Journal of Allergy and Clinical Immunology

To the editorO_ST_ABSBackgroundC_ST_ABSAnaphylaxis is an acute and potentially life-threatening hypersensitivity reaction often involving the cardiovascular system. Circulating microRNAs (miRNAs/miR), including those carried by extracellular vesicles (EVs), are emerging biomarkers that display regulatory functions in allergy. This study aims to investigate the role of miR-29a in anaphylaxis. MethodsMiR-29a (3p and 5p) levels were assessed by qPCR from acute and baseline samples of serum and EVs from 70 patients with food- and drug-mediated anaphylaxis. EVs purification was confirmed by Western blot, electron microscopy, and NanoSight. MiR-29a-3p target genes were studied in silico using systems biology analysis (SBA). Moreover, miR-29a levels were evaluated in vitro in endothelial cells (ECs) exposed to anaphylactic mediators. Additionally, a panel of endothelial glycocalyx (eGCX)-associated mRNA was analyzed after transfection with a miR-29a-3p inhibitor. ResultsPatients with food-induced anaphylaxis exhibited reduced miR-29a-3p levels in both serum and EVs during the acute reaction. In contrast, miR-29a-5p levels were decreased in serum but not in EVs. No significant modulation of either miRNA was observed in drug-induced anaphylaxis. SBA of miR-29a-3p identified molecular pathways, biological processes and functional networks associated with eGCX remodelling. Intracellular levels of miR-29a-3p were modulated in vitro in ECs following exposure to anaphylactic mediators. Inhibition of miR-29a-3p significantly reduced ESM1 expression. ConclusionsThe miR-29a-3p levels are decreased in serum and EVs from patients with acute food-induced anaphylaxis, suggesting its potential as a promising biomarker. Moreover, a role for miR-29a-3p in eGCX integrity under anaphylactic conditions was demonstrated, potentially regulating ESM1. Key MessageMiR-29a-3p is selectively reduced in serum and extracellular vesicles during acute food-induced anaphylaxis and may regulate endothelial glycocalyx-related pathways, which supports its potential as a novel biomarker and molecular mediator of vascular involvement in anaphylactic reactions.

Asthma is a chronic respiratory illness that causes mild to severe inflammation throughout narrowed airways. During allergic airways inflammation, autophagy prevents extensive lung tissue impairment while inducing a protective anti-pathogen response and macrophages in the lung to maintain homeostasis. Previous studies of autophagy genes and asthma have shown an association with variants in ATG5, but a comprehensive analysis of autophagy related genes and asthma has not been performed. Here we utilize summary statistic data generated from a two-stage genome-wide association study (GWAS) of asthma in the UK Biobank. We examined variants in 21 autophagy related genes and found statistically significant associations for 28 variants in two genes in the discovery dataset and nominally significant replication for 16 of these variants, all annotated to ATG4B. This is the first evidence of an association with variants in ATG4B with asthma which provides a novel potential for future drug development.

Introduction: Although temporal variation is the hallmark of asthma, recommended diagnostic approaches largely rely on single clinic-based measurements. Ambulatory monitoring captures diurnal and day-to-day variability and may therefore enhance diagnostic accuracy. We evaluated the clinical feasibility and potential utility of home spirometry and fractional exhaled nitric oxide (FeNO) monitoring in asthma diagnosis. Methods: Symptomatic, untreated adults with GP-suspected asthma underwent diagnostic tests including bronchodilator reversibility, in-clinic FeNO, blood eosinophil counts and bronchial challenge. Participants measured spirometry and FeNO four times daily over one week; during the second week spirometry were measured twice daily. The reference standard was provided (asthma/not-asthma) by an expert panel of at least two asthma specialists based on clinical history and the results of all in-clinic testing; home spirometry (except for peak expiratory flow) and FeNO measurements were blinded to the panel. Results: Of 67 eligible participants, 51(76%) were recruited, and 38 had asthma confirmed or excluded by the panel. 1058 home spirometry measurements were obtained from 37(73%) participants; 848 home FeNO readings were obtained from 39(76%) participants. Among those completing at least one home measurement, median (IQR) adherence was 66.7(58.6-97.6)% for spirometry and 78.5(51.8-103.6)% for FeNO. Collection of health impact data for economic evaluation was feasible. In participants with a confirmed diagnostic outcome who completed home measurements (FeNO: n=32; spirometry: n=28), the putative home-testing metrics demonstrated high sensitivities at [≥]90% specificity, and outperformed peak expiratory flow diurnal variability. Incorporating home testing into the BTS/NICE/SIGN 2024 diagnostic pathway had the potential to reduce reliance on bronchial challenge testing by 57%. Conclusions: Home spirometry and FeNO testing and the prospective collection of health-economic data in the diagnostic setting were feasible. Home-based testing strategy showed early potential to improve asthma diagnosis and pathway efficiency. These findings support further evaluation through an adequately powered diagnostic accuracy study and health-economic assessment.

Peanut allergy represents a major food-allergy burden, raising concerns about food processing and novel dietary products. Current diagnostics assess primarily allergic endpoints rather than immune mechanisms initiating and maintaining type 2 inflammation, particularly DC2-mediated Th2 polarization. Here, an in vitro autologous monocyte-derived dendritic cell (moDC)-T cell and B cell assay has been established to study immunomodulatory effects induced by unprocessed (P-D) and processed (P-DH (heated) or P-DHG (heated and glycated)) peanut proteins and emerging foods (protein concentrates or whole biomass), related to ex vivo DC2-T cell reactions. CD14+ monocytes, CD4+ T cells and CD19+ B cells were isolated from six peanut-allergic patients PBMCs. MoDCs generated with IL4/GM-CSF were exposed (48h) to type 2 polarizing cytokine (DC2) mix, or DC2 mix combined with the food samples. Next, DC2s were co-cultured with T cells (5d), followed by B cells incubation with DC2/T cell supernatant and food samples (10d). Supernatants and cells were analyzed for Th1/Th2/Th-regulatory (Treg) cells, IgE and IgG profiles. DC2 induced a strong Th2 phenotype and activity, P-D DC2 further enhanced IL13 secretion and %Tregs. P-DH DC2 favored Th2, whereas P-DHG DC2 increased IFN{gamma}, with neither increasing %Treg. All increased CD40L+CD25+ memory Th2 cells. Wheat, whey and seaweed biomass had little effect, whereas algae DC2 showed distinct immunomodulatory, adjuvant-like activity. In conclusion, this autologous in vitro assay captures peanut-specific and generic Th2 responses and reactivity to food samples, supporting its use as additional tool to assess type 2-driving potential and allergenicity of emerging foods and processing methods in peanut-allergic patients. Clinical trial registrationThe current in vitro study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethics Review Committee (METC) of the Erasmus MC (NL79534.078.21 MEC-2021-0905) and registrated at International Clinical Trials Registry Platform (NL-OMON51765). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/705773v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1f42191org.highwire.dtl.DTLVardef@c7e4baorg.highwire.dtl.DTLVardef@1798547org.highwire.dtl.DTLVardef@11d0d96_HPS_FORMAT_FIGEXP M_FIG C_FIG Immune cell illustrations were adapted (1, 2) with permission; permission conveyed through Copyright Clearance Center, Inc. Capsule summaryThe autologous moDC-T cell-B cell in vitro assay may be used to assess whether processing methods or new foods might have intrinsic capacity to affect type 2 inflammation in peanut-allergic patients.

Asthma is the most common chronic respiratory disease of childhood and is strongly associated with genetic variants at the 17q21 locus that increase expression of ORMDL3, a negative regulator of serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in de novo sphingolipid synthesis. Reduced sphingolipid production has been linked to airway hyperreactivity, a key physiological feature of asthma, but the mechanisms connecting altered sphingolipid metabolism to airway dysfunction remain unclear. We examined whether sphingolipid metabolites regulate airway smooth muscle reactivity. Circulating sphingolipids were quantified in children with asthma carrying 17q21 risk alleles and in mice with reduced SPT activity. Functional airway responses were assessed in precision-cut lung slices exposed to sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (Sa1P), and S1P receptor antagonists. Homozygous carriers of the rs7216389 risk allele and SPT-deficient mice displayed an increased S1P-to-Sa1P ratio. In functional assays, Sa1P opposed S1P-induced airway contraction, and increasing Sa1P availability reduced airway hyperresponsiveness. These findings identify the S1P/Sa1P axis as a metabolic rheostat regulating airway smooth muscle tone and suggest that targeting sphingolipid metabolism may offer a therapeutic strategy to mitigate intrinsic airway hyperreactivity in asthma. One sentence summaryAn imbalance between sphingosine-1-phosphate and sphinganine-1-phosphate links the asthma risk locus 17q21 to airway hyperreactivity and reveals sphingolipid metabolism as a potential therapeutic target.

The genetic relationship between asthma and lung function may be dependent on age-at-onset (AAO) of asthma. We investigated whether the shared genetics between asthma AAO and lung function is dependent on AAO. Asthma cases from UK Biobank were subset according to their AAO and genetic correlation was used to obtain genetically homogeneous groups, i.e., [≤]20 (LT20), 20-40, and >40 (GT40) years. Association analysis and fine-mapping were performed to identify shared genetics between AAO groups and lung function. Mediation and quantitative trait locus (QTL) analyses were performed to identify mechanisms underlying shared genetic associations. Chr5, chr6, chr12, and chr17 each had one region that displayed a cross-phenotype replicated association with at least one AAO group and lung function. Overlapping credible sets obtained from fine-mapping were observed on chr5 and chr6. Mediation analyses demonstrated that for each region the proportion mediated through asthma on lung function was larger for asthma LT20 compared to 20-40 and GT40 suggesting that their effects on lung function were more strongly driven by this association. Tissue-specific QTL analysis revealed shared etiology on chr5 may be acting through SLC22A5 and C5orf56 which might play an important role in decreased lung function among individuals with earlier-onset asthma.

Activated T cells increase transferrin-bound iron uptake via the transferrin receptor, also called CD71. We previously demonstrated that targeting CD71 with an antibody to reduce iron update can modify CD4 T cell function, with different effects on TH1, TH17, and regulatory T (Treg) cells. CD71 blocking antibody-treated Tregs had no loss of viability or differentiation, and Foxp3 expression was increased. However, a genetic deletion of Tfrc (the gene for CD71) driven by Foxp3-Cre was reported to cause a lethal autoimmunity. Whether altered immune homeostasis or insufficient early developmental tolerance drive the phenotype of CD71 knockout (KO) Treg mice were unclear. Here, we examined the Foxp3-YFP-Cre KO mouse model and a tamoxifen-inducible KO model in adults to determine the role of CD71 expression in Treg cells. We hypothesized that due to a lack of iron for mitochondrial metabolism, KO Treg adapt to rely heavily on glycolysis and become unstable, promoting pro-inflammatory exTreg cells. This effect was not universal, however, and necropsy analyses revealed tissue-specific inflammation. While the colons of mice with KO Treg cells appeared healthy, skin and lung tissue were severely inflamed. Metabolically, KO Treg cells had a significant decrease in their glycolytic capacity and instead increased oxidation of amino acids and fatty acids. In inflamed skin, which that promotes increased oxidative stress, CD71 expression in Treg cells suppressed tissue inflammation in a model of atopic dermatitis-like disease. These results indicate the CD71-iron axis as a new immunometabolic regulator of Treg cell functions in immune and non-immune organs. Capsule SummaryA loss of the transferrin receptor in Tregs causes severe autoimmunity and here we clarify how Tregs rely on this receptor for iron in specific tissues and disease settings including atopic dermatitis.

BackgroundHuman leukocyte antigen (HLA)-C*06:02 is a major genetic risk factor for psoriasis and understanding the HLA-C*06:02-presented peptide antigen repertoire (immunopeptidome) in the skin of patients is crucial for identifying autoantigens. Yet, no skin immunopeptidome data from patients stratified by their HLA-C*06:02 status exists. ObjectiveWe analysed biopsies from lesional and non-lesional skin of patients with psoriasis vulgaris (n=12), guttate psoriasis (n=8), or from skin of healthy controls (n=16). MethodsHLA class I and class II peptide complexes were isolated by serial immunoprecipitation and HLA-bound peptides identified by liquid chromatography-tandem mass spectrometry. HLA-C*06:02 genotyping was performed by polymerase chain reaction. ResultsOver 99,000 non-redundant peptide ligands were identified across all samples. Substantially more HLA class I and class II peptides were detected in lesional psoriatic skin compared to matched non-lesional and healthy skin. Three peptides predicted to bind HLA-C*06:02, including MRASSFLIV from the known psoriasis marker peptidase inhibitor 3 (PI3), were identified in all lesions of HLA-C*06:02-positive patients but were rarely detected or absent in HLA-C*06:02-negative patient lesional skin and not detected at all in unaffected skin. Keratinocyte differentiation-associated protein (KRTDAP) was a notable source of lesion-specific HLA class II ligands contributing three out of six peptides detected in more than half of the lesional samples. ConclusionActive psoriatic lesions display an altered and expanded immunopeptidome compared to unaffected skin. We have identified numerous unreported, lesion-specific HLA-bound peptides and their source proteins. These findings offer insights into the pathobiology of psoriasis and provide a resource for future functional studies. CAPSULE SUMMARYA selection of immunopeptides is presented exclusively in lesional skin of HLA-C*06:02+ patients with psoriasis that may represent antigenic drivers of disease.

BackgroundGroup 2 innate lymphoid cells (ILC2s) are key effector cells of type-2 immunity. A subset of ILC2s expresses KIT (CD117), which display increased phenotypic plasticity and were previously linked to severe asthma and psoriasis. However, the molecular mechanisms promoting a KIT+ ILC2 state remain poorly understood. ObjectiveDefine the molecular basis for the enhanced plasticity of KIT+ ILC2s and identify signals that induce this phenotype, including links with immune disease susceptibility. MethodsWe combine bulk as well as single-cell transcriptome (RNA-seq) and epigenome (ATAC-seq) with in vitro culture assays using primary human KIT+ or KITneg ILC2s and multipotent ILC precursors (ILCPs). Epigenomic data were integrated with genetic risk variants for major human immune diseases. ResultsMulti-omics analyses revealed that KIT+ ILC2s maintain a unique hybrid character marked by expression and open chromatin of genes linked to both ILCP and ILC2 biology. KIT+ ILC2s showed extensive epigenomic priming at gene loci related to naive lymphocyte biology, tissue homing, and ILC3 effector functions, including IL17 and IL23R - explaining why KIT+ ILC2s are poised to adopt an ILC3-like phenotype. Genetic risk variants for asthma and autoimmunity are enriched in the poised epigenome of KIT+ ILC2s. Common {gamma}-chain cytokines IL-2 and IL-7 induced a KIT+ phenotype in KITneg ILC2s through STAT5 activation. ConclusionsOur study defines KIT+ ILC2s as a developmentally immature state carrying a precursor-like epigenome that promotes phenotypic plasticity and is linked to immune disease susceptibility. Importantly, we identify STAT5-mediated cytokine signals as candidates for therapeutic targeting of KIT+ ILC2s.

Mixed granulocytic asthma (MGA) is a severe Th2-low endotype, characterized by high Th17/neutrophilic burden and exacerbated airway remodeling. Both features confer resistance to inhaled corticosteroids, and typical asthma treatments. Thus, MGA is an enormous public health burden. Gaps in knowledge include how Th17 cells induce pathological tissue remodeling, and how Th17 differentiation occurs in response to allergens. We generated a Th2-low murine model of asthma that recapitulates major features of human MGA namely, heightened airway reactivity to methacholine, Th17/neutrophilic inflammation, airway remodeling, and resistance to corticosteroid treatment. Two specific biomarkers enriched in human MGA, the TNF superfamily member 14 (aka LIGHT), and the mitochondrial oxidative phosphorylation (OXPHOS) pathway, are upregulated in this model. We show OXPHOS promotes the metabolic reprograming of Th17 cells, to produce LIGHT that controls airway remodeling. Mechanistically, OXPHOS regulates ROR{gamma}t expression and the subsequent transcriptional network to program survival and differentiation of Th17 cells, whereas LIGHT drives airway remodeling by activating the MMP9-dependent TGF{beta} pathway. Additionally, OXPHOS+Th17 cells promote the expression of osteopontin necessary for fibroblast activation. LIGHT antagonistic blockade reduces airway remodeling, whereas OXPHOS chemical inhibition reduces Th17 cells and neutrophilia. Importantly, the dual blockade of LIGHT and OXPHOS reverses all features of MGA and reciprocally increase the numbers of Treg cells. Thus, the dual blockade of LIGHT and OXPHOS constitutes a promising target for clinical interventions in human MGA, possibly extending to other Th17-driven fibrotic diseases.

Background: Extreme-phenotype comparisons allowed the discovery of novel asthma genetic risk loci. However, this approach remains unexplored in epigenome-wide association studies (EWAS). We aimed to identify bulk and cell-specific methylation markers of asthma with severe exacerbations across diverse ancestry groups. Methods: We conducted a meta-EWAS of 739,543 CpGs in whole blood among 1,192 African American and Latino pediatric populations, comparing non-asthmatics and asthma exacerbators. Genome-wide CpGs were followed up for replication in a meta-analysis across 1,516 ethnically diverse participants and in a cross-tissue evaluation of 393 nasal samples. We conducted differentially methylated region (DMRs), cell-type-deconvoluted, and quantitative trait loci analyses (whole-genome sequencing n=1,668; RNA-seq n=1,209). We examined enrichment in traits, pathways, and druggable genes, and analyzed DNAm predictors of plasma proteins and aging. Results: DNAm at 505 CpGs and 119 DMRs in whole blood were associated with asthma exacerbations (p<9x10-8, {lambda}=1.05). We replicated 25 CpGs in blood cells, cross-validated 7 in nasal samples, and detected 42 cell-specific DNAm markers mainly driven by T cells. DNAm at 134 CpGs was associated with gene expression in whole blood, including 118 associations with T-cell receptor genes, and 446 CpGs were regulated by [&ge;]1 genetic variant. We found enrichment for previous associations with environmental exposures, immune disorders, immune and inflammatory pathways, and druggable genes by developmental drugs. 21 methylation-predicted plasma proteins, involved in host defense, and one lung aging clock were associated with asthma exacerbations. Conclusions: The first meta-EWAS of extreme asthma phenotypes identified hundreds of novel DNAm markers, suggesting novel methylation biomarkers and candidate drugs for asthma and supporting the role of T cells.

Atopic dermatitis (AD) evolves from initial type 2 immunity-driven inflammation to chronic mixed responses by poorly understood mechanisms. To investigate how the prolonged activation of the usually IL-4/IL-13-induced transcription factor STAT6 in keratinocytes impacts on the development and subtype of AD, we generated a new mouse model in which a constitutively active form of STAT6 is selectively expressed in keratinocytes. These K14Cre+STAT6vt/vt mice spontaneously developed AD-like skin lesions characterized by Staphylococcus aureus colonization, neutrophilic inflammation, and pruritus starting at the age of 12-14 weeks. Treatment with antibiotics mitigated pathology, indicating that it is microbiota-driven. Comparison of human AD gene expression data with the transcriptome of skin biopsies from K14Cre+STAT6vt/vt mice revealed features shared with chronic AD, including genes associated with neutrophil and keratinocyte activation. Furthermore, heterozygous K14Cre+STAT6vt/wt mice developed a mixed eosinophilic and neutrophilic skin inflammation with exacerbated pathology compared to wild-type controls in an induced model of atopic dermatitis, compatible with chronic AD. These results indicate that persistent STAT6 activity in keratinocytes facilitates S. aureus outgrowth on the skin, promotes a type 1-/type 3-biased immune response, and is sufficient to mimic the transition from acute type 2 immunity-to chronic type 1-/type 3-immunity-dominated AD.

Eosinophilic esophagitis (EoE) is a leading cause of chronic esophageal dysfunction driven by immune-mediated inflammation. Peak eosinophil count (PEC) in esophageal biopsies is routinely used to assess disease activity, and its associated molecular mechanisms have been well studied. However, PEC only partially captures overall disease severity, which is comprehensively captured by the Index of Severity for Eosinophilic Esophagitis (I-SEE). In contrast to PEC, the molecular and cellular programs associated with the I-SEE-defined disease severity, particularly in children, remain poorly understood. We integrated bulk transcriptomic profiling of pediatric esophageal biopsies with clinical severity metrics and a matched single-cell transcriptomic reference. Increasing severity was associated with a shift from type 2 inflammatory activation toward epithelial stress, cytoskeletal and junctional disruption, metabolic dysfunction, and extracellular matrix remodeling. Single-cell-informed analyses identified that proliferating and transitional epithelial cell states were strongly associated with higher I-SEE scores and exhibited impaired differentiation, heightened metabolic and oxidative stress responses, and structural remodeling programs not captured by bulk transcriptomic analyses alone. These findings reposition epithelial remodeling, rather than eosinophil burden alone, as a central molecular correlate of disease severity in pediatric EoE and provide a framework for improved disease stratification and therapeutic intervention.

Psoriasis is a chronic immune-mediated inflammatory skin disease characterized by excessive keratinocyte proliferation, immune cell infiltration and dysregulated inflammatory signaling. Despite the availability of biologic therapies targeting inflammatory cytokines, many patients experience incomplete responses or relapse, highlighting the need to better understand molecular regulators of cutaneous inflammation. CD109 is a glycosylphosphatidylinositol (GPI)-anchored protein previously identified by our lab as a co-receptor and negative regulator of Transforming Growth Factor-{beta} (TGF-{beta}) signaling that inhibits fibrotic responses. Emerging evidence suggests that CD109 also modulates immune and inflammatory pathways. In this study, we investigated whether epidermal CD109 overexpression influences cutaneous inflammatory responses. Transgenic (TG) mice overexpressing CD109 under the keratin-14 (K14) promoter were used to restrict transgene expression to the epidermis. TG and wild-type (WT) littermates were subjected to lipopolysaccharide (LPS)-induced skin inflammation. CD109 TG mice exhibited significantly reduced immune cell recruitment, including macrophages and neutrophils, along with decreased expression of the pro-inflammatory mediators IL-1 and MCP-1/CCL2 compared with WT mice. Transcriptomic analysis of primary keratinocytes revealed downregulation of multiple inflammatory signaling pathways in CD109-overexpressing cells, including TNF-/NF-{kappa}B, IL-2/STAT5, IFN-{gamma}, IFN-, and IL-6/JAK/STAT3 pathways. Together, these findings demonstrate that epidermal CD109 overexpression attenuates cutaneous inflammatory responses by suppressing key inflammatory signaling networks and limiting immune cell recruitment, suggesting that CD109 may represent an important regulator of inflammatory signaling in the skin and a potential target for inflammatory skin diseases such as psoriasis.

Autophagy has been implicated in several lung diseases, either protecting tissues or driving pathology. Hypersensitivity pneumonitis (HP) is a complex inflammatory lung disease, and autophagy is heavily involved in regulating inflammation. The role of autophagy in HP remains unclear. The aim of our study was to understand the role of autophagy in HP pathogenesis. GFP-LC3 transgenic mice were exposed intranasally to Saccharopolyspora rectivirgula (SR) to induce HP and follow autophagy activation in the lung. Then, we take advantage of our Atg4b-deficient mouse model to assess how autophagy disruption impacts lung inflammation in response to SR antigen challenge. Increased autophagy activation was observed in epithelial and inflammatory cells after SR antigen exposure in GFP-LC3 transgenic lungs. GFP-LC3 puncta colocalized with ATG4B and ATG5 in epithelial and inflammatory cells after antigen exposure. Autophagy impairment limits the inflammatory response after SR antigen exposure in the lungs from the Atg4b-deficient mice when compared to WT mice. To evaluate whether lipopolysaccharide (LPS) exacerbates the inflammatory response in the Atg4b-deficient, a SR+LPS combined treatment was developed and we discovered that LPS aggravates the SR-induced HP in WT but not in Atg4b-deficient mice. Reduced HP severity in Atg4b-deficient mice was associated with decreased expression of NFkB, CCL1, CCL25, CXCL1, TNFR1, IL-13, and IL-17A, diminished CD4+ T cell recruitment and expansion, reduced M2-like macrophages, and decreased granuloma and iBALT development. Our findings highlight autophagy as a critical driver in HP pathogenesis and as a potetial target for novel theraphy development.

miR-378a-3p has been reported to be upregulated in the lesional skin of patients with atopic dermatitis (AD); however, its function in AD remains unclear. Here, we demonstrate that miR-378a-3p expression is induced by IL-4 and live Staphylococcus aureus (S. aureus) in normal human epidermal keratinocytes (NHEKs) cultured in proliferative conditions or in a 3D epidermal culture model. Transcriptomic profiling and gene set enrichment analysis of miR-378a-3p-transfected NHEKs revealed positive enrichment of inflammatory response pathways alongside downregulation of genes associated with epidermal development. More specifically, miR-378a-3p enhanced expression of multiple NF-{kappa}B-dependent inflammatory mediators, accompanied by increased phosphorylation of p65, indicating activation of canonical NF-{kappa}B pathway. Notably, miR-378a-3p concomitantly reduced the expression of several NF-{kappa}B family members and upstream adaptor molecules, supporting a model in which miR-378a-3p promotes canonical NF-{kappa}B activity through coordinated modulation of multiple components within the NF-{kappa}B regulatory network. In NHEKs exposed to live S. aureus, miR-378a-3p significantly increased the secretion of IL-1{beta}, IL-1Ra, and IL-8, indicating that miR-378a-3p may amplify innate immune responses triggered by S. aureus colonization in AD. Collectively, these findings identify miR-378a-3p as a positive regulator of keratinocyte inflammatory responses that may contribute to AD exacerbation, particularly in the context of S. aureus colonization.

BackgroundImpulse oscillometry is a noninvasive pulmonary function test performed during quiet breathing and requires minimal patient cooperation. It is useful for detecting small airway disease and provides increased sensitivity for diagnosing asthma in younger children who may have difficulty completing standard spirometry. Bronchodilator testing, a standard assessment of airflow obstruction reversibility, is recommended in patients with suspected asthma who present obstructive airflow patterns. ObjectiveTo evaluate impulse oscillometry parameters before and after bronchodilator administration across different age groups and to examine the relationship between age and airway resistance in patients with clinician-diagnosed asthma. MethodsThis retrospective study included patients with clinician-diagnosed asthma who demonstrated obstructive airflow patterns and a positive bronchodilator response. Participants were grouped by age: younger than 6 years, 6 to 20 years, and older than 20 years. Key impulse oscillometry parameters--airway resistance at 5 Hz, airway resistance at 20 Hz, the difference between these values, and resonance frequency--were collected and compared across groups. A positive bronchodilator response was defined as a reduction in airway resistance of more than 30% in individuals younger than 18 years and more than 40% in adults. ResultsA total of 225 patients (123 males and 102 females) were included, with a median age of 6 years. At baseline, the median airway resistance at 5 Hz was 175.34% of the reference value (95% CI, 171.66-178.62), and airway resistance at 20 Hz was 121.68% (95% CI, 118.73-127.12). The median difference between these values was 52.32% (95% CI, 49.89-57.14), and the median resonance frequency was 5.11 Hz (95% CI, 4.62-5.35). After bronchodilator administration, airway resistance at 5 Hz decreased to 123.56% (95% CI, 119.07-126.77), corresponding to a median reduction of 52.8% (95% CI, 49.48-56.08; P < 0.0001). Age demonstrated a moderate positive correlation with airway resistance at 20 Hz (r = 0.51, P < 0.001). ConclusionsProximal airway resistance increases with age among patients with asthma, suggesting age-related differences in airway inflammation. Impulse oscillometry combined with bronchodilator assessment provides a practical approach for evaluating airflow reversibility and enhances diagnostic accuracy in suspected asthma.

DNA methylation is a stable epigenetic mark that critically influences the phenotype of immune cells. Identifying differentially methylated regions within immune cell lineages supports their phenotypic and functional characterization, leading to a better understanding of lineage-specific transcriptional regulation. Here, we performed a genome-wide methylation analysis of human innate lymphoid cells (ILCs), which allowed us to define specific epigenetic marker regions for ILC1, ILC2, and ILC3. These regions were associated with genes that have well-described functions in ILCs, such as TBX21 in ILC1, GATA3 and MAF in ILC2, RORC and IL23R in ILC3, but were also found in genetic loci that have not been previously associated with ILCs. In-depth analysis of ILC2-related marker regions within the HPGDS and NRROS gene loci confirmed their critical role in transcriptional regulation and suggested a novel role for NRROS in ILC2. Genome-wide methylation analysis of ILC2, derived from the blood of juvenile donors with atopy or asthma led to the identification of several disease-specific epigenetic regions associated with genes such as GIMAP4 and PTGS2. Together, our study not only provides novel epigenetic marker regions in human ILCs and confirms the functional role of ILC2-related markers, but also identifies promising markers for studying allergies in humans.

Background Recent guidelines differ in how fractional exhaled nitric oxide (FeNO) is used to diagnose school-age asthma, either as one of several tests with a cut-off at 25 ppb or as a single rule-in test at 35 ppb. Evidence on its diagnostic performance and clinical utility in subgroups remain limited. Methods We analysed data from 1,979 school-age children in the Swiss Paediatric Airway Cohort referred for suspected asthma. We investigated FeNO performance with diagnosis by paediatric pulmonologists as reference standard using receiver operating characteristics curves, selected cut-offs and simulated predictive values across different prevalence. Subgroup analyses considered allergic sensitisation with allergic rhinitis and current inhaled corticosteroid (ICS) use. Results In the overall cohort (asthma diagnosis 70%), FeNO showed poor discrimination for asthma (AUC 0.66; 95% CI 0.64-0.68) with an optimal cut-off at 22 ppb. At 25 and 35 ppb, sensitivity was low (43%, 95% CI 40-46; 31%, 95% CI 29-34) and specificity moderate to high (84%, 95% CI 77-84; 90%, 95% CI 87-92). Positive predictive value at 35 ppb was 88% and was 57% when simulated at a prevalence of 30%. FeNO had no diagnostic value in non-sensitised children and lower performance in sensitised children with allergic rhinitis than in those without (AUC 0.59 vs 0.68). Current ICS use did not influence performance. Conclusion FeNO has limited diagnostic performance as a stand-alone test for school-age asthma, and underlying asthma prevalence and allergic characteristics should be considered in the interpretation.

BackgroundGastroesophageal reflux disease (GERD) is highly prevalent in systemic sclerosis (SSc) and frequently persists despite proton pump inhibitor (PPI) therapy. However, the mechanisms underlying PPI-refractory GERD in SSc remain incompletely understood. MethodsWe conducted a singlel7lcentre, retrospective study of adults with SSc who underwent ambulatory pH-multichannel intraluminal impedance (pH/MII) monitoring while receiving twicel7ldaily PPI therapy (2021-2025). Esophageal motility (highl7lresolution manometry, HREM) and gastric emptying scintigraphy were integrated to examine associations between gastro-esophageal dysmotility and reflux phenotypes. ResultsThirty patients were included, of whom 67% had PPI-refractory reflux symptoms and 33% were undergoing pre-lung transplantation evaluation. Refractory GERD was present in 29/30 patients (97%) based on Lyon 2.0 classification, with conclusive evidence in 53% and borderline evidence in 43%. Esophageal dysmotility was identified in 80%, most commonly absent contractility (67%), and was associated with impaired reflux clearance, reflected by longer acid clearance times (2.20 [1.15-3.75] vs 1.15 [0.43-1.90] min) and prolonged reflux episode duration (16.60 [4.38-40.63] vs 1.95 [0.53-20.43] min). Gastric dysmotility was identified in 60.7% and was associated with an increased reflux episode burden (51.00 [30.00-81.50] vs 25.00 [21.00-54.00] episodes/24h). ConclusionsPPIl7lrefractory GERD is nearly universal in this SSc cohort and reflects heterogeneous, quantifiable abnormalities across the foregut, including impaired esophageal clearance and increased reflux burden related to gastric retention. These findings support integrated physiologic evaluation to define reflux mechanisms, inform risk stratification (including lung transplantation), and guide targeted, mechanism-based therapies beyond acid suppression.