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).

Urinary eosinophil-derived neurotoxin is associated with reduced lung function in pediatric asthma.

INTRODUCTION: Eosinophil-derived neurotoxin (EDN) is a biomarker for eosinophilic activation. Urinary (u) EDN may allow non-invasive monitoring of asthma, but clinical recommendations are lacking. We assessed the potential of uEDN as a marker of disease activity in pediatric asthma. METHODS: We assessed urine samples of 371 children from the German ALLIANCE study cohort, from which we had: 169 preschool wheezers (<6 years), 80 asthmatics (≥6 years), and 122 healthy controls using the ImmunoCAP™ EDN Assay. Creatinine (Cr)-adjusted uEDN values were analyzed using correlations, association tests, (non) parametric statistics, multiple linear, and multivariable regression. RESULTS: uEDN/uCr values were higher in atopic versus non-atopic preschool-aged subjects (p = .035) and associated with the sum of allergen-specific IgE in younger (r = 0.24, p = .003), and older subjects (r = 0.23, p = .043). uEDN/uCr was marginally a good determinant for atopy (p = .078, for subjects aged <6 years, and p = .058 for subjects ≥6 years). Children with the T2-high phenotype had higher uEDN/uCr (p < .001) versus T2-low-irrespective of using uEDN/uCr or blood eosinophils in combination to allergen sIgE for disease phenotyping. uEDN/uCr significantly correlated with reduced lung function among asthmatics (FEV1 z-scores: r = -0.30, p = .007, and FEV1/FVC z-scores: r = -0.24, p = .038). Using multivariable modeling, uEDN/uCr was an independent determinant of FEV1 (p = .038), and to a lesser extent, FEV1/FVC (p = .080). CONCLUSIONS: uEDN/uCr may serve as a non-invasive biomarker for clinical features such as lung function in pediatric asthma. We highlight the utility of uEDN/uCr as a biomarker that can be easily assessed using widely available robust diagnostic immunoassays.

Validation of the Asthma Severity Scoring System (ASSESS) in the ALLIANCE Cohort.

BACKGROUND: The Asthma Severity Scoring System (ASSESS) quantifies asthma severity in adolescents and adults. Scale performance in children younger than 12 years is unknown. OBJECTIVE: To validate the ASSESS score in the All Age Asthma Cohort and explore its use in children younger than 12 years. METHODS: Scale properties, responsiveness, and known-group validity were assessed in 247 children (median age, 11 years; interquartile range, 8-13 years) and 206 adults (median age, 52 years; interquartile range, 43-63 years). RESULTS: Overall, measures of internal test consistency and test-retest reliability were similar to the original data of the Severe Asthma Research Program. Cronbach α was 0.59 in children aged 12 to 18 years and 0.73 in adults, reflecting the inclusion of multiple and not-always congruent dimensions to the ASSESS score, especially in children. Analysis of known-group validity confirmed the discriminatory power, because the ASSESS score was significantly worse in patients with poor asthma control, exacerbations, and increased salbutamol use. In children aged 6 to 11 years, test-retest reliability was inferior compared with that in adults and adolescents (Cronbach α, 0.27) mostly because of a less lung function impairment in children with asthma of this age group. Known-group validity, however, confirmed good discriminative power regarding severity-associated variables similar to adolescents and adults. CONCLUSIONS: Test-retest reliability and validity of the ASSESS score was confirmed in the All Age Asthma Cohort. In children aged 6 to 11 years, internal consistency was inferior compared with that in older patients with asthma; however, test validity was good and thus encourages age-spanning usage of the ASSESS score in all patients 6 years or older.

Tracheostomy decannulation in children: a proposal for a structured approach on behalf of the working group chronic respiratory insufficiency within the German-speaking society of pediatric pulmonology.

The number of children with tracheostomies with and without home mechanical ventilation has grown continuously in recent years. For some of these children, the need for tracheostomy resolves and the child can be weaned from the tracheal cannula. Choosing the optimal time point for decannulation after elaborated prior diagnostic work-up needs careful consideration. The decannulation process requires an interdisciplinary team; however, these specialized structures for the experienced care of these children with tracheostomy are not available in all areas. The Working Group on Chronic Respiratory Insufficiency in the German Speaking Pediatric Pneumology Society (GPP) developed these recommendations to guide through a decannulation process. Initial evaluation of decannulation feasibility starts in the outpatient clinic with a detailed history, examination, and a speaking valve trial and is followed by an inpatient workup including sleep study, airway endoscopy and possibly modifications of the tracheal cannula. Downsizing the tracheal cannula allows a stepwise controlled weaning prior to removal of the tracheal cannula. After shrinking of the tracheostomy, the final surgical closure is performed.  Conclusion: An algorithm with diagnostic and therapeutic procedures for a safe and successful decannulation process is proposed. What is Known: • In children tracheostomy decannulation is a complex process that requires careful preparation and surveillance. What is New: • This statement of the German speaking society of pediatric pulmonology provides an expert practice guidance on the decannulation procedure and the value of one-way speaking valves.

Quality Control of Nitrogen Multiple Breath Washout in a Multicenter Pediatric Asthma Study.

BACKGROUND: Nitrogen multiple breath washout (N2MBW) is a lung function test increasingly used in small airway diseases. Quality criteria have not yet been globally implemented and time-consuming retrospective overreading is necessary. Little data has been published on children with recurrent wheeze or asthma from multicentered studies. METHODS: Children with wheeze or asthma and healthy controls were included in the longitudinal All Age Asthma Cohort (ALLIANCE). To assess ventilation inhomogeneity, N2MBW tests were performed in five centers from 2013 until 2020. All N2MBW tests were centrally overread by one center. Multiple washout procedures (trials) at the visit concluded to one test occasion. Tests were accepted if trials were technically sound (started correctly, terminated correctly, no leak, regular breathing pattern) and repeatable within one test occasion. Signal misalignment was retrospectively corrected. Factors that may impact test quality were analyzed, such as experience level. RESULTS: N2MBW tests of n=561 participants were analyzed leading to n=949 (68.3%) valid tests of n=1,390 in total. Inter-center test acceptability ranged from 27.6% to 77.8%. End-of-test criterion and leak were identified to be the most common reasons for rejection. Data loss and uncorrectable signal misalignment led to rejection of 58% of trials in one center. In preschool children, significant improvement of test acceptability was found longitudinally (χ2(8)=18.6; p=0.02). CONCLUSION: N2MBW is feasible in a multicenter asthma study in children. However, the quality of this time-consuming procedure is dependent on experience level of staff in preschool children and still requires retrospective overreading for all age groups.

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.

Complete Tracheal Ring Deformity, Recurrent Pneumothoraces and Pleuropulmonary Blastoma in a Child: Coincidence or Common Genetic Cause?

Complete tracheal ring deformity (CTRD) is a rare abnormality of unknown etiology characterized by circumferentially continuous cartilaginous tracheal rings leading to variable degrees of tracheal stenosis with or without additional heart and lung malformations. Pleuropulmonary blastomas (PPB) are rare malignant mesenchymal tumors, which occur almost exclusively in young children. Pathogenic germline DICER1 variants are associated with PPB but also with other tumors like rhabdomyosarcoma or syndromic diseases like GLOW (Global developmental delay, lung cysts, overgrowth and Wilms tumor) syndrome. Here, we report a case with CTRD and recurrent pneumothoraces who additionally developed PPB on the genetic background of a pathogenic DICER1 variant.

Regulatory B cells control airway hyperreactivity and lung remodeling in a murine asthma model.

BACKGROUND: Asthma is a widespread, multifactorial chronic airway disease. The influence of regulatory B cells on airway hyperreactivity (AHR) and remodeling in asthma is poorly understood. OBJECTIVE: Our aim was to analyze the role of B cells in a house dust mite (HDM)-based murine asthma model. METHODS: The influence of B cells on lung function, tissue remodeling, and the immune response were analyzed by using wild-type and B-cell-deficient (µMT) mice and transfer of IL-10-proficient and IL-10-deficient B cells to µMT mice. RESULTS: After HDM-sensitization, both wild-type and µMT mice developed AHR, but the AHR was significantly stronger in µMT mice, as confirmed by 2 independent techniques: invasive lung function measurement in vivo and examination of precision-cut lung slices ex vivo. Moreover, airway remodeling was significantly increased in allergic µMT mice, as shown by enhanced collagen deposition in the airways, whereas the numbers of FoxP3+ and FoxP3- IL-10-secreting regulatory T cells were reduced. Adoptive transfer of IL-10-proficient but not IL-10-deficient B cells into µMT mice before HDM-sensitization attenuated AHR and lung remodeling. In contrast, FoxP3+ regulatory T cells were equally upregulated by transfer of IL-10-proficient and IL-10-deficient B cells. CONCLUSION: Our data in a murine asthma model illustrate a central role of regulatory B cells in the control of lung function and airway remodeling and may support future concepts for B-cell-targeted prevention and treatment strategies for allergic asthma.

IL-17 regulates DC migration to the peribronchial LNs and allergen presentation in experimental allergic asthma.

IL-17 is associated with different phenotypes of asthma, however, it is not fully elucidated how it influences induction and maintenance of asthma and allergy. In order to determine the role of IL-17 in development of allergic asthma, we used IL-17A/F double KO (IL-17A/F KO) and WT mice with or without neutralization of IL-17 in an experimental allergic asthma model and analyzed airway hyperresponsiveness, lung inflammation, T helper cell polarization, and DCs influx and activation. We report that the absence of IL-17 reduced influx of DCs into lungs and lung draining LNs. Compared to WT mice, IL-17A/F KO mice or WT mice after neutralization of IL-17A showed reduced airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and IgE levels. DCs from draining LNs of allergen-challenged IL-17A/F KO mice showed a reduction in expression of migratory and costimulatory molecules CCR7, CCR2, MHC-II, and CD40 compared to WT DCs. Moreover, in vivo stimulation of adoptively transferred antigen-specific cells was attenuated in lung-draining LNs in the absence of IL-17. Thus, we report that IL-17 enhances airway DC activation, migration, and function. Consequently, lack of IL-17 leads to reduced antigen-specific T cell priming and impaired development of experimental allergic asthma.

Improved protocol for simultaneous analysis of leukocyte subsets and epithelial cells from murine and human lung.

PURPOSE: To study and isolate lung cells by flow cytometry, enzymatic digestion and generation of single cell suspensions is required. This significantly influences expression of cellular epitopes and protocols need to be adapted for the best isolation and subsequent analysis of specific cellular subsets. MATERIALS AND METHODS: We optimized protocols for the simultaneous isolation and characterization of specific human and murine lung cell types. For alveolar epithelial cells (AEC), a primarily dispase based digestion method and for leukocytes, a primarily collagenase based technique was adapted. Protocols were applied in parallel in either single experimental mice or human lung specimens. RESULTS: Optimized dispase/DNase digestion yielded a high percentage of Epcam+CD45-CD31- AEC as assessed by flow cytometry. Epcam+CD45-CD3-CD11b-CD11c-CD16/32-CD19-CD31-F4/80- AEC were readily sortable with high purity and typical morphology and function upon in vitro stimulation with lipopolysaccharide or respiratory-syncytial-virus (RSV) infection. To analyze lung leukocytes, specimens were digested with an adapted collagenase/DNase protocol yielding high percentages of viable leukocytes with typical morphology, function, and preserved subset specific leukocyte markers. Both protocols could be applied simultaneously in a single experimental mouse post mortem. Application of both digestion methods in primary human lung specimens yielded similar results with high proportions of Epcam+CD45- human AEC after dispase/DNase digestion and preservation of human T cell epitopes after collagenase/DNase digestion. CONCLUSION: The here described protocols were optimized for the simple and efficient isolation of murine and human lung cells. In contrast to previously described techniques, they permit simultaneous in-depth characterization of pulmonary epithelial cells and leukocyte subsets such as T helper, cytotoxic T, and B cells from one sample. As such, they may help to comprehensively and sustainably characterize murine and human lung specimens and facilitate studies on the role of lung immune cells in different respiratory pathologies.

The role of sleep diagnostics and non-invasive ventilation in children with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a degenerative motor neurone disorder causing progressive muscular weakness. Without assisted ventilation or novel therapies, most children with SMA type 1 die before the second year of life due to respiratory failure as the respiratory muscles and bulbar function are severely affected. Active respiratory treatment (mechanically assisted cough, invasive or non-invasive ventilation) has improved survival significantly in recent decades, but often at the cost of becoming ventilator dependent. The advent of a new oligonucleotide based therapy (Nusinersen) has created new optimism for improving motor function. However, the long-term effect on respiratory function is unclear and non-invasive respiratory support will remain an important part of medical management in patients with SMA. This review summarises the existing knowledge about sleep-disordered breathing and respiratory failure in patients with SMA, especially type 1, as well as the evidence of improved outcome and survival in patients treated with non-invasive or invasive ventilation. Practical considerations and ethical concerns are delineated with discussion on how these may be affected by the advent of new therapies such as Nusinersen.

Reduced PRF1 enhancer methylation in children with a history of severe RSV bronchiolitis in infancy: an association study.

BACKGROUND: Acute lower respiratory tract infection is the commonest disease affecting children under five worldwide. Respiratory syncytial virus (RSV) is among the most common causative pathogens. Epidemiological data suggest an association between severe viral respiratory infections in infancy and increased incidence of childhood wheeze and asthma. DNA methylation is involved in immune cell differentiation and identity. It provides an avenue for environmental influences on the genome and therefore has potential as a marker for sustained effects of infectious insults. In this study we investigated the association between DNA methylation patterns in the perforin gene (PRF1) in childhood and a history of hospitalisation for severe RSV disease in the first two years of life. METHODS: In this retrospective study, we explored patterns of whole blood DNA methylation at a methylation sensitive region of the proximal PRF1 enhancer in a group of children with a record of hospitalisation for severe RSV disease during infancy (n = 43) compared to healthy controls matched for age and sex with no similar hospitalisation history, no allergy and no persistent wheeze (n = 43). Univariate and bivariate conditional logistic regression analyses were conducted to test the association between PRF1 enhancer methylation and record of hospitalisation for RSV disease. RESULTS: Children with a record of hospitalisation for severe RSV bronchiolitis demonstrated markedly lower levels of DNA methylation at two cytosine-phosphate-guanine dinucleotide (CpG) loci of the PRF1 proximal enhancer, corresponding to a signal transducer and activator of transcription 5 (STAT5) responsive element, compared to controls, adjusted odds ratios of 0.82 (95% confidence interval [CI] 0.71, 0.94) and 0.73 (95% CI 0.58, 0.92) for each 1% increase in DNA methylation. Smoking in the household showed a significant influence on DNA methylation at the assayed positions. CONCLUSIONS: Our findings support an association between childhood DNA methylation patterns in PRF1 and a record of severe RSV infection in infancy. Longitudinal studies are required to establish the utility of PRF1 methylation as a marker of severe RSV disease.

Pediatric severe asthma with fungal sensitization is mediated by steroid-resistant IL-33.

BACKGROUND: The mechanism underlying severe asthma with fungal sensitization (SAFS) is unknown. IL-33 is important in fungus-induced asthma exacerbations, but its role in fungal sensitization is unexplored. OBJECTIVE: We sought to determine whether fungal sensitization in children with severe therapy-resistant asthma is mediated by IL-33. METHODS: Eighty-two children (median age, 11.7 years; 63% male) with severe therapy-resistant asthma were included. SAFS (n = 38) was defined as specific IgE or skin prick test response positivity to Aspergillus fumigatus, Alternaria alternata, or Cladosporium herbarum. Clinical features and airway immunopathology were assessed. Chronic exposure to house dust mite and A alternata were compared in a neonatal mouse model. RESULTS: Children with SAFS had earlier symptom onset (0.5 vs 1.5 years, P = .006), higher total IgE levels (637 vs 177 IU/mL, P = .002), and nonfungal inhalant allergen-specific IgE. Significantly more children with SAFS were prescribed maintenance oral steroids (42% vs 14%, P = .02). SAFS was associated with higher airway IL-33 levels. In neonatal mice A alternata exposure induced higher serum IgE levels, pulmonary IL-33 levels, and IL-13(+) innate lymphoid cell (ILC) and TH2 cell numbers but similar airway hyperresponsiveness (AHR) compared with those after house dust mite exposure. Lung IL-33 levels, IL-13(+) ILC numbers, TH2 cell numbers, IL-13 levels, and AHR remained increased with inhaled budesonide during A alternata exposure, but all features were significantly reduced in ST2(-/-) mice lacking a functional receptor for IL-33. CONCLUSION: Pediatric SAFS was associated with more oral steroid therapy and higher IL-33 levels. A alternata exposure resulted in increased IL-33-mediated ILC2 numbers, TH2 cell numbers, and steroid-resistant AHR. IL-33 might be a novel therapeutic target for SAFS.

Single cell RNA sequencing reveals distinct clusters of Irf8-expressing pulmonary conventional dendritic cells.

A single population of interferon-regulatory factor 8 (Irf8)-dependent conventional dendritic cell (cDC type1) is considered to be responsible for both immunogenic and tolerogenic responses depending on the surrounding cytokine milieu. Here, we challenge this concept of an omnipotent single Irf8-dependent cDC1 cluster through analysis of pulmonary cDCs at single cell resolution. We report existence of a pulmonary cDC1 cluster lacking Xcr1 with an immunogenic signature that clearly differs from the Xcr1 positive cDC1 cluster. The Irf8+Batf3+Xcr1- cluster expresses high levels of pro-inflammatory genes associated with antigen presentation, migration and co-stimulation such as Ccr7, Cd74, MHC-II, Ccl5, Il12b and Relb while, the Xcr1+ cDC1 cluster expresses genes corresponding to immune tolerance mechanisms like Clec9a, Pbx1, Cadm1, Btla and Clec12a. In concordance with their pro-inflammatory gene expression profile, the ratio of Xcr1- cDC1s but not Xcr1+cDC1 is increased in the lungs of allergen-treated mice compared to the control group, in which both cDC1 clusters are present in comparable ratios. The existence of two distinct Xcr1+ and Xcr1- cDC1 clusters is furthermore supported by velocity analysis showing markedly different temporal patterns of Xcr1- and Xcr1+cDC1s. In summary, we present evidence for the existence of two different cDC1 clusters with distinct immunogenic profiles in vivo. Our findings have important implications for DC-targeting immunomodulatory therapies.

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.

In vitro neutrophil migration is associated with inhaled corticosteroid treatment and serum cytokines in pediatric asthma.

Background: Different asthma phenotypes are driven by molecular endotypes. A Th1-high phenotype is linked to severe, therapy-refractory asthma, subclinical infections and neutrophil inflammation. Previously, we found neutrophil granulocytes (NGs) from asthmatics exhibit decreased chemotaxis towards leukotriene B4 (LTB4), a chemoattractant involved in inflammation response. We hypothesized that this pattern is driven by asthma in general and aggravated in a Th1-high phenotype. Methods: NGs from asthmatic nd healthy children were stimulated with 10 nM LTB4/100 nM N-formylmethionine-leucyl-phenylalanine and neutrophil migration was documented following our prior SiMA (simplified migration assay) workflow, capturing morphologic and dynamic parameters from single-cell tracking in the images. Demographic, clinical and serum cytokine data were determined in the ALLIANCE cohort. Results: A reduced chemotactic response towards LTB4 was confirmed in asthmatic donors regardless of inhaled corticosteroid (ICS) treatment. By contrast, only NGs from ICS-treated asthmatic children migrate similarly to controls with the exception of Th1-high donors, whose NGs presented a reduced and less directed migration towards the chemokines. ICS-treated and Th1-high asthmatic donors present an altered surface receptor profile, which partly correlates with migration. Conclusions: Neutrophil migration in vitro may be affected by ICS-therapy or a Th1-high phenotype. This may be explained by alteration of receptor expression and could be used as a tool to monitor asthma treatment.

Impact of imposed social isolation and use of face masks on asthma course and mental health in pediatric and adult patients with recurrent wheeze and asthma.

BACKGROUND: There is currently a dramatic increase in the number of COVID-19 cases worldwide, and further drastic restrictions in our daily life will be necessary to contain this pandemic. The implications of restrictive measures like social-distancing and mouth-nose protection on patients with chronic respiratory diseases have hardly been investigated. METHODS: Our survey, was conducted within the All Age Asthma Cohort (ALLIANCE), a multicenter longitudinal observational study. We assessed the effects of COVID-19 imposed social isolation and use of facial masks, on asthma course and mental health in patients with asthma and wheezing. RESULTS: We observed a high rate of problems associated with using facemasks and a significant reduction in the use of routine medical care. In addition to unsettling impacts, such as an increase in depression symptoms in adults, an astonishing and pleasing effect was striking: preschool children experienced an improvement in disease condition during the lockdown. This improvement can be attributed to a significant reduction in exposure to viral infections. CONCLUSION: Long-term observation of this side effect may help improve our understanding of the influence of viral infections on asthma in early childhood.

Differential expression patterns of glycosphingolipids and C-type lectin receptors on immune cells in absence of functional regulatory T cells.

BACKGROUND: Glycosylation is a common and complex type of protein posttranslational modification. Altered glycosylation of immunoglobulins in autoimmune diseases has led to the "altered glycan hypothesis" postulating existence of a unique glycan signature on immune cells and extracellular proteins characterized by site-specific relative abundances of individual glycan structures and glycosylation patterns. However, it is not clear how glycosylation on leukocyte subpopulations differ between states of health or inflammation. HYPOTHESIS: Glycosphingolipid patterns on immune cells of forkhead-box-P3-deficient scurfy mice differs from those on wild-type immune cells. METHODS: T cells and dendritic cells were isolated from spleens of either wild-type or age-matched scurfy mice. Glycosphingolipids of CD4+ T cells and splenic dendritic cells from wild-type and scurfy mice were then analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). In addition, flow cytometry and ChipCytometry were used to access expression patterns of various C-type lectin receptors on antigen-presenting cells from various organs of both wild-type and scurfy mice. RESULTS: We, hereby report differential expression of glycosphingolipids in health and under inflammatory conditions as reflected in wild-type and scurfy mice. Furthermore, we observed that the absence of functional regulatory T cells correlated with elevated expression of CLEC-7A and CD205 but a reduction in levels of CLEC12A and CD206 on antigen-presenting cells. CONCLUSION: We hereby show that the absence of functional regulatory T cells affects expression pattern and quantities of glycosphingolipids on immune cells. Thus, glycosphingolipids could serve as biomarkers for mapping genetical and homeostatic perturbances such as those resulting from a diseased condition.

FoxP3 deficiency causes no inflammation or neurodegeneration in the murine brain.

Regulatory T cells (Treg) maintain immunological self-tolerance and their functional or numerical deficits are associated with progression of several neurological diseases. We examined the effects of Treg absence on the structure and integrity of the unchallenged murine brain. When compared to control, Treg-deficient FoxP3sf mutant mice showed no differences in brain size, myelin amount and oligodendrocyte numbers. FoxP3sf strain displayed no variations in quantity of neurons and astrocytes, whereas microglia numbers were slightly reduced. We demonstrate lack of neuroinflammation and parenchymal responses in the brains of Treg-deficient mice, suggesting a minor Treg role in absence of blood-brain barrier breakdown.