BACKGROUND:Preschool wheeze is heterogeneous and only a subset of children progresses to persistent asthma. We hypothesized that early-life wheeze reflects divergent airway epithelial maturation trajectories associated with distinct mucosal immune programs. METHODS:Nasal epithelial transcriptomes from 265 children and young adults (79 healthy, 81 wheezers, 105 asthmatics; median age 10.4 [1.1-20.3] years) from the All Age Asthma Cohort (ALLIANCE) cohort were analyzed using a hypothesis-driven candidate approach based on predefined cytokine axes. Transcriptional signatures were mapped to epithelial cells by scRNA-sequencing. Asthma-outcome associations were assessed in age- and sensitization-adjusted multivariate models. RESULTS:The candidate approach identified three antagonistic epithelial immune programs: an interferon-associated (E1; IDO1, CSF3, CCL20), a type-2-associated (E2; POSTN, CCL26, CST1), and a type-17-associated program (E3; IL36G, KLK7, KRTDAP). These programs were detected across basal, ciliated, and secretory epithelial cells. In early childhood (1-3 years), wheezers predominantly exhibited E1/E3-dominant epithelial profiles (n = 36). Age-group-specific comparisons showed higher E2 and lower E1/E3 expression in wheezers aged 4-6 years (n = 38) than in those aged 1-3 years (n = 36). E2 dominance correlated with IgE, FeNO, and eosinophilia (p < 0.01). In multivariate models assessing asthma outcome at age 6, E1 expression was inversely associated with asthma persistence (OR = 0.15, p < 0.05), whereas E3 expression was associated with disease progression (OR = 4.93, p < 0.05). The combined epithelial signature discriminated asthma outcomes with high accuracy (AUC = 0.90), independent of allergic sensitization. CONCLUSION:Early-life wheeze reflects distinct airway epithelial immune programs characterized by age-associated patterns in epithelial immune activity. Nasal epithelial transcriptional signatures capture molecular trajectories associated with asthma persistence and may enable early risk stratification. TRIAL REGISTRATION:All-Age-Asthma (ALLIANCE) cohort: clinicaltrials.gov: NCT02496468; adult arm: NCT02419274.

Abstract Early-life wheezing in children has been associated with microbial alterations along the gut-airway axis, yet studies simultaneously investigating bacterial communities in both compartments remain scarce. The aim of this cross-sectional exploratory pilot study (n=25) was to characterize and compare nasal and stool bacterial communities in preschool children aged 1–4 years with recurrent wheezing and healthy controls using 16S rRNA gene metabarcoding. Across participants, nasal and stool bacteriomes were highly individualized and taxonomically diverse. Overall richness, evenness, and community composition did not differ significantly between healthy children and wheezers in either compartment. However, wheezers displayed markedly higher within-group variability, particularly in nasal communities. Stratification based on microbiome similarity to healthy samples revealed increased Moraxella and reduced commensal genera including Prevotella spp. and Veillonella , along with lower richness and evenness (all p<0.001) in nasal samples with divergent bacterial communities. Stool alterations were more subtle but included trends toward reduced Bacteroides , Faecalibacterium , and Alistipes in wheezers more divergent from healthy controls. Community assembly in both compartments was largely governed by stochastic processes but accompanied by less complex and more fragmented bacterial interaction networks in wheezing children. Cross-compartment correlations were also altered, most prominently involving stool Lactococcus showing stronger and more numerous correlations with nasal taxa in wheezers than in healthy controls. Divergent wheezers exhibited distinct modular network structure and cross-compartment profiles, consistent with a differentiated microbial organization. Together, these findings suggest compartment-specific differences in microbial interaction patterns across the gut–airway axis in early-life wheezing, despite limited differences in overall community diversity. Take home message Preschool wheezers showed fragmented gut–airway microbial networks and Moraxella -associated airway community stratification despite limited differences in overall diversity.

Abstract Background The relevance of high-sensitivity C-reactive protein (hsCRP), a marker of low-grade systemic inflammation, remains unclear with regard to its association with severity and clinical outcomes in wheeze/asthma. We aimed to assess the role of hsCRP across different phenotypes and severity levels. Methods We studied children with preschool wheeze (≥ 2 episodes), and patients with GINA-defined asthma (school-age/adult) compared with healthy controls (HCs) in the well-characterized ALLIANCE (All Age Asthma Cohort) study. HsCRP was measured (AU5800®-CRP-Latex test) in 944 study participants (pediatric: n = 728; adult: n = 216) at baseline. Age-stratified analyses (age groups 0–5, 6–18, ≥ 18 years) of standardized log 10 -transformed hsCRP concentrations (age, sex, BMI, site) were performed using univariable tests and regression models. The validated ASSESS score and its dimensions (exacerbations, lung function, inhaled corticosteroids, symptom control) were primary outcomes. Results Adult patients with asthma showed higher hsCRP than HCs (OR 2.22, 95% CI 1.56–3.24). Across all ages, hsCRP increased with clinical severity of wheeze/asthma. The ASSESS score correlated positively with hsCRP in patients aged ≥ 6 years ( R = 0.19, p = 0.007 ). hsCRP was increased in school-age asthmatics with prior exacerbations (OR 1.37, 95% CI 1.01–1.87), and in adult asthmatics with impaired lung function ( R = 0.2, p = 0.013). Inhaled corticosteroid use was associated with lower hsCRP in preschool wheezers (OR 0.66, 95% CI 0.50–0.85) but higher levels in adults (OR 1.99, 95% CI 1.02–4.09). Conclusions HsCRP was increased in adult asthmatics compared to HCs and was associated with several severity-related clinical characteristics. ICS use was associated with higher hsCRP levels in adults, potentially reflecting greater disease severity, whereas ICS use in preschool wheezers was associated with lower hsCRP levels. These age-dependent effects may mirror varying disease courses across the lifespan and progression of asthma. The association of hsCRP with asthma severity in child- and adulthood may indicate its potential relevance for the course of disease and monitoring clinical outcomes. Future longitudinal studies are needed to assess, whether hsCRP may support therapy monitoring. Trial registration ClinicalTrials.gov; Pediatric arm: NCT02496468, Registration date: 03 July 2015; Adult arm: NCT02419274, Registration date: 14 April 2015.

Early-life wheezing in children has been associated with microbial alterations along the gut-airway axis, yet studies simultaneously investigating bacterial communities in both compartments remain scarce. The aim of this cross-sectional exploratory pilot study (n = 25) was to characterize and compare nasal and stool bacterial communities in preschool children aged 1–4 years with recurrent wheezing and healthy controls using 16S rRNA gene metabarcoding. Across participants, nasal and stool bacteriomes were highly individualized and taxonomically diverse. Overall richness, evenness, and community composition did not differ significantly between healthy children and wheezers in either compartment. However, wheezers displayed markedly higher within-group variability, particularly in nasal communities. Stratification based on microbiome similarity to healthy samples revealed increased Moraxella and reduced commensal genera including Prevotella spp. and Veillonella, along with lower richness and evenness (all p < 0.001) in nasal samples with divergent bacterial communities. Stool alterations were more subtle but included trends toward reduced Bacteroides, Faecalibacterium, and Alistipes in wheezers more divergent from healthy controls. Community assembly in both compartments was largely governed by stochastic processes but accompanied by less complex and more fragmented bacterial interaction networks in wheezing children. Cross-compartment correlations were also altered, most prominently involving stool Lactococcus showing stronger and more numerous correlations with nasal taxa in wheezers than in healthy controls. Divergent wheezers exhibited distinct modular network structure and cross-compartment profiles, consistent with a differentiated microbial organization. Together, these findings suggest compartment-specific differences in microbial interaction patterns across the gut–airway axis in early-life wheezing, despite limited differences in overall community diversity.

BACKGROUND:Childhood asthma and wheezing disorders are heterogeneous conditions that may be reflected by distinct immune response patterns. In this study, we investigated whether CD3/CD28-stimulated cytokine responses differ between children with asthma, preschool wheeze, and healthy controls. METHODS:Baseline samples from 511 children in the All Age Asthma cohort (ALLIANCE; 196 children with asthma, 181 with preschool wheeze, and 134 healthy controls) were analyzed. Whole blood was stimulated with anti-CD3/CD28 antibodies for 48 h, and 37 cytokines were quantified using multiplex immunoassays. Cytokine co-variation patterns were assessed using principal component analysis (PCA). Co-regulated cytokine modules were identified using CytoMod, and associations with clinical characteristics were examined using linear and logistic regression models. RESULTS:Two cytokines, sCD30 and sCD163, showed significant age-related decreases (β = -.07 (95% CI -0.09 to -0.06), padj = 1.2e-14; and β = -.03 (95% CI -0.04 to -0.01), padj = .018, respectively). After correction for multiple testing, no individual cytokine differed significantly between children with asthma, preschool wheeze, or healthy controls. PCA demonstrated highly conserved cytokine co-variation patterns across groups (Spearman's rho >.9 for the first principal component loadings). Modular analysis identified six distinct co-regulatory modules after adjustment for background cytokine levels. Associations between module expression and clinical characteristics were generally modest. Module 1, comprising cytokines associated with tissue remodeling and IL-6 signaling, was inversely associated with asthma in children aged ≥6 years (OR = 0.63 (95% CI 0.46-0.85), padj = .017). However, this association attenuated in age-matched sensitivity analyses (OR = 0.72 (95% CI 0.51-0.99), padj = .281), suggesting residual age-related confounding. CONCLUSIONS:CD3/CD28-stimulated cytokine responses show highly conserved T-cell activation networks across pediatric asthma, preschool wheeze, and health, with limited ability to distinguish between clinical groups at the single-cytokine level. Nevertheless, modular network analyses identify coordinated immune patterns associated with clinical features, highlighting the potential value of immune profiling in refining biological phenotyping and advancing the understanding of immune heterogeneity in pediatric airway disease.

Rationale: An obesogenic milieu in early life is a risk factor for childhood obesity, but the impact on asthma phenotypes is unclear. Growing evidence suggests that obesity-related meta-inflammation, in part linked to high Interleukin-6 (IL-6), may contribute to the development of lung disease in early life. Therefore, we now investigated obesity-related airway aging processes in (i) a longitudinal pediatric asthma cohort study and (ii) a complementary, intergenerational murine model of perinatal obesity. Methods: (i) 221 children with doctor-diagnosed asthma/wheeze and 33 controls from the german All-Age Asthma Cohort (ALLIANCE) were investigated. Maternal and children's metabolic parameters [BMI and Body-Fat Fraction (BFF)] were correlated with symptom control, hospital admission rate, baseline and longitudinal zFEV1 and age of asthma onset. In 120 asthmatics and controls, methylome profiling with a methylome-derived epigenetic clock model (mDNAge) was performed in respiratory epithelial cells (REC) of asthmatic children. In 324 study participants (asthmatics and controls) a cytokine cluster analysis was conducted to test for association with BMI and clinical variables. (ii) In a murine perinatal obesity model, we analyzed aging-associated processes in bronchial epithelial cells (BEC) via immunofluorescent staining after postnatal pharmacological inhibition of IL-6 cis- and trans-signaling using anti-IL-6 monoclonal antibody or soluble gp130Fc, respectively. Results: (i) In the ALLIANCE cohort, maternal BMI during pregnancy was higher in the subsequent asthmatics than in controls (p<0,05) and related to an increased rate of childhood hospital admissions (p<0,05). Moreover, BFF in early life was higher in children, who developed wheezing early in life (p < 0,01). Increased BFF was associated with lower baseline FEV1 and worse symptom control, especially in wheezers (p<0,01). On a molecular level, assessment of cytokine modules in CD3/CD28 stimulated whole blood of asthmatic children identified a cytokine cluster that correlated with high BMI (p<0,001) and lower lung function trajectory (p<0,01) in asthmatics. Furthermore, lower levels of soluble gp130 (sgp130) were associated with high BMI (p<0,001) and low symptom control in pediatric asthma (p<0,01). This high BMI-related meta-inflammation in asthmatic children with overweight was linked to higher mDNAge of RECs (p<0,01). (ii) In the murine model, targeting IL-6 trans-signaling protected against premature aging (e.g. DNA damage response) in BECs after perinatal obesity. Conclusion: Our results highlight the impact of perinatal obesity-associated meta-inflammation on asthma severity and onset in childhood. Obesity-driven premature aging of RECs could be a pathomechanism for epithelial dysfunction, triggering obesity-related asthma.

Introduction Asthma is distinctly sexually dimorphic. Young males report higher asthma rates than females. This reverses at puberty, where females present increased incidence, reduced therapeutic effectiveness and worse asthma symptoms. The underlying molecular mechanisms causing this remain unknown. Biological DNA methylation (DNAm) clocks correlate with worse outcomes in chronic diseases such as asthma. The DNAm profile of males and females with asthma in the context of DNAm aging (DNAge) is yet to be explored. As such, we aimed to characterise and explore the DNA methylation profile associated with DNAge and biological sex in asthma. Methods Nasal brushings from the paediatric ALLIANCE cohort (n = 46F/74M; asthma = 73/ healthy = 31/ wheeze = 16; avg age = 11 yr range = 3 – 20 yr) were collected and DNAm processed and analysed by Illumina EPIC array using R software (v4.1; watermelon, minfi). DNAge was calculated using the established skinHorvath clock. The association between DNAge, sex, symptom severity or frequency of inhaled corticosteroid (ICS) use was analysed. Differential methylation analysis (DMA) comparing pathway enrichment analyses was completed using R packages (limma, minfi, methylGSA). Differential DNAm significance was determined at a false discovery rate (FDR) < 0.05. Results Highly symptomatic females with asthma are enriched for an accelerated DNAge compared to their male counterparts (p = 0.03). DNAge-accelerated females present increased asthma symptom frequency compared to DNAge-decelerated females and both male groups (p < 0.0001) despite equivalent ICS use. Focussing on highly symptomatic asthmatics, DMA comparing accelerated vs normal DNAged females and males revealed 459 differentially methylated sites (FDR < 0.05). Pathway analysis reported enrichment for Toll-like receptor, MAPK and Wnt signalling pathways as well as interferon type I, virus defence and mitochondrial response pathways (FDR < 0.05). Conclusions Here, we highlight a unique subset of female asthma patients who experience more asthma symptoms despite an equivalent use of ICS. In addition, this DNAged female sub-cohort carries a distinct DNAm profile from the male counterparts, with enrichment for pathologically relevant signalling and cellular differentiation pathways. This may present a novel foundation and support for the use of the DNAge clock as a tool for the characterisation of asthma patient sub-groups.

BACKGROUND:The triple cystic fibrosis transmembrane conductance regulator modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) rapidly improves airway and systemic inflammation in people with cystic fibrosis. However, longitudinal effects on systemic inflammation and their relationship to lung function remain unknown. METHODS:In this prospective, observational, multicentre study, we analysed peripheral blood neutrophil counts, C-reactive protein (CRP) and six pro-inflammatory serum cytokines in a cohort of 198 people with cystic fibrosis aged ≥6 years at baseline and follow-up visits 3, 12 and 24 months after initiation of ETI, compared to 74 age-matched healthy control participants. RESULTS:Neutrophil counts and CRP, granulocyte colony-stimulating factor, interleukin (IL)-1β, IL-6 and IL-8 were reduced to 71%, 40%, 41%, 63%, 46% and 81% of median baseline values, respectively, after 3 months of therapy (all p<0.05), whereas monocyte chemotactic protein-1 reached 82% of baseline levels at 12 months only (p<0.05). Change from baseline to 3 months correlated with improvements in percent predicted forced expiratory volume for all systemic inflammation parameters except IL-8 (Spearman's r -0.17 to -0.42, p<0.05). All cytokines reached healthy control levels at or before 24 months. Decreased inflammation levels were sustained until 24 months for all parameters (p<0.05) except IL-6. CONCLUSIONS:Our results demonstrate that ETI exerts rapid and sustained effects on systemic inflammation associated with lung function improvements in children, adolescents and adults with cystic fibrosis in a real-world, post-approval setting. However, our data also show that individual markers of systemic inflammation remain at levels above those of healthy controls, particularly in certain subgroups, suggesting persistence or resurgence of residual systemic inflammation.

QUESTION:Epidemiological studies suggest that respiratory viral infections are major triggers of asthma exacerbations, and clinical studies have suggested the involvement of an increased interleukin-6 (IL-6) release. What is the pathophysiological role of IL-6 in asthma exacerbation, and which mechanisms lead to enhanced IL-6 release? MATERIALS AND METHODS:Exacerbations of ovalbumin-induced experimental allergic asthma were elicited in wild-type and IL-6-deficient mice by intranasal (i.n.) application of poly(I:C). Airway inflammation, cytokine expression and release, mucus production and airway hyperresponsiveness were measured. IL-6 was neutralised by i.n. anti-IL-6 antibody application. The human bronchial epithelial cell line, BEAS-2B, was stimulated with poly(I:C) and infected with human rhinovirus-16 in vitro, followed by quantification of IL6 gene expression and DNA methylation. Genome-wide DNA methylation was assessed in bronchial epithelial cells from adults with asthma (cohort I, n = 54) and in nasal epithelial cells from children and adults in the All-Age-Asthma cohort (ALLIANCE, n = 53 and n = 108 respectively). RESULTS:Poly(I:C)-induced experimental exacerbations in mice were preceded and paralleled by exaggerated IL-6 release in the airway epithelium, with IL-6 neutralisation completely preventing experimental exacerbations. Repetitive infection/stimulation with RV16 or poly(I:C) resulted in training of the IL-6 release in human respiratory epithelial cells. In patients, hypomethylation at the IL6 gene methylation was associated with high IL6 expression and future exacerbations. ANSWER:An exaggerated IL-6 release is required for exacerbation of experimental asthma, potentially the result of viral PAMP-induced immune training of airway epithelial cells. Additionally, patients with asthma carrying the epigenetic signature of a trained IL-6 response exacerbate more frequently. These findings open new avenues to identify and treat exacerbation-prone patients.

Allergy and asthma are defined by local type-2 immunity at the airway barrier. However, whether immunity type-specific epithelial phenotypes can predict the persistence of asthma at school-age, remains elusive. We analyzed nasal immunophenotypes (nasotypes; NT) by nasal transcriptomics from 265 children (79 healthy, 81 wheezers, 105 asthmatic) from the ALL-Age Asthma Cohort (ALLIANCE; DZL), median age 10.4 [1.1-20.3]. E-signatures were assigned in single-cell transcriptomics (scRNASeq) of primary 3D-airway cultures. Correlation analysis of secreted genes showed antagonistic expression between IFNG-correlated E1-NT (IDO,CSF3,CCL20), IL4-correlated E2-NT (POSTN,CCL26,CST1) and IL17C-correlated E3-NT (IL36G,KLK7,KRTDAP). E1/E2/E3 transcriptional programs were assigned to distinct cells in scRNASeq. E2-NT was highly expressed in asthmatic children (age>6) and correlated with type-2 parameters (total/specific IgE, FeNO, eosinophilia). Wheezers showed an early E1/E3-signature (age 1-3; N = 36), while E2-signature was activated from age 4 (N = 38). Multivariate models adjusted for sensitization and age (AUC=0.87) revealed E1-NT as protective (OR = 0.29;P<0.05) and E3-NT as risk factor (OR = 2.03;P<0.05) for asthma development at age 6. The assessment of the here identified three nasotypes, activated age- and disease-dependently, predicted to a high degree (AUC=0.87) the persistence of asthma with E1 showing protection from progression, but E3 independently predicting progression of disease. Supported by the German Center of Lung Research (DZL) funded by the Federal Ministry of Education and Research (BMBF), the Bavarian State Office for Health and Food Safety (LGL; K1-2497-GLB-20-V4) of the Bavarian State Ministry of Health and Care and by grants from Deutsche Forschungsgemeinschaft RTG2668 (Project A5, Project-ID: 435874434) to C.B. Schmidt-Weber. Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Introduction: There is growing evidence that small airway disease indicated by ventilation heterogeneity plays an important role in subgrouip of children with bronchial asthma. Yet clinical implications of conducting N2 Multiple Breath Washout (N2MBW) measurements remain unclear. This study aims to evaluate the role of Lung clearance index (LCI) in the diagnosis of childhood asthma and in identifying children at risk for exacerbations and a potentially more severe course of disease. Materials and Methods: Children with preschool wheeze (6 mo.- 5 ys, min. 2 episodes of wheeze within past 12 months), or asthma (age 6-17 ys, doctor‘s diagnosis of asthma according to current guideline), and healthy age matched controls were included from the multicentre longitudinal All Age Asthma Cohort (ALLIANCE). Exacerbations were defined as increased intake of short-acting-beta-agonists (SABA) in the last twelve month. N2MBW was performed according to standard operating procedures using Spiroware and LungSim software. The upper limit of normal for LCI2,5% was set to 7.1. N2MBW, clinical data and spirometry were obtained during follow-up over up to 8 years. Results: We included 91 children with wheeze (6,6 years median age, 3.2-10.7I), 171 with asthma (13 years median age, 6.5-21.8) and 53 controls (13 years median age, 4.7-20.3). 51/364 (14 %) asthmatic patients, 44/141 (31.2%) wheezers and 2/53 (3.8%) healthy controls had abnormal LCI results. LCI2.5% baseline measurements had a high specificity (96.2%) to discriminate between asthmatic and healthy children, but low sensitivity (20.3%). The majority of asthmatic children with abnormal LCI2.5% 69% (24/35) had a normal FEV1 z-score. Children with evidence of more frequent exacerbations preceding baseline measurements presented with significantly higher LCI2.5% (p = 0.014, 6.68 – 6.86 LCI2.5%), and comparable FEV1 z-score (p=0.3). Mixed linear models identified eosinophil counts (beta= 0.15; 0.05-0.24CI; p=0.0043) to predict baseline LCI2.5% after adjusting for confounders, like BMI, FEV1, age, gender and center specific effects. Discussion: Our data shows that N2MBW might help to confirm asthma in symptomatic children with normal spirometry. Furthermore, it could help to identify a special phenotype with an increased T2 inflammation, increased exacerbation frequency and potentially more severe course of the disease.

Background: Obesity early in life affects the clinical course of asthma. Maternal obesity is a risk factor for childhood obesity, but the impact on asthma phenotypes is unclear. Our previously reported findings in the All-Age Asthma Cohort (ALLIANCE) study showed a clinical association between (a) maternal BMI and asthma severity in the offspring, (b) high body-mass-index (BMI) and high bodyfat-fraction (BFF) in early life and a worse symptom control of asthma, earlier onset of symptoms and lower lung function. Additionally, (c) methylome profiling with methylome-derived epigenetic clock in nasal respiratory epithelial cells (REC) of asthmatic children showed premature epithelial cell aging in overweight asthmatics. Therefore, we now investigated obesity-related inflammation, including the pathways of adipocytokine Interleukin-6 (IL-6) in (i) the ALLIANCE cohort and (ii) a murine model of perinatal obesity with pharmacological IL-6 intervention.

Introduction: Asthma is the most common chronic condition in children, with the majority of asthma cases associated with an allergic phenotype. Sensitization rates against house dust mite (HDM) are high in children with atopy and asthma or preschool wheeze. Many patients develop early HDM sensitization with increasing number of specific IgE against HDM molecules associated with a higher symptom burden. However, it is still unclear which factors drive HDM polysensitization.