loops between FEV 1 -associated variants in TDRD9 and the promoter region of the PPP1R13B gene, a stimulator of p53-mediated apoptosis. Expression of PPP1R13B in airway epithelial cells was significantly associated the FEV 1 risk alleles (p = 1.3x10 -5 ; β = 0.12, 95% CI = 0.06–0.17). These combined results highlight a potential novel mechanism for reduced lung function in urban youth resulting from both genetics and smoking exposure.

Mepolizumab (anti-IL5) reduces asthma exacerbations in urban children. We previously utilized nasal transcriptomics to identify inflammatory pathways (gene co-expression modules) associated with exacerbations despite this therapy. To understand mepolizumab's precise impact on these pathways, we assess gene co-expression and loss of correlation, "decoherence," using differential co-expression network analyses. 290 urban children (6-17 years) with exacerbation-prone asthma and blood eosinophils ≥150/microliter were randomized (1:1) to q4 week placebo or mepolizumab injections added to guideline-based care for 52 weeks. Nasal lavage samples were collected before and during treatment for RNA-sequencing. Differential co-expression of gene networks was evaluated to assess interactions and regulatory aspects of type-2 and eosinophilic airway inflammation. Mepolizumab, but not placebo, significantly reduced the overall expression of an established type-2 inflammation gene co-expression module (fold change=0.77, p=0.002) enriched for eosinophil, mast cell, and epithelial IL-13 response genes (242 genes). Mepolizumab uncoupled co-expression of genes in this pathway. During mepolizumab, but not placebo treatment, there was significant loss of correlation among eosinophil-specific genes including RNASE2 (EDN), RNASE3 (ECP), CLC, SIGLEC8, and IL5RA contrasting a reciprocal increase in correlation among mast cell-specific genes (TPSAB1, CPA3, FCER1A), T2 cytokines (IL4, IL5, and IL13), and POSTN. These results suggest mepolizumab disrupts the regulatory interactions of gene co-expression among airway eosinophils, mast cells and epithelium by interrupting transcription regulation in eosinophils with enhancement in mast cell and epithelial inflammation. This paradoxical effect may contribute to an incomplete reduction of asthma exacerbations and demonstrates how differential co-expression network analyses can identify targets for more precise therapies.