An integrated molecular risk score early in life for subsequent childhood asthma risk.

BACKGROUND: Numerous children present with early wheeze symptoms, yet solely a subgroup develops childhood asthma. Early identification of children at risk is key for clinical monitoring, timely patient-tailored treatment, and preventing chronic, severe sequelae. For early prediction of childhood asthma, we aimed to define an integrated risk score combining established risk factors with genome-wide molecular markers at birth, complemented by subsequent clinical symptoms/diagnoses (wheezing, atopic dermatitis, food allergy). METHODS: Three longitudinal birth cohorts (PAULINA/PAULCHEN, n = 190 + 93 = 283, PASTURE, n = 1133) were used to predict childhood asthma (age 5-11) including epidemiological characteristics and molecular markers: genotype, DNA methylation and mRNA expression (RNASeq/NanoString). Apparent (ap) and optimism-corrected (oc) performance (AUC/R2) was assessed leveraging evidence from independent studies (Naïve-Bayes approach) combined with high-dimensional logistic regression models (LASSO). RESULTS: Asthma prediction with epidemiological characteristics at birth (maternal asthma, sex, farm environment) yielded an ocAUC = 0.65. Inclusion of molecular markers as predictors resulted in an improvement in apparent prediction performance, however, for optimism-corrected performance only a moderate increase was observed (upto ocAUC = 0.68). The greatest discriminate power was reached by adding the first symptoms/diagnosis (up to ocAUC = 0.76; increase of 0.08, p = .002). Longitudinal analysis of selected mRNA expression in PASTURE (cord blood, 1, 4.5, 6 years) showed that expression at age six had the strongest association with asthma and correlation of genes getting larger over time (r = .59, p < .001, 4.5-6 years). CONCLUSION: Applying epidemiological predictors alone showed moderate predictive abilities. Molecular markers from birth modestly improved prediction. Allergic symptoms/diagnoses enhanced the power of prediction, which is important for clinical practice and for the design of future studies with molecular markers.

From preschool wheezing to asthma: Immunological determinants.

Asthma represents a chronic respiratory disease affecting millions of children worldwide. The transition from preschool wheezing to school-age asthma involves a multifaceted interplay of various factors, including immunological aspects in early childhood. These factors include complex cellular interactions among different immune cell subsets, induction of pro-inflammatory mediators and the molecular impact of environmental factors like allergens or viral infections on the developing immune system. Furthermore, the activation of specific genes and signalling pathways during this early phase plays a pivotal role in the manifestation of symptoms and subsequent development of asthma. Early identification of the propensity or risk for asthma development, for example by allergen sensitisation and viral infections during this critical period, is crucial for understanding the transition from wheeze to asthma. Favourable immune regulation during a critical 'window of opportunity' in early childhood can induce persistent changes in immune cell behaviour. In this context, trained immunity, including memory function of innate immune cells, has significant implications for understanding immune responses, potentially shaping long-term immunological outcomes based on early-life environmental exposures. Exploration of these underlying immune mechanisms that drive disease progression will provide valuable insights to understand childhood asthma development. This will be instrumental to develop preventive strategies at different stages of disease development for (i) inhibiting progression from wheeze to asthma or (ii) reducing disease severity and (iii) uncovering novel therapeutic strategies and contributing to more tailored and effective treatments for childhood asthma. In the long term, this shall empower healthcare professionals to develop evidence-based interventions that reduce the burden of asthma for children, families and society overall.

What Have Mechanistic Studies Taught Us About Childhood Asthma?

Childhood asthma is a chronic heterogeneous syndrome consisting of different disease entities or phenotypes. The immunologic and cellular processes that occur during asthma development are still not fully understood but represent distinct endotypes. Mechanistic studies have examined the role of gene expression, protein levels, and cell types in early life development and the manifestation of asthma, many under the influence of environmental stimuli, which can be both protective and risk factors for asthma. Genetic variants can regulate gene expression, controlled partly by different epigenetic mechanisms. In addition, environmental factors, such as living space, nutrition, and smoking, can contribute to these mechanisms. All of these factors produce modifications in gene expression that can alter the development and function of immune and epithelial cells and subsequently different trajectories of childhood asthma. These early changes in a partially immature immune system can have dramatic effects (e.g., causing dysregulation), which in turn contribute to different disease endotypes and may help to explain differential responsiveness to asthma treatment. In this review, we summarize published studies that have aimed to uncover distinct mechanisms in childhood asthma, considering genetics, epigenetics, and environment. Moreover, a discussion of new, powerful tools for single-cell immunologic assays for phenotypic and functional analysis is included, which promise new mechanistic insights into childhood asthma development and therapeutic and preventive strategies.

Timing of Blood Sample Processing Affects the Transcriptomic and Epigenomic Profiles in CD4+ T-cells of Atopic Subjects.

Optimal pre-analytical conditions for blood sample processing and isolation of selected cell populations for subsequent transcriptomic and epigenomic studies are required to obtain robust and reproducible results. This pilot study was conducted to investigate the potential effects of timing of CD4+ T-cell processing from peripheral blood of atopic and non-atopic adults on their transcriptomic and epigenetic profiles. Two heparinized blood samples were drawn from each of three atopic and three healthy individuals. For each individual, CD4+ T-cells were isolated from the first blood sample within 2 h (immediate) or from the second blood sample after 24 h storage (delayed). RNA sequencing (RNA-Seq) and histone H3K27 acetylation chromatin immunoprecipitation sequencing (ChIP-Seq) analyses were performed. A multiplicity of genes was shown to be differentially expressed in immediately processed CD4+ T-cells from atopic versus healthy subjects. These differences disappeared when comparing delayed processed cells due to a drastic change in expression levels of atopy-related genes in delayed processed CD4+ T-cells from atopic donors. This finding was further validated on the epigenomic level by examining H3K27 acetylation profiles. In contrast, transcriptomic and epigenomic profiles of blood CD4+ T-cells of healthy donors remained rather unaffected. Taken together, for successful transcriptomics and epigenomics studies, detailed standard operation procedures developed on the basis of samples from both healthy and disease conditions are implicitly recommended.

IgA+ memory B-cells are significantly increased in patients with asthma and small airway dysfunction.

BACKGROUND: Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS: In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS: Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS: With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

T2-high asthma phenotypes across lifespan.

RATIONALE: In adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children. OBJECTIVES: To define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages. METHODS: In the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28. MEASUREMENTS AND MAIN RESULTS: Based on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: "atopy-only", "eosinophils-only", "T2-high" (eosinophilia + atopy) and "T2-low" (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (p<0.0001) and, from school age onwards, by increased production of IL-5 after anti-CD3/CD28 stimulation of whole blood. CONCLUSIONS: Using easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

17q12-21 risk-variants influence cord blood immune regulation and multitrigger-wheeze.

BACKGROUND: Childhood wheeze represents a first symptom of asthma. Early identification of children at risk for wheeze related to 17q12-21 variants and their underlying immunological mechanisms remain unknown. We aimed to assess the influence of 17q12-21 variants and mRNA expression at birth on the development of wheeze. METHODS: Children were classified as multitrigger/viral/no wheeze until six years of age. The PAULINA/PAULCHEN birth cohorts were genotyped (n = 216; GSA-chip). mRNA expression of 17q21 and innate/adaptive genes was measured (qRT-PCR) in cord blood mononuclear cells. Expression quantitative trait loci (eQTL) and mediation analyses were performed. Genetic variation of 17q12-21 asthma-single nucleotide polymorphisms (SNPs) was summarized as the first principal component (PC1) and used to classify single SNP effects on gene expression as (locus)-dependent/independent eQTL SNPs. RESULTS: Core region risk variants (IKZF3, ZPBP2, GSDMB, ORMDL3) were associated with multitrigger wheeze (OR: 3.05-5.43) and were locus-dependent eQTL SNPs with higher GSDMA, TLR2, TLR5, and lower TGFB1 expression. Increased risk of multitrigger wheeze with rs9303277 was in part mediated by TLR2 expression. Risk variants distal to the core region were mainly locus-independent eQTL SNPs with decreased CD209, CD86, TRAF6, RORA, and IL-9 expression. Distinct immune signatures in cord blood were associated either with multitrigger wheeze (increased innate genes, e.g., TLR2, IPS1, LY75) or viral wheeze (decreased NF-κB genes, e.g., TNFAIP3 and TNIP2). CONCLUSION: Locus-dependent eQTL SNPs (core region) associated with increased inflammatory genes (primarily TLR2) at birth and subsequent multitrigger wheeze indicate that early priming and imbalance may be crucial for asthma pathophysiology. Locus-independent eQTL SNPs (mainly distal region, rs1007654) may be involved in the initiation of dendritic cell activation/maturation (TRAF6) and interaction with T cells (CD209, CD86). Identifying potential mechanistic pathways at birth may point to critical key points during early immune development predisposing to asthma.

Fetomaternal immune cross talk modifies T-cell priming through sustained changes to DC function.

BACKGROUND: Prenatal exposure to infections can modify immune development. These environmental disturbances during early life potentially alter the incidence of inflammatory disorders as well as priming of immune responses. Infection with the helminth Schistosoma mansoni is widely studied for its ability to alter immune responsiveness and is associated with variations in coinfection, allergy, and vaccine efficacy in endemic populations. OBJECTIVE: Exposure to maternal schistosomiasis during early life, even without transmission of infection, can result in priming effects on offspring immune responses to bystander antigenic challenges as related to allergic responsiveness and vaccination, with this article seeking to further clarify the effects and underlying immunologic imprinting. METHODS: Here, we have combined a model of chronic maternal schistosomiasis infection with a thorough analysis of subsequent offspring immune responses to allergy and vaccination models, including viral challenge and steady-state changes to immune cell compartments. RESULTS: We have demonstrated that maternal schistosomiasis alters CD4+ responses during allergic sensitization and challenge in a skewed IL-4/B-cell-dominant response to antigenic challenge associated with limited inflammatory response. Beyond that, we have uncovered previously unidentified alterations to CD8+ T-cell responses during immunization that are dependent on vaccine formulation and have functional impact on the efficacy of vaccination against viral infection in a murine hepatitis B virus model. CONCLUSION: In addition to steady-state modifications to CD4+ T-cell polarization and B-cell priming, we have traced these modified CD8+ responses to an altered dendritic cell phenotype sustained into adulthood, providing evidence for complex priming effects imparted by infection via fetomaternal cross talk.

A strategy for high-dimensional multivariable analysis classifies childhood asthma phenotypes from genetic, immunological, and environmental factors.

BACKGROUND: Associations between childhood asthma phenotypes and genetic, immunological, and environmental factors have been previously established. Yet, strategies to integrate high-dimensional risk factors from multiple distinct data sets, and thereby increase the statistical power of analyses, have been hampered by a preponderance of missing data and lack of methods to accommodate them. METHODS: We assembled questionnaire, diagnostic, genotype, microarray, RT-qPCR, flow cytometry, and cytokine data (referred to as data modalities) to use as input factors for a classifier that could distinguish healthy children, mild-to-moderate allergic asthmatics, and nonallergic asthmatics. Based on data from 260 German children aged 4-14 from our university outpatient clinic, we built a novel multilevel prediction approach for asthma outcome which could deal with a present complex missing data structure. RESULTS: The optimal learning method was boosting based on all data sets, achieving an area underneath the receiver operating characteristic curve (AUC) for three classes of phenotypes of 0.81 (95%-confidence interval (CI): 0.65-0.94) using leave-one-out cross-validation. Besides improving the AUC, our integrative multilevel learning approach led to tighter CIs than using smaller complete predictor data sets (AUC = 0.82 [0.66-0.94] for boosting). The most important variables for classifying childhood asthma phenotypes comprised novel identified genes, namely PKN2 (protein kinase N2), PTK2 (protein tyrosine kinase 2), and ALPP (alkaline phosphatase, placental). CONCLUSION: Our combination of several data modalities using a novel strategy improved classification of childhood asthma phenotypes but requires validation in external populations. The generic approach is applicable to other multilevel data-based risk prediction settings, which typically suffer from incomplete data.