Measurement is an essential activity in the field of allergy and due to the subjective nature of the results that are being measured, it is increasingly necessary to have valid, reproducible, and reliable methods. There are some reviews or task forces that present different methods or techniques to develop a validation study, which may cause some confusion. With this review, we intend to bring together and synthesize some of the information found, to help researchers and clinicians to interpret or understand the varied nomenclature read in the literature. Before any data collection, a translation of the questionnaire is needed. Psychometry involves the application of statistical techniques to test the measurement properties of an instrument. There are several measures to evaluate an instrument, the main ones being: classical test theory and modern test theory. Regarding the classical test theory, the key psychometric characteristics are scale structure, accuracy (validity), precision (reliability) and responsiveness. Modern test theory models are techniques to assess the psychometric characteristics of an instrument, focused on the dimensionality of the questionnaire. Responsiveness, validity, and precision are interlinked. Following these steps ensures the development of validated questionnaires that provide valuable insights into allergy prevalence, impact, and management.

Background and objective Exposure to inorganic dust and particulate matter (PM) disrupts the integrity and function of the airway epithelial barrier (AEB). Herein, the immune response and epithelial barrier integrity disruption effect of respirable ex vivo airway tissue models. Methods Particles at an average size of 1 µm, referred to as ex vivo. Computational fluid dynamics simulations were performed to assess shear stress profiles under different flow conditions. WST-1, LDH, SEM, ELISA, IF and qRT-PCR analyses were carried out to evaluate cell survivability, morphology, barrier integrity and inflammation. Results In AEB-on-a-chip platform, the exposure to PM2.5 dusts disrupted AEB integrity via decreasing cell adhesion-barrier markers such as ZO-1, Vinculin, ACE2 and CD31, impaired cell viability and increased the expression levels of proinflammatory markers; IFNs, IL-6, IL-1s, TNF-α, CD68, CD80, and Inos, mostly under dynamic conditions. Besides, decreased tissue viability, impaired tissue integrity via decreasing of Vinculin, ACE2, β-catenin and E-cadherin, and also proinflammatory response with elevated CD68, IL-1α , IL-6, IFN-Ɣ, Inos and CD80 markers, were observed after PM2.5 dust exposure in ex vivo tissue. Conclusion The disruption of the barrier integrity and subacute immune response upon silica PM2.5 exposures is nicely demonstrated by both AEB-on-a-chip platform emulating dynamic forces in the body and resident immune cells containing ex vivo bronchial biopsy slices. Airborne dust pollutants pose a significant future respiratory hazard for human airways under climate change conditions and Lung-on-a-Chip Models will serve as reliable substitutes in this context.

Background: Peanut allergy is among the most severe and common food allergies. The diagnosis has a significant impact on the quality of life for patients and their families. An effective management approach depends on accurate, safe, and easily implementable diagnostic methods. We previously developed a cell-based assay using Hoxb8 mast cells (Hoxb8 MCs) aimed at improving clinical allergy diagnosis. In this study we assessed its diagnostic performance by measuring blinded sera from a prospectively enrolled and pre-validated peanut allergy cohort. Methods: Hoxb8 MCs were passively sensitized with sera from peanut-allergic and peanut tolerant children and adolescents (n=96). Degranulation of Hoxb8 MCs was quantified upon stimulation with dose-titrated peanut extract by means of flow cytometry, using CD107a as activation marker. The results from the Hoxb8 mast cell activation test (Hoxb8 MAT) were compared to established diagnostic assays such as the skin prick test (SPT), specific IgE (sIgE) levels, and the basophil activation test (BAT). Additionally, serum samples from BAT non-responders were assessed with the Hoxb8 MAT. Results: Hoxb8 MAT displayed a robust dose-dependent activation to peanut extract, with a cut-off value of ≤5.2% CD107a positive cells. The diagnostic accuracy was highest at allergen concentrations ≥100 ng/ml, with an area under the receiver operating characteristic curve (AUROC) of 0.97, 93% sensitivity, and 96% specificity, outperforming traditional SPT and sIgE tests. When compared to BAT, Hoxb8 MAT exhibited comparable diagnostic efficacy. However, sera from BAT non-responders were accurately classified into allergics and non-allergics by the Hoxb8 MAT. Conclusions: The Hoxb8 MAT demonstrated a very good diagnostic precision in patients prospectively assessed for peanut allergy comparable to the fresh blood based BAT. It also demonstrated its value for accurate classification of BAT non-responders into allergic and non-allergic individuals. Further investigations into its utility in the routine clinical setting are warranted.

Background: Limited information is available on the use of omalizumab (OMA) updosing since its introduction as a second-line therapy in chronic spontaneous urticaria (CSU) in 2014. Practical guidelines from health authorities are lacking, and the specific characteristics of patients requiring higher doses remain unknown. Our objectives were to characterize the patterns of OMA updosing (defined as changes in dose and/or injection intervals), to identify the predictive factors associated with updosing, and to improve CSU management. Methods: We conducted a prospective, multicentric, real-life, observational study, including patients diagnosed with CSU and starting OMA. The data were collected at 0, 3, 6 and 9 months. The primary endpoint was the frequency of OMA updosing at 3 months. The secondary endpoints included an analysis of updosed patients’ profile, and an assessment of OMA efficacy and safety. Results: We included 153 patients. Twenty percent of patients at M3 were updosed, and 27% in total during the 9-month follow-up. Practitioners mainly chose to increase the frequency of injections (66%). At baseline, the updosed patients were more likely, to have inducible urticaria (50% vs. 33%, p=0.046), more severe CSU (UCT 3 vs. 4, p<0.001; DLQI 14 vs. 11, p=0.040), a lower lymphocyte count (1894 vs. 2100, p=0.026) and IgE below 70 UI/mL (65% vs. 45%, p=0.039). The side effects of OMA were not more frequent after updosing. Conclusion: One in five patient underwent updosing within just three months. OMA updosing is frequent in particular in cases of severe disease, inducible urticaria and low IgE blood levels.

Subcutaneous adipose tissue (SAT) is the deepest component of the three-layered cutaneous integument. While mesenteric adipose tissue-based immune processes have gained recognition in the context of the metabolic syndrome, SAT has been traditionally considered primarily for energy storage, with less attention to its immune functions. SAT harbors a reservoir of immune and stromal cells that significantly impact metabolic and immunologic processes not only in the skin, but even on a systemic level. These processes include wound healing, cutaneous and systemic infections, immunometabolic and autoimmune diseases, inflammatory skin diseases, as well as neoplastic conditions. A better understanding of SAT immune functions in different processes, could open avenues for novel therapeutic interventions. Targeting SAT may not only address SAT-specific diseases but also offer potential treatments for cutaneous or even systemic conditions. This review aims to provide a comprehensive overview on SAT’s structure and functions, highlight recent advancements in understanding its role in both homeostatic and pathological conditions within and beyond the skin, and discuss the main questions for future research in the field.

Background Approximately 70 % of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g. hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) in vitro and in vivo. Methods CD4 + T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor- specific restimulation the phenotype, proliferation and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. Results After IL-10 DC priming and allergen-specific restimulation (Bet or Cor) non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder T H2 cell responses in vitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). In vivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms and immune responses. Conclusion Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

Single-cell transcriptomics reveals the alteration of peripheral blood mononuclear cells in AR patient challenge by allergen

Background: Various biomarkers are used to define peanut allergy (PA). We aimed to observe changes in PA resolution and persistence over time comparing biomarkers in PA and peanut sensitised but tolerant (PS) children in a population-based cohort. Methods: Participants were recruited from the EAT and EAT-On studies, conducted across England and Wales and were generally well exclusively breastfed babies recruited at 3 months old and followed up until 11 years old. Clinical characteristics, skin prick test (SPT), sIgE to peanut and peanut components and mast cell activation tests (MAT) were assessed at 12m, 36m and 7-11y. Results: The prevalence of PA was 2.1% with only 1 child having PA resolution at 7-11y. PA children had larger SPT size, higher peanut-sIgE, Ara h 2-sIgE and MAT (all p<0.001) compared to PS children at 36m and 7-11y. SPT, peanut-sIgE, Ara h 2-sIgE and MAT between children with persistent PA, new PA, outgrown PA and PS were statistically significant at both 36m and 7-11y (p<0.001). Those with persistent PA had SPT, peanut-sIgE and Ara h 2-sIgE that increased over time and MAT which was highest at 36m. New PA children had increased SPT and peanut-sIgE from 36m to 7-11y, but MAT remained low. PS children had low biomarkers across time. Conclusions: In this cohort, few children outgrow or develop new PA between 36m and 7-11y. Children with PA have significantly higher SPT, peanut-sIgE, Ara h 2-sIgE and MAT compared to PS children, evident from 12-36m of age.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype, and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment, and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

Transcriptomic Profiles of Well-Differentiated Airway Epithelial Cells in Response to Environmental Triggers of Asthma ExacerbationAntonella Marrocco1, Jennifer A. Mitchel1, Margaret Parker2, Maureen McGill1, Robert P. Chase2, Scott T. Weiss2, Diane R. Gold1,2, Peter J. Castaldi2, Jin-Ah Park1, Joanne Sordillo34Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA2Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.3Division of Chronic Disease Research Across the Lifecourse (CoRAL), Department of Population Medicine, Harvard Medical School and Harvard Pilgrim Health Care Institute, Boston, MA, USA.Corresponding Author: Joanne E. SordilloEmail : [email protected]:Landmark Center West 401 Park Dr., 4th Floor Boston, MA 02115The authors have no conflicts of interest to disclose.Funding: R01HL148152, P30ES000002, T32HL007118, Francis Family FoundationKeywords: Bronchial epithelial cells, gene expression, RNAseq, asthma

The gut microbiome is indispensable for the host physiological functioning. Yet, the impact of non-nutritious dietary compounds on the human gut microbiota and the role of the gut microbes in their metabolism and potential adverse biological effects have been overlooked. Identifying potential hazards and benefits would contribute to protecting and harnessing the gut microbiome’s role in supporting human health. We discuss the evidence on the potential detrimental impact of certain food additives and microplastics on the gut microbiome and health endpoints, with a focus on underlying mechanisms and causality. We provide recommendations for the incorporation of gut microbiome science in food risk assessment and identify knowledge and tools needed to fulfill the gaps. The incorporation of gut microbiome endpoints to safety assessments, together with well-established toxicity and mutagenicity studies, might better inform the risk assessment of certain contaminants in food, and/or food additives.

Background: The use of allergy tests to guide dietary exclusions for disease control in children with atopic dermatitis (AD) is controversial. We undertook a consensus exercise on how to interpret skin prick test (SPT) results and dietary history for cow’s milk, hen’s egg, wheat and soya in children <2 years old with AD. Methods: Fourteen clinicians from general practice, paediatrics, paediatric dermatology, paediatric allergy and paediatric dietetics from UK and Ireland took part in an online modified Delphi study. Over three rounds, participants gave their anonymous opinions and received individualised and group feedback. The findings were discussed in an online workshop. Results: Of 14 symptoms, 12 were identified as relevant to immediate and 7 to delayed allergy. Regarding SPTs, there was consensus over which allergens to use for wheat and soya but not cow’s milk or hen’s egg; for all study foods, wheal size was determined as 0-1 mm negative, ≥5mm sensitised , but between 2-4 mm categorisation varied by food. During the final workshop, consensus was reached on dietary advice should be given according to SPT results and dietary history. Conclusion: We attained consensus on how SPTs combined with dietary history for four common food allergens should be interpreted in young children under two years of age with AD. These pragmatic recommendations may support clinician education, consistency of decision-making and future research.

The skin barrier is vital for protection against environmental threats including insults caused by skin-resident microbes. Dysregulation of this barrier is a hallmark of atopic dermatitis (AD) and ichthyosis, with variable consequences for host immune control of colonizing commensals and opportunistic pathogens. While Malassezia is the most abundant commensal fungus of the skin, little is known about the host control of this fungus in inflammatory skin diseases. Here we show that in barrier-impaired skin, Malassezia acquires enhanced fitness and overt growth properties. By using four distinct and complementary murine models of atopic dermatitis and ichthyosis we provide evidence that structural and metabolic changes in the dysfunctional epidermal barrier environment provide increased accessibility and an altered lipid profile, to which the lipid-dependent yeast adapts for enhanced nutrient assimilation. These findings reveal fundamental insights into the implication of the mycobiota in the pathogenesis of common skin barrier disorders.

Following the European Forum for Research and Education in Allergy and Airway Diseases (EUFOREA) treatment algorithm for chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), patients suffering from severe uncontrolled CRSwNP are recommended to receive oral corticosteroids, (revision) sinus surgery, systemic biologicals, and/or aspirin treatment after desensitization (ATAD). Given the major differences in indications, outcomes, practical considerations, risks and costs of these key pillars of treatment, there is a growing need to define criteria for each treatment option and list the clinically relevant and major considerations for them. This EUFOREA document therefore provides an expert panel overview of the expected outcomes, specific considerations and (contra)indications of the five major treatment arms of severe uncontrolled CRSwNP: oral corticosteroids, primary and revision sinus surgery, biological treatment, and ATAD. This overview of treatment considerations is needed to allow physicians and patients to consider the different options in the context of providing optimal and personalized care for severe uncontrolled CRSwNP. In conclusion, the five major treatment options for severe uncontrolled CRSwNP have intrinsic advantages, specific indications, and considerations that are of importance to the patient, the physician, and the society. This EUFOREA statement supports the unmet need to define criteria for the indication of every treatment pillar of CRSwNP.

Sputum cytokines associated with raised FeNO after anti-IL5 biologic therapy in severe asthma.To the Editor,Biomarkers such as circulating absolute eosinophil count, % of eosinophils in sputum, and fraction of exhaled nitric oxide (FeNO) are predictors of response to anti-inflammatory therapy for asthma. Failure to normalize FeNO with high doses of corticosteroids are likely to be related to cytokines and chemokines such as IL-5, IL-4, IL-13, eotaxin and TARC derived from eosinophils and other Th2 cells, and alarmins such as TSLP and IL-33 from sources such as the airway epithelium(1). All Anti-IL5 biologics suppress eosinophils in sputum. However benralizumab (anti-IL5RMab) has greater effect in the severe prednisone-dependent patients than mepolizumab/reslizumab (anti-IL5 neutralizing Mab)(2,3). While raised eosinophil count is a predictor of clinical response to anti-IL5 biologics, raised FeNO is often not(4). However, they reduce FeNO to variable levels(4,5) suggesting that FeNO is partly regulated by cytokines derived from eosinophils in the airway(1). The cytokines in sputum associated with raised FeNO in prednisone-dependent severe asthmatics treated with effective anti-eosinophil drugs are not known.In this retrospective observational study, we measured cytokines in sputum using an automated ELISA reader (EllaTM, Protein Simple, R&D Systems, BioTechne, Minneapolis) at baseline and after 4 months of treatment with either benralizumab or mepolizumab/reslizumab and compared the levels of cytokines in those whose FeNO remained high after treatment. Raised FeNO was defined by FeNO >40ppb and an increase of at least 16ppb from pre-treatment value. The study was approved by Hamilton Integrated Research Ethics Board (#11227, 5037), and all patients gave written informed consent. The cytokines assayed were IL-5, IL-4 and IL-13 (Th2 inflammation) and IL-1β, IL-6, IL-10, IL-12p70, IL-15 IL-17A, IL-18, IL-33, IFNγ and TNFα (Th1/Th17 inflammation, Table e3). Details of baseline demographics, methods and statistics are shown in the online supplement.Paired measurements were made in 30 patients who received benralizumab, and 10 each who received mepolizumab/reslizumab. Overall, as previously reported(5), FeNO levels were not significantly reduced by anti-IL5 treatment (median FeNO pre-treatment 29 [5-156] vs FeNO post treatment 37 [6-280]; p=0.25; Figure e1). This change in FeNO did not correlate with a reduction of sputum eosinophils (r=-0.24; p=0.16).Among 15 patients, FeNO remained raised after treatment (Table 1). On average, IL-4 and IL-13 were the only cytokines significantly higher in the sputum of these patients compared to those in whom the FeNO values normalized (Figure 1, Figure e2). Within this group, there were patients with raised IL-4 (31%) and IL-13 (15%) and those with normal IL-4/IL-13. A small proportion of those with normal IL4/13 had raised levels of IL-18 or IL-1β (20%). Residual eosinophilic airway inflammation was significantly more present in patients with raised FeNO (30.8% vs 8.1%; P=0.04; Table e2). Patients with raised FeNO remained to have poor asthma control with an ACQ>1.5, however this did not significantly differ from those with a normalized FeNO (ACQ 1.7±0.9 vs 1.4±1.1; P=0.36; Table 1).This study, despite its limitation of retrospective design and small numbers, provide novel information on the cytokine profile in the airways of severe prednisone-dependent eosinophilic asthma patients whose FeNO remain high after anti-IL5 treatment. This is a common clinically encountered situation. Our observations suggest that IL-4/IL-13 are the cytokines most associated with this phenomenon. This may be due to the airway eosinophilia being uncontrolled or due to a non-eosinophilic source of these cytokines. However, there could be non-IL-4/IL-13 related increase in FeNO that may be due to inflammasome activation and through non-Th2 cytokine pathways that may raise the possibility of airway infections or autoimmune activation(6). This has important clinical implication. These patients may not show adequate response to switching to anti-IL4R Mab if their asthma remains uncontrolled. This needs to be evaluated prospectively.References:Couillard S, Shrimanker R, Chaudhuri R, et al. Fractional Exhaled Nitric Oxide Nonsuppression Identifies Corticosteroid-Resistant Type 2 Signaling in Severe Asthma. Am J Respir Crit Care Med 2021; 204: 731-734.Mukherjee M, Forero DF, Tran S, Boulay ME, et al. Suboptimal treatment response to anti-IL-5 monoclonal antibodies in severe eosinophilic asthmatics with airway autoimmune phenomena. Eur Respir J 2020 Oct 8;56(4):2000117. doi: 10.1183/13993003.00117-2020.Mukherjee M, Bhalla A, Venegas-Garrido C, et al. Benralizumab attenuates blood and airway eosinophilia in severe asthmatics with inadequate response to anti-IL-5 neutralizing antibodies [abstract]. Eur Respir J 2022; 60 (suppl 66): 3994; DOI: 10.1183/13993003.Hearn AP, Kavanagh J, d’Ancona G, et al. The relationship between Feno and effectiveness of mepolizumab and benralizumab in severe eosinophilic asthma. J Allergy Clin Immunol Pract 2021; 9: 2093-2096.e1.Nair P, Kjarsgaard M, Armstrong S, Efthimiadis A, O’Byrne PM, Hargreave FE. Nitric oxide in exhaled breath is poorly correlated to sputum eosinophils in patients with prednisone-dependent asthma. J Allergy Clin Immunol 2010; 126: 404-6.Donnelly LE, Barnes PJ. Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells. Am J Respir Cell Mol Biol 2002; 26: 144-51.AuthorsPieter-Paul Hekking 1,2, Kayla Zhang1, Carmen Paz Venegas Garrido 1, Raquel Lopez-Rodriguez 1,3, Melanie Kjarsgaard1, Manali Mukherjee 1, Parameswaran Nair 11. Division of Respirology, Department of Medicine, St. Joseph’s Healthcare Hamilton, McMaster University, Hamilton, Ontario, Canada.2. Department of Respiratory Diseases, STZ Centre of Excellence for Asthma & COPD, Franciscus Gasthuis & Vlietland Hospital, Rotterdam, the Netherlands.3. Department of Allergy, Lucus Augusti Hospital, Lugo, SpainCorrespondenceDr Parameswaran NairFirestone Institute for Respiratory HealthSt Joseph’s Healthcare Hamilton50 Charlton Avenue EastHamilton, Ontario, L8N4A6, CanadaTel: 905-522-1155 x 35044Fax: 905-521-6183E-mail: [email protected]

Background: IgE-mediated cow’s milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which β-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies. Methods: We report a longitudinal in vivo murine model, in which 2 mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n=6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further supported by fecal microbiome-metabolome data from our population of IgE-CMA (n=24) and healthy (n=23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes. Results: During sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. The latter was reflected in a disturbed sphingolipid metabolism. We confirmed microbial dysbiosis, and its causal effect on metabolic alterations in our patient cohort, which was accompanied by metabolic signatures of low-grade inflammation. Conclusion: Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.

Background Thymic stromal lymphopoietin (TSLP), a pleiotropic cytokine mainly expressed by epithelial cells, plays a key role in asthma pathobiology. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP), overlapping the lfTSLP C-terminus. Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play central roles in different asthma phenotypes. Methods Immunofluorescence and Western blot were employed to localize intracellular TSLP. Limited proteolysis and mass spectrometry allowed the identification of cleavage sites of TSLP caused by tryptase and chymase. ELISA assays were employed to measure TSLP and VEGF-A. Results TSLP was detected in highly purified (≥ 99%) macrophages isolated from human lung and subcellularly localized in the cytoplasm by confocal microscopy and Western blot. IL-4 and lipopolysaccharide induced the release of TSLP from HLMs. HLMCs contain and release tryptase and chymase that specifically cleaved TSLP. Mass spectrometric analyses of TSLP treated with tryptase showed the production of 1-97 and 98-132 fragments. Chymase treatment of TSLP generated two peptides 1-36 and 37-132. HLM activation by lfTSLP induced VEGF-A, the most potent angiogenic factor, release. The four TSLP fragments generated by tryptase and chymase failed to activate HLMs. sfTSLP neither activated HLMs nor interfered with activating property of lfTSLP on HLMs. Conclusions Given the close proximity between mast cells and macrophages in the human lung, our results illuminate a new circuit between HLMs and mast cells. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

Background Activation of mast cells through IgE results in secretion and shedding of mast cell proteins and in vivo models suggest that these processes are governed by IgE antibody affinity. Methods We passively sensitized cultured primary human mast cells with recombinant human IgE clones with either high or low affinity for Der p 2, with a 200-fold affinity difference, and activated them with recombinant allergen. Activation was assessed by CD63 upregulation and PGD 2 secretion. Supernatants collected from mast cells activated for 0, 3, 6 and 24 hours were assessed for PGD 2 and inflammatory mediators on the OLINK platform at repeated time points. Results CD63 upregulation and PGD 2 synthesis scaled with affinity, as did secretion of cytokines like IL-8 and IL-13. Secretion of chemokines like CCL3 and CCL4 appeared to depend less on affinity, whereas shedding of surface markers CD40, SLAMF4 and CD5, and secretion of intracellular markers SIRT2 and CASP-8, were elevated by stimulation through low affinity IgE compared with high affinity IgE, illustrating differential responses dependent on the affinity of IgE. Conclusion Cytokine secretion and shedding of surface receptors of sensitized, cultured primary human mast cells is differentially regulated depending on the affinity of IgE for the Der p 2 allergen and may shape the chronic response to repeated allergic activation.