Background: This initiative aimed to elucidate the clinical relevance of type 2 (T2) inflammation as a driver of asthma, atopic dermatitis, chronic rhinitis, chronic rhinosinusitis with nasal polyps (CRSwNP) and eosinophilic esophagitis. Methods: A steering committee (SC) conducted a non-systematic literature search to inform the design of a Delphi questionnaire including 23 consensus statements, which was circulated to 30 experts including the SC. Experts rated their agreement with each statement on a 9-point Likert scale and provided optional feedback that was used to develop a second Delphi questionnaire. On 22 October 2020, a meeting was held to discuss the conclusions from the questionnaires and explore how this initiative may impact the management of patients with T2 inflammation-driven disease. Post meeting, a consensus statement on the role of T2 inflammation in eosinophilic esophagitis was circulated to the experts. Results: It was agreed that T2 inflammation may be an underlying driver of asthma, atopic dermatitis, chronic rhinitis, CRSwNP and eosinophilic esophagitis, and that the published evidence suggests that these diseases overlap. Some of this overlap may include related multimorbid conditions driven by T2 inflammation. Thus, in patients with multiple T2 inflammation-driven diseases, a cross-speciality approach is warranted to provide effective care. A question guide with input from relevant experts was proposed, to identify comorbidities and facilitate appropriate holistic patient management. Conclusions: These consensus recommendations should be used as a framework to further understand the extent of T2 inflammation-driven multi-organ disease and to improve the holistic management and care of these patients.

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.

Background. Because of altered airway microbiome in asthma, we analysed the bacterial species in sputum of patients with severe asthma. Methods. Whole genome sequencing was performed on induced sputum from non-smoking (SAn) and current or ex-smoker (SAs/ex) severe asthma patients, mild/moderate asthma (MMA) and healthy controls (HC). Data was analysed by asthma severity, inflammatory status and transcriptome-associated clusters (TACs). Results. α-diversity at the species level was lower in SAn and SAs/ex, with an increase in Haemophilus influenzae and Moraxella catarrhalis, and Haemophilus influenzae and Tropheryma whipplei, respectively, compared to HC. In neutrophilic asthma, there was greater abundance of Haemophilus influenzae and Moraxella catarrhalis and in eosinophilic asthma, Tropheryma whipplei was increased. There was a reduction in α-diversity in TAC1 and TAC2 that expressed high levels of Haemophilus influenzae and Tropheryma whipplei, and Haemophilus influenzae and Moraxella catarrhalis, respectively, compared to HC. Sputum neutrophils correlated positively with Moraxella catarrhalis and negatively with Prevotella, Neisseria and Veillonella species and Haemophilus parainfluenzae. Sputum eosinophils correlated positively with Tropheryma whipplei which correlated with pack-years of smoking. α- and β-diversities were stable at one year. Conclusions. Haemophilus influenzae and Moraxella catarrhalis were more abundant in severe neutrophilic asthma and TAC2 linked to inflammasome and neutrophil activation, while Haemophilus influenzae and Tropheryma whipplei were highest in SAs/ex and in TAC1 associated with highest expression of IL-13 Type 2 and ILC2 signatures with the abundance of Tropheryma whipplei correlating positively with sputum eosinophils. Whether these bacterial species drive the inflammatory response in asthma needs evaluation.

Non-steroidal anti-inflammatory drugs (NSAIDs) and other eicosanoid pathway modifiers are among the most ubiquitously used medications in the general population. Their broad anti-inflammatory, antipyretic and analgesic effects are applied against symptoms of respiratory infections, including SARS-CoV-2, as well as in other acute and chronic inflammatory diseases that often coexist with allergy and asthma. However, the current pandemic of COVID-19 also revealed the gaps in our understanding of their mechanism of action, selectivity and interactions not only during viral infections and inflammation, but also in asthma exacerbations, uncontrolled allergic inflammation, and NSAIDs-exacerbated respiratory disease (NERD). In this context, the consensus report summarises currently available knowledge, novel discoveries and controversies regarding the use of NSAIDs in COVID-19, and the role of NSAIDs in asthma and viral asthma exacerbations. We also describe here novel mechanisms of action of leukotriene receptor antagonists (LTRAs), outline how to predict responses to LTRA therapy and discuss a potential role of LTRA therapy in COVID-19 treatment. Moreover, we discuss interactions of novel T2 biologicals and other eicosanoid pathway modifiers on the horizon, such as prostaglandin D2 antagonists and cannabinoids, with eicosanoid pathways, in context of viral infections and exacerbations of asthma and allergic diseases. Finally, we identify and summarise the major knowledge gaps and unmet needs in current eicosanoid research.

Serum pregnancy-associated plasma protein A (PAPPA) as a predictor of eosinophilic Type-2 high asthmaTo the Editor,Pregnancy-associated plasma protein A (PAPPA), a metalloproteinase that cleaves insulin-like growth factor (IGF)-binding proteins (IGFBPs) to increase IGF availability, is expressed systemically in pregnant women but also in other tissues (1). Higher serum PAPPA levels are reported in patients with newly-diagnosed asthma (1) and allergic rhinitis compared to healthy controls and are decreased following omalizumab treatment (2). We determined whether PAPPA could represent a novel biomarker for Type-2 (T2) asthma by exploring the relationship between asthma severity and phenotypes of severe asthma and PAPPA gene and protein expression (3).We recruited 288 severe non-smoking asthma (NSA), 102 smokers and ex-smokers with severe asthma (SSA), 86 mild/moderate non-smoking asthmatics (MMA) and 95 healthy non-smoking controls (HC) from the U-BIOPRED cohort (NCT01976767) (4) (Table S1 ). Transcriptomic and proteomic profiling of blood and sputum samples and specific serum periostin ELISA were performed (3). Gene set variation analysis (GSVA) was used to calculate the enrichment score (ES) of 34 genes that were upregulated following in vitro stimulation of primary human bronchial epithelial cells with IL-13 (T2_IL-13_IVS) (3). Eosinophilic inflammation was defined by sputum eosinophilia >1.49% (3). Local Ethics Committees of the recruiting centres approved the study and all participants gave written informed consent.Sputum cell PAPPA mRNA was elevated in NSA compared to SSA, MMA and HC subjects particularly in granulocytic asthmatics and in the transcriptomic-associated cluster (TAC)1; an eosinophilic cluster (5) (Figure 1A-C ). This was more pronounced with sputum PAPPA protein analysis according to asthma severity, in eosinophilic and mixed granulocytic asthmatics and in T2-high asthmatics identified by the T2_IL-13_IVS signature (Figure 1D-F ).PAPPA mRNA expression in blood cells was similar across asthma severities, blood granulocytes and molecular phenotypes (Supplementary Figure 1A-C ). However, serum PAPPA protein levels supported the discrimination seen in sputum with significant elevation seen in SA compared to HC, in eosinophilic and mixed granulocytic asthma and in T2-high asthma (SupplementaryFigure 1D-F ).Sputum eosinophil percentages were significantly correlated with sputum (r=0.88, p=10-6) and serum (r=0.41, p=10-6) PAPPA protein levels. Overall, sputum PAPPA protein gave a greater distinction between asthma severity, granulocyte composition and T2-high asthma than with serum although fewer samples were available.These results were validated in sputum from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study (6) (Supplementary Figure S2 ). Elevated PAPPA protein in the serum and sputum of severe asthmatics and in eosinophilic compared to non- eosinophilic subjects was seen (SupplementaryFigure S2A-D ). In addition, sputum PAPPA mRNA levels were also elevated in eosinophilic versus non-eosinophilic asthma in the ADEPT cohort (Supplementary Figure S2E ).The ES score of the T2_IL-13_IVS gene signature in bronchial brushings was significantly, but weakly, correlated with blood eosinophil counts (r=0.329, p=10-6), serum PAPPA (r=0.356, p=10-6), but not with serum periostin levels (r=0.07, p-value=0.48). In contrast, the T2 IL-13 IVS ES score was strongly correlated with sputum PAPPA levels (r=0.72, p=10-3). Sputum PAPPA protein levels also significantly correlated with markers of remodelling such as MMP10 (r=0.646, p<10-6) and MET (r=0.429, p<10-6).Receiver-operating characteristics (ROC) curve analysis was performed for sputum eosinophilia (Supplementary Table S2 ). The area under the ROC curve (AUC) for serum indicated that there was no good predictor although blood eosinophilia was the best (0.79) being marginally better than serum PAPPA and exhaled NO (Figure 2A ). In contrast, sputum PAPPA was an excellent predictor of sputum eosinophilia (0.98), better than blood eosinophilia and exhaled nitric oxide levels (Figure 2B ).Therefore, sputum PAPPA is an excellent biomarker for sputum eosinophilia and for T2-high asthma whilst serum PAPPA is as effective as blood eosinophilia in predicting high sputum eosinophil levels and with T2-high asthma.

Background: Immunohistochemical analysis of granule-associated proteases has revealed that human lung mast cells constitute a heterogeneous population of cells, with distinct subpopulations identified. However, a systematic and comprehensive analysis of cell-surface markers to study human lung mast cell heterogeneity has yet to be performed. Methods: Human lung mast cells were obtained from lung lobectomies, and the expression of 332 cell-surface markers was analyzed using flow cytometry and the LEGENDScreenTM kit. Markers that exhibited high variance were selected for additional analyses to reveal whether they were correlated and whether discrete mast cell subpopulations were discernable. Results: We identified the expression of 102 surface markers on human lung mast cells. Several markers showed high continuous variation in expression within the mast cell population. Six of these markers were correlated: SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR. The expression of these markers was also correlated with the size and granularity of mast cells. However, no marker produced an expression profile consistent with a bi- or multimodal distribution. Conclusions: LEGENDScreen analysis identified more than 100 cell-surface markers on mast cells, including 23 that, to the best of our knowledge, have not been previously described on human mast cells. Several of the newly described markers are known to be involved in sensing the microenvironment, and their identification can shed new light on mast cell functions. The exhaustive expression profiling of the 332 surface markers failed to detect distinct mast cell subpopulations. Instead, we demonstrate the continuous nature of human lung mast cell heterogeneity.

Background. Patients with severe asthma may have a greater risk of dying from COVID-19 disease. Angiotensin converting enzyme-2 (ACE2) and the enzyme proteases, transmembrane protease serine 2 (TMPRSS2) and FURIN, are needed for viral attachment and invasion into host cells. Methods. We examined microarray mRNA expression of ACE2, TMPRSS2 and FURIN in sputum, bronchial brushing and bronchial biopsies of the European U-BIOPRED cohort. Clinical parameters and molecular phenotypes, including asthma severity, sputum inflammatory cells, lung functions, oral corticosteroid (OCS) use, and transcriptomic-associated clusters, were examined in relation to gene expression levels. Results. ACE2 levels were significantly increased in sputum of severe asthma compared to mild-moderate asthma. In multivariate analyses, sputum ACE2 levels were positively associated with OCS use and male gender. Sputum FURIN levels were significantly related to neutrophils (%) and the presence of severe asthma. In bronchial brushing samples, TMPRSS2 levels were positively associated with male gender and body mass index, whereas FURIN levels with male gender and blood neutrophils. In bronchial biopsies, TMPRSS2 levels were positively related to blood neutrophils. The neutrophilic molecular phenotype characterised by high inflammasome activation expressed significantly higher FURIN levels in sputum than the eosinophilic Type 2-high or the pauci-granulocytic oxidative phosphorylation phenotypes. Conclusion. Levels of ACE2 and FURIN may differ by clinical or molecular phenotypes of asthma. Sputum FURIN expression levels were strongly associated with neutrophilic inflammation and with inflammasome activation. This might indicate the potential for a greater morbidity and mortality outcome from SARS-CoV-2 infection in neutrophilic severe asthma.