17q21 Variants Disturb Mucosal Host Defense in Childhood Asthma.

Rationale: The strongest genetic risk factor for childhood-onset asthma, the 17q21 locus, is associated with increased viral susceptibility and disease-promoting processes.Objectives: To identify biological targets underlying the escalated viral susceptibility associated with the clinical phenotype mediated by the 17q21 locus.Methods: Genome-wide transcriptome analysis of nasal brush samples from 261 children (78 healthy, 79 with wheezing at preschool age, 104 asthmatic) within the ALLIANCE (All-Age-Asthma) cohort, with a median age of 10.0 (range, 1.0-20.0) years, was conducted to explore the impact of their 17q21 genotype (SNP rs72163891). Concurrently, nasal secretions from the same patients and visits were collected, and high-sensitivity mesoscale technology was employed to measure IFN protein levels.Measurements and Main Results: This study revealed that the 17q21 risk allele induces a genotype- and asthma/wheeze phenotype-dependent enhancement of mucosal GSDMB expression as the only relevant 17q21-encoded gene in children with preschool wheeze. Increased GSDMB expression correlated with the activation of a type-1 proinflammatory, cell-lytic immune, and natural killer signature, encompassing key genes linked to an IFN type-2-signature (IFNG, CXCL9, CXCL10, KLRC1, CD8A, GZMA). Conversely, there was a reduction in IFN type 1 and type 3 expression signatures at the mRNA and protein levels.Conclusions: This study demonstrates a novel disease-driving mechanism induced by the 17q21 risk allele. Increased mucosal GSDMB expression is associated with a cell-lytic immune response coupled with compromised airway immunocompetence. These findings suggest that GSDMB-related airway cell death and perturbations in the mucosal IFN signature account for the increased vulnerability of 17q21 risk allele carriers to respiratory viral infections during early life, opening new options for future biological interventions.The All-Age-Asthma (ALLIANCE) cohort is registered at www.clinicaltrials.gov (pediatric arm, NCT02496468).

Proinflammatory pattern in the lower airways of non-asthmatic obese adolescents.

BACKGROUND: Obesity is known to be a pro-inflammatory condition affecting multiple organs. Obesity as a systemic pro-inflammatory state, might be associated with bronchial inflammation in non-smoking adolescents with a BMI ≥ 30 kg/m2 without evidence of concomitant chronic diseases. MATERIALS AND METHODS: We studied non-asthmatic obese patients (n = 20; median age 15.8 years; BMI 35.0 kg/m2) compared to age matched healthy control subjects (n = 20; median age 17.5 years; BMI 21.5 kg/m2). Induced sputum differential cell counts and sputum mRNA levels were assessed for all study subjects. Serum levels of CRP, IL-6, and IL-8 were measured. Further, IL-5, IL-6, IL-8, IL-13, IL-17, TNF-α, IFN-γ, and IP-10 protein levels were analyzed in induced sputum was. RESULTS: Serum CRP levels, sputum inflammatory cell load and sputum eosinophils differed significantly between obese and non-obese subjects, for sputum neutrophils, a correlation was shown with BMI ≥ 30 kg/m2. Differences were also observed for sputum mRNA expression of IL6, IL8, IL13, IL17, IL23, and IFN-γ, as well as the transcription factors T-bet, GATA3, and FoxP3. CONCLUSIONS: Increased bronchial inflammation, triggered by systemic or local inflammatory effects of obesity itself, may account for the higher rates of airway disease in obese adolescents.

Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

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.

Bronchial inflammation biomarker patterns link humoral immunodeficiency with bronchiectasis-related small airway dysfunction.

BACKGROUND: The progression of chronic destructive lung disease in patients with humoral immunodeficiency (ID) and concomitant development of bronchiectasis is difficult to prevent. Lung function tests in these patients typically show bronchial obstruction of the small airways in combination with increased air trapping in the distal airways, which is consistent with small airway dysfunction. OBJECTIVE: The objective was to assess the grade of chronic lower airway inflammation and small airway dysfunction from induced sputum and the corresponding local pro-inflammatory mediator pattern to discriminate patients affected by bronchiectasis-related Small Airway Dysfunction (SAD). METHODS: In a prospective design, 22 patients with ID (14 CVID, 3 XLA, 3 hyper-IgM syndrome, 1 hyper-IgE syndrome and low IgG levels due to treatment with rituximab and 1 SCID after BMT and persistent humoral defect) and 21 healthy controls were examined. Lung function, Fraction Expiratory Nitric Oxide (FeNO) and pro-inflammatory cytokine levels were compared in subsets of patients with (ID + BE) and without bronchiectasis (ID) pre-stratified using high-resolution computed tomography (HRCT) scans and control subjects. RESULTS: Analysis of induced sputum showed significantly increased total cell counts and severe neutrophilic inflammation in ID. The concomitant SAD revealed higher total cell numbers compared to ID. Bronchial inflammation in ID is clearly mirrored by pro-inflammatory mediators IL-1ß, IL-6 and CXCL-8, whilst TNF-α revealed a correlation with lung function parameters altered in the context of bronchiectasis-related Small Airway Dysfunction. CONCLUSIONS: In spite of immunoglobulin substitution, bronchial inflammation was dominated by neutrophils and was highly increased in patients with ID + BE. Notably, the pro-inflammatory cytokines in patients with ID were significantly increased in induced sputum. The context-dependent cytokine pattern in relation to the presence of concomitant bronchiectasis associated with SAD in ID patients could be helpful in delimiting ID patient subgroups and individualizing therapeutic approaches.

Secretoglobins in the big picture of immunoregulation in airway diseases.

The proteins of the secretoglobin (SCGB) family are expressed by secretory tissues of barrier organs. They are embedded in immunoregulatory and anti-inflammatory processes of airway diseases. This review particularly illustrates the immune regulation of SCGBs by cytokines and their implication in the pathophysiology of airway diseases. The biology of SCGBs is a complex topic of increasing importance, as they are highly abundant in the respiratory tract and can also be detected in malignant tissues and as elements of immune control. In addition, SCGBs react to cytokines, they are embedded in Th1 and Th2 immune responses, and they are expressed in a manner dependent on cell maturation. The big picture of the SCGB family identifies these factors as critical elements of innate immune control at the epithelial barriers and highlights their potential for diagnostic assessment of epithelial activity. Some members of the SCGB family have so far only been superficially examined, but have high potential for translational research.

IL-37 regulates allergic inflammation by counterbalancing pro-inflammatory IL-1 and IL-33.

BACKGROUND: Children with asthma have impaired production of interleukin (IL) 37; in mice, IL-37 reduces hallmarks of experimental allergic asthma (EAA). However, it remains unclear how IL-37 exerts its inhibitory properties in asthma. This study aimed to identify the mechanism(s) by which IL-37 controls allergic inflammation. METHODS: IL-37 target cells were identified by single-cell RNA-seq of IL-1R5 and IL-1R8. Airway tissues were isolated by laser-capture microdissection and examined by microarray-based gene expression analysis. Mononuclear cells (MNC) and airway epithelial cells (AECs) were isolated and stimulated with allergen, IL-1ß, or IL-33 together with recombinant human (rh) IL-37. Wild-type, IL-1R1- and IL-33-deficient mice with EAA were treated with rhIL-37. IL-1ß, IL-33, and IL-37 levels were determined in sputum and nasal secretions from adult asthma patients without glucocorticoid therapy. RESULTS: IL-37 target cells included AECs, T cells, and dendritic cells. In mice with EAA, rhIL-37 led to differential expression of >90 genes induced by IL-1ß and IL-33. rhIL-37 reduced production of Th2 cytokines in allergen-activated MNCs from wild-type but not from IL-1R1-deficient mice and inhibited IL-33-induced Th2 cytokine release. Furthermore, rhIL-37 attenuated IL-1ß- and IL-33-induced pro-inflammatory mediator expression in murine AEC cultures. In contrast to wild-type mice, hIL-37 had no effect on EAA in IL-1R1- or IL-33-deficient mice. We also observed that expression/production ratios of both IL-1ß and IL-33 to IL-37 were dramatically increased in asthma patients compared to healthy controls. CONCLUSION: IL-37 downregulates allergic airway inflammation by counterbalancing the disease-amplifying effects of IL-1ß and IL-33.

Immunological effects of adjuvanted low-dose allergoid allergen-specific immunotherapy in experimental murine house dust mite allergy.

BACKGROUND: Native allergen extracts or chemically modified allergoids are routinely used to induce allergen tolerance in allergen-specific immunotherapy (AIT), although mechanistic side-by-side studies are rare. It is paramount to balance optimal dose and allergenicity to achieve efficacy warranting safety. AIT safety and efficacy could be addressed by allergen dose reduction and/or use of allergoids and immunostimulatory adjuvants, respectively. In this study, immunological effects of experimental house dust mite (HDM) AIT were investigated applying high-dose HDM extract and low-dose HDM allergoids with and without the adjuvants microcrystalline tyrosine (MCT) and monophosphoryl lipid A (MPL) in a murine model of HDM allergy. METHODS: Cellular, humoral, and clinical effects of the different AIT strategies were assessed applying a new experimental AIT model of murine allergic asthma based on physiological, adjuvant-free intranasal sensitization followed by subcutaneous AIT. RESULTS: While low-dose allergoid and high-dose extract AIT demonstrated comparable potency to suppress allergic airway inflammation and Th2-type cytokine secretion of lung-resident lymphocytes and draining lymph node cells, low-dose allergoid AIT was less effective in inducing a potentially protective IgG1 response. Combining low-dose allergoid AIT with MCT or MCT and dose-adjusted MPL promoted Th1-inducing mechanisms and robust B-cell activation counterbalancing the allergic Th2 immune response. CONCLUSION: Low allergen doses induce cellular and humoral mechanisms counteracting Th2-driven inflammation by using allergoids and dose-adjusted adjuvants. In light of safety and efficacy improvement, future therapeutic approaches may use low-dose allergoid strategies to drive cellular tolerance and adjuvants to modulate humoral responses.

Inflammatory macrophage memory in nonsteroidal anti-inflammatory drug-exacerbated respiratory disease.

BACKGROUND: Nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (N-ERD) is a chronic inflammatory condition, which is driven by an aberrant arachidonic acid metabolism. Macrophages are major producers of arachidonic acid metabolites and subject to metabolic reprogramming, but they have been neglected in N-ERD. OBJECTIVE: This study sought to elucidate a potential metabolic and epigenetic macrophage reprogramming in N-ERD. METHODS: Transcriptional, metabolic, and lipid mediator profiles in macrophages from patients with N-ERD and healthy controls were assessed by RNA sequencing, Seahorse assays, and LC-MS/MS. Metabolites in nasal lining fluid, sputum, and plasma from patients with N-ERD (n = 15) and healthy individuals (n = 10) were quantified by targeted metabolomics analyses. Genome-wide methylomics were deployed to define epigenetic mechanisms of macrophage reprogramming in N-ERD. RESULTS: This study shows that N-ERD monocytes/macrophages exhibit an overall reduction in DNA methylation, aberrant metabolic profiles, and an increased expression of chemokines, indicative of a persistent proinflammatory activation. Differentially methylated regions in N-ERD macrophages included genes involved in chemokine signaling and acylcarnitine metabolism. Acylcarnitines were increased in macrophages, sputum, nasal lining fluid, and plasma of patients with N-ERD. On inflammatory challenge, N-ERD macrophages produced increased levels of acylcarnitines, proinflammatory arachidonic acid metabolites, cytokines, and chemokines as compared to healthy macrophages. CONCLUSIONS: Together, these findings decipher a proinflammatory metabolic and epigenetic reprogramming of macrophages in N-ERD.

COL4A3 is degraded in allergic asthma and degradation predicts response to anti-IgE therapy.

BACKGROUND: Asthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3). OBJECTIVE: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. METHODS: The serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test. RESULTS: Serum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5). CONCLUSION: C4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

Sputum microRNA-screening reveals Prostaglandin EP3 receptor as selective target in allergen-specific immunotherapy.

BACKGROUND: Several microRNAs (miRs) have been described as potential biomarkers in liquid biopsies and in the context of allergic asthma, while therapeutic effects on the airway expression of miRs remain elusive. In this study, we investigated epigenetic miR-associated mechanisms in the sputum of grass pollen-allergic patients with and without allergen-specific immunotherapy (AIT). METHODS: Induced sputum samples of healthy controls (HC), AIT-treated and -untreated grass pollen-allergic rhinitis patients with (AA) and without asthma (AR) were profiled using miR microarray and whole-transcriptome microarray analysis of the same samples. miR targets were predicted in silico and used to identify inverse regulation. Local PGE2  levels were measured using ELISA. RESULTS: Two hundred and fifty nine miRs were upregulated in the sputum of AA patients compared with HC, while only one was downregulated. The inverse picture was observed in induced sputum of AIT-treated patients: while 21 miRs were downregulated, only 4 miRs were upregulated in asthmatics upon AIT. Of these 4 miRs, miR-3935 stood out, as its predicted target PTGER3, the prostaglandin EP3 receptor, was downregulated in treated AA patients compared with untreated. The levels of its ligand PGE2 in the sputum supernatants of these samples were increased in allergic patients, especially asthmatics, and downregulated after AIT. Finally, local PGE2  levels correlated with ILC2 frequencies, secreted sputum IL-13 levels, inflammatory cell load, sputum eosinophils and symptom burden. CONCLUSIONS: While profiling the sputum of allergic patients for novel miR expression patterns, we uncovered an association between miR-3935 and its predicted target gene, the prostaglandin E3 receptor, which might mediate AIT effects through suppression of the PGE2 -PTGER3 axis.

Spotlight on microRNAs in allergy and asthma.

In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post-transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.

Allergen-specific immunotherapy induces the suppressive secretoglobin 1A1 in cells of the lower airways.

BACKGROUND: While several systemic immunomodulatory effects of allergen-specific immunotherapy (AIT) have been discovered, local anti-inflammatory mechanisms in the respiratory tract are largely unknown. We sought to elucidate local and epithelial mechanisms underlying allergen-specific immunotherapy in a genome-wide approach. METHODS: We induced sputum in hay fever patients and healthy controls during the pollen peak season and stratified patients by effective allergen immunotherapy or as untreated. Sputum was directly processed after induction and subjected to whole transcriptome RNA microarray analysis. Nasal secretions were analyzed for Secretoglobin1A1 (SCGB1A1) and IL-24 protein levels in an additional validation cohort at three defined time points during the 3-year course of AIT. Subsequently, RNA was extracted and subjected to an array-based whole transcriptome analysis. RESULTS: Allergen-specific immunotherapy inhibited pro-inflammatory CXCL8, IL24, and CCL26mRNA expression, while SCGB1A1, IL7, CCL5, CCL23, and WNT5BmRNAs were induced independently of the asthma status and allergen season. In our validation cohort, local increase of SCGB1A1 occurred concomitantly with the reduction of local IL-24 in upper airways during the course of AIT. Additionally, SCGB1A1 was identified as a suppressor of epithelial gene expression. CONCLUSIONS: Allergen-specific immunotherapy induces a yet unknown local gene expression footprint in the lower airways that on one hand appears to be a result of multiple regulatory pathways and on the other hand reveals SCGB1A1 as novel anti-inflammatory mediator of long-term allergen-specific therapeutic intervention in the local environment.

Noninvasive and minimally invasive techniques for the diagnosis and management of allergic diseases.

Allergic diseases of the (upper and lower) airways, the skin and the gastrointestinal tract, are on the rise, resulting in impaired quality of life, decreased productivity, and increased healthcare costs. As allergic diseases are mostly tissue-specific, local sampling methods for respective biomarkers offer the potential for increased sensitivity and specificity. Additionally, local sampling using noninvasive or minimally invasive methods can be cost-effective and well tolerated, which may even be suitable for primary or home care sampling. Non- or minimally invasive local sampling and diagnostics may enable a more thorough endotyping, may help to avoid under- or overdiagnosis, and may provide the possibility to approach precision prevention, due to early diagnosis of these local diseases even before they get systemically manifested and detectable. At the same time, dried blood samples may help to facilitate minimal-invasive primary or home care sampling for classical systemic diagnostic approaches. This EAACI position paper contains a thorough review of the various technologies in allergy diagnosis available on the market, which analytes or biomarkers are employed, and which samples or matrices can be used. Based on this assessment, EAACI position is to drive these developments to efficiently identify allergy and possibly later also viral epidemics and take advantage of comprehensive knowledge to initiate preventions and treatments.

An exhausted phenotype of TH 2 cells is primed by allergen exposure, but not reinforced by allergen-specific immunotherapy.

BACKGROUND: Studies show that proallergic TH 2 cells decrease after successful allergen-specific immunotherapy (AIT). It is likely that iatrogenic administration of allergens drives these cells to exhaustion due to chronic T-cell receptor stimulation. This study aimed to investigate the exhaustion of T cells in connection with allergen exposure during AIT in mice and two independent patient cohorts. METHODS: OVA-sensitized C57BL/6J mice were challenged and treated with OVA, and the development of exhaustion in local and systemic TH 2 cells was analyzed. In patients, the expression of exhaustion-associated surface markers on TH 2 cells was evaluated using flow cytometry in a cross-sectional grass pollen allergy cohort with and without AIT. The treatment effect was further studied in PBMC collected from a prospective long-term AIT cohort. RESULTS: The exhaustion-associated surface markers CTLA-4 and PD-1 were significantly upregulated on TH 2 cells upon OVA aerosol exposure in OVA-allergic compared to non-allergic mice. CTLA-4 and PD-1 decreased after AIT, in particular on the surface of local lung TH 2 cells. Similarly, CTLA-4 and PD-1 expression was enhanced on TH 2 cells from patients with allergic rhinitis with an even stronger effect in those with concomitant asthma. Using an unbiased Louvain clustering analysis, we discovered a late-differentiated TH 2 population expressing both markers that decreased during up-dosing but persisted long term during the maintenance phase. CONCLUSIONS: This study shows that allergen exposure promotes CTLA-4 and PD-1 expression on TH 2 cells and that the dynamic change in frequencies of exhausted TH 2 cells exhibits a differential pattern during the up-dosing versus the maintenance phases of AIT.

Clinical relevance of Aspergillus fumigatus sensitization in cystic fibrosis.

RATIONALE: The clinical relevance of sensitization to Aspergillus (A) fumigatus in cystic fibrosis (CF) is unclear. Some researchers propose that specific A fumigatus IgE is an innocent bystander, whereas others describe it as the major cause of TH-2-driven asthma-like disease. OBJECTIVES: Lung function parameters in mild CF patients may be different in patients with and without A fumigatus sensitization. We aimed to ascertain whether allergen exposure to A fumigatus by bronchial allergen provocation (BAP) induces TH-2 inflammation comparable to an asthma-like disease. METHODS: A total of 35 patients, aged 14.8 ± 8.5 years, and 20 healthy controls were investigated prospectively. The patients were divided into two groups: group 1 (n = 18): specific (s)IgE negative, and group 2 (n = 17): sIgE positive (≥0.7 KU/L) for A fumigatus. Lung function, exhaled NO, and induced sputum were analysed. All sensitized patients with an FEV1 > 75% (n = 13) underwent BAP with A fumigatus, and cell counts, and the expression of IL-5, IL-13, INF-γ, and IL-8 as well as transcription factors T-bet, GATA-3, and FoxP3, were measured. RESULTS: Lung function parameters decreased significantly compared to controls, but not within the CF patient group. After BAP, 8 of 13 patients (61%) had a significant asthmatic response and increased eNO 24 hours later. In addition, marked TH-2-mediated inflammation involving eosinophils, IL-5, IL-13, and FoxP3 became apparent in induced sputum cells. CONCLUSION: Our study demonstrated the clinical relevance of A fumigatus for the majority of sensitized CF patients. A distinct IgE/TH-2-dominated inflammation was found in induced sputum after A fumigatus exposure.

Activin-A Is a Pro-Inflammatory Regulator in Type-2-Driven Upper Airway Disease.

BACKGROUND: Allergic upper airway disease involves pro-inflammatory type-2 cytokines such as IL-5 and regulatory tissue repair mediators, in particular transforming growth factor (TGF)-ß1. The TGF-ß-superfamily member activin-A displays multiple biological functions and shares certain signalling pathways with TGF-ß1. We aimed to examine the coregulation of mucosal activin-A and TGF-ß1 in acute allergic and chronic Th2-driven upper airway disease. METHODS: We investigated mucosal cytokine expression profiles and kinetics using RT-PCR after nasal allergen challenges in patients with seasonal allergic rhinitis. Furthermore, we analysed mucosal specimens from patients with chronic upper airway disease with nasal polyps using ELISPOTs and confocal microscopy. In addition, we stimulated nasal mucosa ex vivo from patients with nasal polyps as well as primary nasal cell cultures from healthy donors. RESULTS: Mucosal activin-A expression revealed increasing correlation with IL-5 and TGF-ß1 at 0.25, 6, and 24 h, respectively, and was significantly upregulated at 6 h after allergen challenge. The correlated expression was found to be more pronounced in chronic disease with nasal polyps, showing substantially (48-fold) increased activin-A-producing cells in nasal polyps by ELISPOT, while submucosal downstream signalling as determined by confocal microscopy was decreased. Ex vivo stimulations of nasal tissue suggested that activin-A and TGF-ß1 mutually regulate each other's expression at the mRNA level and, when combined, enhance IL-5 expression. CONCLUSION: Activin-A in allergic upper airway disease acts as a pro-inflammatory mediator and TGF-ß1 modifier. Our data in the upper airways oppose the view of potentially anti-inflammatory properties in contrast to lymphatic compartments.

Age dictates a steroid-resistant cascade of Wnt5a, transglutaminase 2, and leukotrienes in inflamed airways.

BACKGROUND: Airway remodeling is a detrimental and refractory process showing age-dependent clinical manifestations that are mechanistically undefined. The leukotriene (LT) and wingless/integrase (Wnt) pathways have been implicated in remodeling, but age-specific expression profiles and common regulators remained elusive. OBJECTIVE: We sought to study the activation of the LT and Wnt pathways during early- or late-onset allergic airway inflammation and to address regulatory mechanisms and clinical relevance in normal human bronchial epithelial cells (NHBEs) and nasal polyp tissues. METHODS: Mice were sensitized with house dust mite (HDM) allergens from days 3, 15, or 60 after birth. Remodeling factors in murine bronchoalveolar lavage fluid, lung tissue, or human nasal polyp tissue were analyzed by means of Western blotting, immunoassays, or histology. Regulatory mechanisms were studied in cytokine/HDM-stimulated NHBEs and macrophages. RESULTS: Bronchoalveolar lavage fluid LT levels were increased in neonatal and adult but reduced in juvenile HDM-sensitized mice. Lungs of neonatally sensitized mice showed increased 5-lipoxygenase levels, whereas adult mice expressed more group 10 secretory phospholipase A2, Wnt5a, and transglutaminase 2 (Tgm2). Older mice showed colocalization of Wnt5a and LT enzymes in the epithelium, a pattern also observed in human nasal polyps. IL-4 promoted epithelial Wnt5a secretion, which upregulated macrophage Tgm2 expression, and Tgm2 inhibition in turn reduced LT release. Tgm2, group 10 secretory phospholipase A2, and LT enzymes in NHBEs and nasal polyps were refractory to corticosteroids. CONCLUSION: Our findings reveal age differences in LT and Wnt pathways during airway inflammation and identify a steroid-resistant cascade of Wnt5a, Tgm2, and LTs, which might represent a therapeutic target for airway inflammation and remodeling.

Biomarkers in Allergic Airway Disease: Simply Complex.

Clinical diagnoses of allergic airway disease need confirmation of a relevant sensitization to allergens. Serum-based component-resolved diagnosis has become increasingly popular and allows the precise assessment of specific IgE to molecular allergens. Molecular allergen diagnosis has improved our understanding of disease phenotypes in allergic patients, including cross-reactive patterns and food allergy. Beyond sensitization profiles, characterization of the (endo-)type and severity of a local allergic disease is useful for targeted therapy with biologics in advanced allergic airway disease. Surrogate markers of treatment efficacy are increasingly under validation. Prognostic and predictive biomarkers will represent a promising option for stratified prevention strategies.