Direct cleavage and activation of gasdermin B by asthma trigger allergensTo the Editor:Recent fine-mapping studies have pointed to gasdermn B (GSDMB ) as a potential asthma susceptibility gene in 17q21 locus, the strongest and most highly replicated signal in genome-wide association studies1. The GSDMB protein is a member of the gasdermin family that, when cleaved, triggers an inflammatory cell death known as pyroptosis2. Caspase-1 and granzyme A have been shown to cut GSDMB at specific sites to release the N-terminal fragment of the protein (GSDMB-NT) that has the ability to induce pyroptosis in cells, including airway epithelial cells3,4. These findings suggest that the role of GSDMB in asthma lies in its ability to be activated through cleavage to induce pyroptosis; however, it remains unclear whether GSDMB cleavage and activation occur in the context of asthma.Common asthma trigger allergens often possess protease activities that cause airway epithelial injury and inflammation5,6. We thus tested whether the allergens directly cleave GSDMB. Incubation of extracts from house dust mite (HDM), a common asthma trigger, with lysates from human bronchial epithelial cells, which express endogenous GSDMB3, resulted in GSDMB cleavage as evidenced by the appearance of a smaller protein around 17kD (Figure 1A). Since the GSDMB antibody used in the Western blotting targets the C-terminus of the protein, the 17kD protein band likely represents the C-terminal GSDMB fragment. Such GSDMB cleavage was also observed when lysates from cells expressing C-terminal-FLAG-tagged GSDMB were mixed with HDM extract (Figure 1B). Furthermore, mold or cockroach extract also cleaved tagged GSDMB (Figure 1C). The cleavage of GSDMB protein by all allergen extracts resulted in a single product of similar size (about 17 kD), suggesting a specific cutting site.To identify the cleavage site, we incubated recombinant full-length GSDMB with HDM extract and resolved the cleaved protein products on SDS-PAGE (Figure 1D). We excised the putative 17 kD C-terminal fragment (GSDMB-CT, Figure 1D) and determined the N-terminal amino acid sequence of the fragment via Edman sequencing (Supplemental Figure S1, Figure 1E). Despite some ambiguities, the first ten amino acid residues of the 17 kD GSDMB-CT largely map to position 245 to 254 (SLGSEDSRNM) of the full length GSDMB protein (Figure 1E). This result indicates that GSDMB was cleaved immediately after the lysine residue at position 244 (K244). Interestingly, granzyme A also cuts GSDMB at the same K244 site4. To confirm K244 as the site of cleavage, we mutated lysine 244 to alanine (K244A) in GSDMB and tested whether the mutant protein can be cleaved by HDM. As shown by Western blotting, HDM was able to cleave wild type (WT) GSDMB but failed to cleave K244A GSDMB as evidenced by the absence of the 17 kD fragment (Figure 1F).The cleavage of GSDMB by HDM is expected to release an N-terminal fragment of 244 amino acids (GSDMB-NT-K244) (Figure 2A). We next tested whether GSDMB-NT-K244 triggers pyroptosis. Transfection of GSDMB-NT-K244 induced cell morphological changes characteristic of pyroptosis, including rounding up and detachment (Figure 2B). LDH release assay confirmed increased toxicity in these cells (~3.4 fold) as compared to cells transfected with the full-length GSDMB (Figure 2C). Consistent with our previous finding on GSDMB-NT shortened by a functional asthma-associated splice variant3, transfection of a truncated GSDMB-NT from the variant (NT-K231var) did not induce pyroptosis (Figure 2B,C).While future studies are needed to identify the specific proteases within the allergen extracts that cleave GSDMB, our current study demonstrates that asthma triggers such as HDM can directly cleave and activate GSDMB, thus providing biochemical evidence linking GSDMB-mediated pyroptosis to asthma.
Progressive knowledge of allergenic structures resulted in a broad availability of allergenic molecules for diagnosis. Component resolved diagnosis allowed a better understanding of patient sensitization patterns, facilitating allergen immunotherapy decisions. In parallel to the discovery of allergenic molecules, there was a progressive development of a regulation framework that affected both in vitro diagnostics and Allergen Immunotherapy products. With a progressive understanding of underlying mechanisms associated to Allergen immunotherapy and an increasing experience of application of molecular diagnosis in daily life, we focus in analyzing the evidences of the value provided by molecular allergology in daily clinical practice, with a focus on Allergen Immunotherapy decissions.
Although there is a considerable body of knowledge about allergen immunotherapy (AIT), there is a lack of data on the reliability of real-world evidence (RWE) in AIT and consequently, a lack of information on how AIT effectively works in real life. To address the current unmet need for an appraisal of the quality of RWE in AIT, the European Academy of Allergy and Clinical Immunology Methodology Committee recently initiated a systematic review of observational studies of AIT, which will use the RELEVANT tool and the Grading of Recommendations Assessment, Development and Evaluation approach (GRADE) to rate the quality of the evidence base as a whole. The next step will be to develop a broadly applicable, pragmatic “real-world” database using systematic data collection. Based on the current RWE base, and perspectives and recommendations of authorities and scientific societies, a hierarchy of RWE in AIT is proposed, which places pragmatic trials and registry data at the positions of highest level of evidence. There is a need to establish more AIT registries that collect data in a cohesive way, using standardised protocols. This will provide an essential source of real-world data that can be easily shared, promoting evidence-based research and quality improvement in study design and clinical decision-making.
In addition to known allergens, other proteins in pollen can aid the development of an immune response in allergic individuals. The contribution of the “unknown” protein allergens is apparent in phylogenetically related species where, despite of high homology of the lead allergens, the degree of allergenic potential can vary greatly. The aim of this study was to identify other potentially allergenic proteins in pollen of three common and highly related allergenic tree species: birch (Betula pendula), hazel (Corylus avellana) and alder (Alnus glutinosa). For that purpose, we carried out a comprehensive, comparative proteomic screening of the pollen from the three species. In order to maximize protein recovery and coverage, different protein extraction and isolation strategies during sample preparation were employed. As a result, we report 2500 - 3000 identified proteins per each of the pollen species. Identified proteins were further used for a number of annotation steps, providing insight into differential distribution of peptidases, peptidase inhibitors and other potential allergenic proteins across the three species. Moreover, we carried out functional enrichment analyses that, interestingly, corroborated high species similarity in spite of their relatively distinct protein profiles. We provide to our knowledge first insight into proteomes of two very important allergenic pollen types, hazel and alder, where not even transcriptomics data is available, and compared them to birch. Datasets from this study can be readily used as protein databases, and as such serve as basis for further functional studies.
To the Editor,The proportion of the population with allergic diseases has increased rapidly in recent decades1, 2. In addition to affecting the quality of life, a significant economic burden of these diseases was transferred to society and the national health care system1. China is a large country with a rapidly developing economy, wide geography, and diverse climate and lifestyles, which may lead to significantly regional differences in the distribution of allergens. Although a series of studies have explored the prevalence of allergen sensitization in China, the majority of them focus on one part of geography in China3-5. In 2009, a study6 was conducted to estimate the prevalence of common aeroallergens among patients with allergic asthma and/or rhinitis in mainland China. Although the study investigated the differences of the prevalence in different regions of China, it divided China into only four geographical regions, which may neglect detailed information about the characteristics of sensitization prevalence in different places in China. In that study, the skin prick test (SPT) was used to detect the sensitization to allergens. The method has low accuracy for positive results because it is heavily affected by certain factors, such as the skill of the tester, reagent used, interpretation of results and so on. Our research has the following different characteristics compared with previous studies: 1) covering a variety of allergic diseases, 2) exploring both aeroallergens and food allergens simultaneously, 3) including a large set of data from all the seven regions of mainland China, and 4) using an internationally recognized method of sIgE testing, ImmunoCAP, to detect sensitization. These advantages may help us obtain more accurate and reliable results and conclusions.Here, we conducted a large multicenter study on the prevalence patterns of serum allergen-specific IgE (sIgE) sensitization to the four most common food allergens (i.e., egg whites, cow’s milk, crab, and shrimp) and five aeroallergens (i.e., house dust mite, German cockroach, tree pollen mix, mold mix, dog dander) among 44156 patients with allergic symptoms in 52 cities from 26 provinces of all the seven geographical regions in mainland China from July 2015 to June 2018. The sIgE sensitization was tested by a certified third-party laboratory service provider with uniform and standardized procedures. This study was approved by the ethics committee of the First Affiliated Hospital of Guangzhou Medical University (Approval number: GYFYY-2017-18). Details about the methods were in the supplementary materials .Our study showed that the overall prevalence of positive sIgE responses to the 9 allergens across mainland China from the highest to the lowest was 33.74% for house dust mites, 24.5% for cockroaches, 19.97% for shrimp, 17.31% for crab, 11.62% for cow’s milk, 10.92% for egg whites, 9.35% for tree pollen mix, 4.02% for dog dander and 3.92% for mold mix (Table 1 ). Our study confirmed that an observation shown in several previous studies based on certain specific areas in China3-5 that the positive cases in sIgE fell mainly in the two low classes (i.e., classes 1 and 2) was also held in all the seven regions in mainland China (Table 1 ).Our study revealed the distinctive patterns in the prevalence of allergen sensitization among regions, gender, age groups and seasons. Geographically, there is a significant difference in the prevalence among regions for all 9 allergens except for the mold mix (Table S1 ). House dust mites were the allergen with the highest prevalence of sensitization in all seven regions, with the highest in South China (40.79%) and the lowest in Northeast China (11.21%). Allergies to German cockroaches had a higher prevalence in southern regions (Southwest China, South China and East China) than in northern regions (North China and Northeast China). The prevalence of sIgE responses to dog dander was the highest in North China and was very close to each other in the southern regions. The prevalence of the egg whites and milk in Central China, East China and South China was higher than in Southwest China, North China and Northeast China, which means that patients living in eastern, coastal and/or southern areas were more sensitive to egg whites and cow’s milk. The prevalence of crab and shrimp sensitization in Southwest China and South China was higher than that in the northern regions (North China and Northeast China). The heatmap (Figure 1 ) displays the distribution of the prevalence of the sIgE response to allergens in different regions of mainland China.The prevalence of sensitization to all nine allergens was higher overall in males than in females (Table 1 and Figure S1 ) although that may not be true in each age group for each allergen as shown in the forest plot in Figure S1 . Our study showed that, whereas the sensitization to egg whites and milk was the highest in children, the sensitization to other allergens tended to be the highest in teenagers and young adults (Figure S2 ). Figure S3displays he prevalence pattern of allergens by months across years. The prevalence of dog dander and mold mix was very stable across months; however, the prevalence of other allergens fluctuated from January to December. The prevalence of house dust mites, German cockroach, shrimp and crab were higher in the summer months (from June to August) than in other months. The prevalence of tree pollen mix was much higher in April and October than in other months.This should be the first large study to investigate the prevalence of allergen sensitization in the patients with allergic symptoms from all the seven geographic regions of mainland China. Based on this study, we found that the prevalence of sIgE sensitization to allergens displayed obvious and distinctive patterns among regions, gender, age groups and seasons. The reasons for these patterns may include lifestyle factors, socioeconomic factors, genetic predispositions, climate, sexual hormones, cross-reactivity and so on3,4,6-9. Please refer to the supplementary materials for the detailed discussion on the factors that influenced these variations. Our findings may help clinicians find effective individualized treatments for unique patient groups and direct researchers to conduct further studies on the epidemiology of allergic diseases.
Background The prevalence of allergy to cat is expanding worldwide. Allergen-specific immunotherapy (AIT) has advantages over symptomatic pharmacotherapy and promises long lasting disease control in allergic patients. However, there is still a need to improve cat AIT regarding efficacy, safety and adherence to the treatment. Here we aim to boost immune tolerance to the major cat allergen Fel d 1 by increasing the anti-inflammatory activity of AIT with the established immunomodulatory adjuvant CpG, but at a higher dose than previously used in AIT. Methods Together with CpG, we used endotoxin-free Fel d 1 as therapeutic allergen throughout the study in a BALB/c model of allergy to Fel d 1, thus mimicking the conditions of human AIT trials. Multidimensional immune phenotyping including mass cytometry was applied to analyze AIT-specific immune signatures. Results We show that AIT with high-dose CpG in combination with endotoxin-free Fel d 1 reverts all major hallmarks of allergy. High dimensional CyTOF analysis of the immune cell signatures initiating and sustaining the AIT effect indicates the simultaneous engagement of both, the pDC-Treg and -B cell axis, with the emergence of a systemic GATA3+ FoxP3hi biTreg population. The regulatory immune signature also suggests the involvement of the anti-inflammatory TNF/TNFR2 signaling cascade in NK and B cells at an early stage and in Tregs later during AIT. Conclusion Our results highlight the potential of CpG adjuvant in a novel formulation to be further exploited for inducing allergen-specific tolerance in patients with cat allergy or other allergic diseases in the future.
Background: Multiplex tests allow for measurement of allergen-specific IgE responses to multiple allergen extracts and components and have several advantages for large cohort studies. Due to significant methodological differences, test systems are difficult to integrate in meta-analyses/systematic reviews since there is a lack of datasets with direct comparison. We aimed to create models for statistical integration of allergen-specific IgE to peanut/tree nut allergens from three IgE-test platforms. Methods: Plasma from Canadian and Austrian children with peanut/tree nut sensitization and a cohort of sensitized, high-risk, pre-school asthmatics (total n=166) were measured with three R&D multiplex IgE test platforms: Allergy Explorer, ALEX (Macro Array Dx), MeDALL-chip (Mechanisms of Development of Allergy) (Thermo Fisher), and EUROLINE (EUROIMMUN). Skin prick test (n=51) and ImmunoCAP (n=62) results for extracts were available in a subset. Regression models (Multivariate Adaptive Regression Splines, local polynomial regression) were applied if >30% of samples were positive to the allergen. Intra-test correlations between PR-10 and nsLTP allergens were assessed. Results: Using two regression methods, we demonstrated the ability to model allergen-specific relationships with acceptable measures of fit (r2=94-56%) for peanut and tree nut sIgE testing at the extract and component-level, in order from highest to lowest: Ara h 2, Ara h 6, Jug r 1, Ana o 3, Ara h 1, Jug r 2, Cor a 9. Conclusion: Our models support the notion that conversion is reasonably possible between sIgE multiplex platforms for allergen extracts and components and may provide options to aggregate data for future meta-analysis.