Faecal microbial transfer and complex carbohydrates mediate protection against COPD.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DESIGN: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation. RESULTS: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. CONCLUSION: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically.

Adverse roles of mast cell chymase-1 in COPD.

BACKGROUND: COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase (hCMA)1 and its orthologue mCMA1/mouse mast cell protease (mMCP)5 are exocytosed from activated mast cells and have adverse roles in numerous disorders, but their role in COPD is unknown. METHODS: We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5-deficient (-/-) mice to evaluate this protease's role and potential for therapeutic targeting in CS-induced experimental COPD. In addition, we used ex vivo/in vitro studies to define mechanisms. RESULTS: The levels of hCMA1 mRNA and CMA1+ mast cells were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated mast cell numbers and mMCP5 protein were increased in lung tissues of wild-type mice with experimental COPD. mmcp5 -/- mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of mast cells from wild-type, but not mmcp5 -/- mice with wild-type lung macrophages increased in tumour necrosis factor (TNF)-α release. It also caused the release of CMA1 from human mast cells, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD. CONCLUSION: CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.

T2-low: what do we know?: Past, present, and future of biologic therapies in noneosinophilic asthma.

T2-low asthma is an often severe asthma subtype with limited treatment options and biologic therapeutics are lacking. Several monoclonal antibodies (mAbs) targeting non-T2 cytokines were previously reported to be ineffective in asthma. These trials often investigated heterogeneous asthma populations and negative outcomes could be related to unsuitable study cohorts. More tailored approaches in selecting participants based on specific biomarkers have been beneficial in treating severe T2-high asthma. Similarly, mAbs previously deemed ineffective bear the potential to be useful when administered to the correct target population. Here, we review individual clinical trials conducted between 2005 and 2021 and assess the suitability of the selected cohorts, whether study end points were met, and whether outcome measures were appropriate to investigate the effectiveness of the respective drug. We discuss potential target groups within the T2-low asthma population and suggest biomarkers that may predict a treatment response. Furthermore, we assess whether biomarker-guided approaches or subgroup analyses were associated with more positive study outcomes. The mAbs directed against alarmins intervene early in the inflammatory cascade and are the first mAbs found to have efficacy in T2-low asthma. Several randomized controlled trials performed predefined subgroup analyses that included T2-low asthma. Subgroup analyses were associated with positive outcomes and were able to reveal a stronger response in at least 1 subgroup. A better understanding of T2-low subgroups and specific biomarkers is necessary to identify the most responsive target population for a given mAb.

An altered sputum macrophage transcriptome contributes to the neutrophilic asthma endotype.

BACKGROUND: Neutrophilic asthma (NA) is a clinically important asthma phenotype, the cellular and molecular basis of which is not completely understood. Airway macrophages are long-lived immune cells that exert important homeostatic and inflammatory functions which are dysregulated in asthma. Unique transcriptomic programmes reflect varied macrophage phenotypes in vitro. We aimed to determine whether airway macrophages are transcriptomically altered in NA. METHODS: We performed RNASeq analysis on flow cytometry-isolated sputum macrophages comparing NA (n = 7) and non-neutrophilic asthma (NNA, n = 13). qPCR validation of RNASeq results was performed (NA n = 13, NNA n = 23). Pathway analysis (PANTHER, STRING) of differentially expressed genes (DEGs) was performed. Gene set variation analysis (GSVA) was used to test for enrichment of NA macrophage transcriptomic signatures in whole sputum microarray (cohort 1 - controls n = 16, NA n = 29, NNA n = 37; cohort 2 U-BIOPRED - controls n = 16, NA n = 47, NNA n = 57). RESULTS: Flow cytometry-sorting significantly enriched sputum macrophages (99.4% post-sort, 44.9% pre-sort, p < .05). RNASeq analysis confirmed macrophage purity and identified DEGs in NA macrophages. Selected DEGs (SLAMF7, DYSF, GPR183, CSF3, PI3, CCR7, all p < .05 NA vs. NNA) were confirmed by qPCR. Pathway analysis of NA macrophage DEGs was consistent with responses to bacteria, contribution to neutrophil recruitment and increased expression of phagocytosis and efferocytosis factors. GSVA demonstrated neutrophilic macrophage gene signatures were significantly enriched in whole sputum microarray in NA vs. NNA and controls in both cohorts. CONCLUSIONS: We demonstrate a pathophysiologically relevant sputum macrophage transcriptomic programme in NA. The finding that there is transcriptional activation of inflammatory programmes in cell types other than neutrophils supports the concept of NA as a specific endotype.

Sputum Gene Expression Reveals Dysregulation of Mast Cells and Basophils in Eosinophilic COPD.

Purpose: The clinical and inflammatory associations of mast cells (MCs) and basophils in chronic obstructive pulmonary disease (COPD) are poorly understood. We previously developed and validated a qPCR-based MC/basophil gene signature in asthma to measure these cells in sputum samples. Here, we measured this gene signature in a COPD and control population to explore the relationship of sputum MCs/basophils to inflammatory and COPD clinical characteristics. Patients and Methods: MC/basophil signature genes (TPSAB1/TPSB2, CPA3, ENO2, GATA2, KIT, GPR56, HDC, SOCS2) were measured by qPCR in sputum from a COPD (n=96) and a non-respiratory control (n=17) population. Comparative analyses of gene expression between the COPD and the control population, and between eosinophilic COPD and non-eosinophilic COPD were tested. Logistic regression analysis and Spearman correlation were used to determine relationships of sputum MC/basophil genes to inflammatory (sputum eosinophil proportions, blood eosinophils) and clinical (age, body mass index, quality of life, lung function, past year exacerbations) characteristics of COPD. Results: MC/basophil genes were increased in COPD versus control participants (CPA3, KIT, GATA2, HDC) and between eosinophilic-COPD and non-eosinophilic COPD (TPSB2, CPA3, HDC, SOCS2). We found all MC/basophil genes were positively intercorrelated. In COPD, MC/basophil genes were associated with eosinophilic airway inflammation (GATA2, TPSB2, CPA3, GPR56, HDC, SOCS2), blood eosinophilia (all genes) and decreased lung function (KIT, GATA2, GPR56, HDC). Conclusion: We demonstrate associations of MCs and basophils with eosinophilic inflammation and lower lung function in COPD. These findings are consistent with prior results in asthma and may represent a new tool for endotyping eosinophilic-COPD.

Time-resolved proteomic profiling of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. METHODS: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. RESULTS: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. CONCLUSION: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies.

Airway monocyte modulation relates to tumour necrosis factor dysregulation in neutrophilic asthma.

BACKGROUND: Dysregulation of tumour necrosis factor-α (TNF-α) signalling is implicated in neutrophilic asthma. TNF-α signalling involves membrane-bound and soluble ligand (TNF-α) and receptors (TNFRs); however, little is known about how these proteins are altered in asthma. We hypothesised that intercompartment-, immune cell- and/or asthma inflammatory phenotype-dependent regulation could relate to TNF dysregulation in neutrophilic asthma. METHODS: Measurements were made in 45 adults with asthma (36 non-neutrophilic, 9 neutrophilic) and 8 non-asthma controls. Soluble TNF-α, TNF receptor 1 (TNFR1) and TNFR2 were quantified in plasma and sputum supernatant by ELISA, and membrane-bound TNF-α/TNFR1/TNFR2 measured on eosinophils, neutrophils, monocytes, and macrophages in blood and sputum by flow cytometry. Marker expression was compared between inflammatory phenotypes and compartments, and relationship of membrane-bound and soluble TNF markers and immune cell numbers tested by correlation. RESULTS: Soluble sputum TNFR1 and TNFR2 were increased in neutrophilic versus non-neutrophilic asthma (p=0.010 and p=0.029). Membrane-bound TNF-α expression was upregulated on sputum versus blood monocytes, while TNFR1 and TNFR2 levels were reduced on airway versus blood monocytes and neutrophils. Soluble TNFR1 and TNFR2 in sputum significantly correlated with the number of airway monocytes (p=0.016, r=0.358 and p=0.029, r=0.327). CONCLUSION: Our results imply that increased sputum soluble TNF receptor levels observed in neutrophilic asthma relate to the increased recruitment of monocytes and neutrophils into the airways and their subsequent receptor shedding. Monocytes also increase TNF-α ligand expression in the airways. These results suggest an important contribution of airway monocytes to the altered inflammatory milieu in neutrophilic asthma.

Necroptosis Signaling Promotes Inflammation, Airway Remodeling, and Emphysema in Chronic Obstructive Pulmonary Disease.

Rationale: Necroptosis, mediated by RIPK3 (receptor-interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like), is a form of regulated necrosis that can drive tissue inflammation and destruction; however, its contribution to chronic obstructive pulmonary disease (COPD) pathogenesis is poorly understood. Objectives: To determine the role of necroptosis in COPD. Methods: Total and active (phosphorylated) RIPK3 and MLKL were measured in the lung tissue of patients with COPD and control subjects without COPD. Necroptosis-related mRNA and proteins as well as cell death were examined in lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3-/- and Mlkl-/- mice to acute and chronic CS exposure were compared with those of wild-type mice. The combined inhibition of apoptosis (with the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone [qVD-OPh]) and necroptosis (with deletion of Mlkl in mice) was assessed. Measurements and Main Results: The total MLKL protein in the epithelium and macrophages and the pRIPK3 and pMLKL in lung tissue were increased in patients with severe COPD compared with never-smokers or smoker control subjects without COPD. Necroptosis-related mRNA and protein levels were increased in the lungs and macrophages in CS-exposed mice and experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation upon acute CS exposure. Ripk3 deficiency reduced airway inflammation and remodeling as well as the development of emphysematous pathology after chronic CS exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodeling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung-cell death. Conclusions: Necroptosis is induced by CS exposure and is increased in the lungs of patients with COPD and in experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodeling, and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD.

Hemopexin: A Novel Anti-inflammatory Marker for Distinguishing COPD From Asthma.

PURPOSE: Systemic inflammatory biomarkers can improve diagnosis and assessment of chronic obstructive pulmonary disease (COPD) and asthma. We aimed to validate an airway disease biomarker panel of 4 systemic inflammatory biomarkers, α2-macroglobulin, ceruloplasmin, haptoglobin and hemopexin, to establish their relationship to airway disease diagnosis and inflammatory phenotypes and to identify an optimized biomarker panel for disease differentiation. METHODS: Participants with COPD or asthma were classified by inflammatory phenotypes. Immunoassay methods were used to measure levels of validation biomarkers in the sera of participants with disease and non-respiratory disease controls. Markers were analyzed individually and in combination for disease differentiation and compared to established biomarkers (C-reactive protein, interleukin-6, and white blood cell/blood eosinophil count). RESULTS: The study population comprised of 141 COPD, 127 severe asthma, 54 mild-moderate asthma and 71 control participants. Significant differences in ceruloplasmin, haptoglobin and hemopexin levels between disease groups and between systemic inflammatory phenotypes were observed. However, no differences were found between airway inflammatory phenotypes. Hemopexin was the best performing individual biomarker and could diagnose COPD versus control participants (area under the curve [AUC], 98.3%; 95% confidence interval [CI], 96.7%-99.9%) and differentiate COPD from asthmatic participants (AUC, 97.0%; 95% CI, 95.4%-98.6%), outperforming established biomarkers. A biomarker panel, including hemopexin, haptoglobin and other established biomarkers, could diagnose asthma versus control participants (AUC, 87.5%; 95% CI, 82.8%-92.2%). CONCLUSIONS: Hemopexin can be a novel biomarker with superior diagnostic ability in differentiating COPD and asthma. We propose an anti-inflammatory axis between the airways and systemic circulation, in which hemopexin is a protective component in airway disease.

Sputum TNF markers are increased in neutrophilic and severe asthma and are reduced by azithromycin treatment.

BACKGROUND: The AMAZES randomized controlled trial demonstrated that long-term low-dose azithromycin treatment reduces exacerbations of poorly controlled asthma, but the therapeutic mechanisms remain unclear. Dysregulation of the inflammatory tumour necrosis factor (TNF) pathway is implicated in asthma and could be suppressed by azithromycin. We aimed to determine the inflammatory and clinical associations of soluble TNF signalling proteins (TNF receptors [TNFR] 1 and 2, TNF) in sputum and serum, and to test the effect of 48 weeks of azithromycin vs placebo on TNF markers. METHODS: Sputum supernatant and serum TNFR1, TNFR2 (n = 142; 75 azithromycin-treated, 67 placebo-treated) and TNF (n = 48; 22 azithromycin-treated, 26 placebo-treated) were measured by ELISA in an AMAZES trial sub-population at baseline and end of treatment. Baseline levels were compared between sputum inflammatory phenotypes, severe/non-severe asthma and frequent/non-frequent exacerbators. Effect of azithromycin on markers was tested using linear mixed models. RESULTS: Baseline sputum TNFR1 and TNFR2 were significantly increased in neutrophilic vs non-neutrophilic asthma phenotypes, while serum markers did not differ. Sputum TNFR1 and TNFR2 were increased in severe asthma and correlated with poorer lung function, worse asthma control and increasing age. Serum TNFR1 was also increased in severe asthma. Sputum and serum TNFR2 were increased in frequent exacerbators. Azithromycin treatment significantly reduced sputum TNFR2 and TNF relative to placebo, specifically in non-eosinophilic participants. CONCLUSIONS: We demonstrate dysregulation of TNF markers, particularly in the airways, that relates to clinically important phenotypes of asthma including neutrophilic and severe asthma. Suppression of dysregulated TNF signalling by azithromycin could contribute to its therapeutic mechanism.

Sputum mast cell/basophil gene expression relates to inflammatory and clinical features of severe asthma.

BACKGROUND: Mast cells (MCs) and basophils are important in asthma pathophysiology, however direct measurement is difficult, and clinical and inflammatory associations in severe asthma are poorly understood. Transcriptomic hallmarks of MCs/basophils may allow their measurement in sputum using gene expression. OBJECTIVES: This study sought to develop and validate a sputum MC/basophil gene signature and investigate its relationship to inflammatory and clinical characteristics of severe asthma. METHODS: A total of 134 candidate MC/basophil genes (identified by the Immunological Genome Project Consortium) were screened in sputum microarray for differential expression among control subjects (n = 18), patients with eosinophilic (n = 29), and patients with noneosinophilic asthma (n = 30). Candidate genes were validated by confirming correlation of gene expression with flow cytometry-quantified sputum MCs and basophils in a separate asthma cohort (n = 20). The validated gene signature was measured in a severe asthma cohort (n = 81), and inflammatory and clinical associations were tested. RESULTS: Through microarray screening and subsequent validation, we found quantitative PCR gene expression of 8 targets correlated with sputum MCs/basophils: TPSAB1/TPSB2, CPA3, ENO2, GATA2, KIT, GPR56, HDC, SOCS2. In severe asthma, MC/basophil genes were associated with eosinophilic airway inflammation (GATA2, TPSB2, CPA3, GPR56, HDC, SOCS2), blood eosinophils (TPSB2, CPA3, GATA2, SOCS2, FCER1A, HDC), fractional exhaled NO (GATA2, SOCS2), decreased lung function (KIT, ENO2), and moderate exacerbation history (GATA2, SOCS2). CONCLUSIONS: Quantitative PCR-based measures reflect varying sputum MC/basophil abundance, demonstrating associations of MCs/basophils with eosinophilic inflammation, spirometry and exacerbation history in severe asthma.

Molecular markers of type 2 airway inflammation are similar between eosinophilic severe asthma and eosinophilic chronic obstructive pulmonary disease.

BACKGROUND: Airway and systemic eosinophilia are important treatable traits in both severe asthma and COPD. The molecular basis of eosinophilia in COPD is poorly understood but could involve type 2 cytokines (IL5, IL13) and prostaglandin D2 (PGD2 ). METHODS: This study included non-obstructive airways disease (OAD) controls (n = 19), a COPD cohort (n = 96) and a severe asthma cohort (n = 84). Demographics, exacerbation history, disease impact (SGRQ) and spirometry were assessed. Participants were categorized as eosinophilic using either sputum eosinophil proportion (≥3%) or blood eosinophil count (≥300/µL). Sputum type 2 inflammatory measures included PGD2 by ELISA and gene expression (qPCR) of IL5, IL13 and the haematopoietic PGD2 synthase (HPGDS). RESULTS: Type 2 markers did not differ across groups except HPGDS mRNA which was highest in non-OAD controls and lowest in COPD. IL5 and IL13 mRNA and PGD2 levels were significantly increased in eosinophilic vs non-eosinophilic severe asthma but did not differ between eosinophilic COPD and eosinophilic severe asthma or non-eosinophilic COPD. HPGDS expression was higher in eosinophilic severe asthma compared with eosinophilic COPD. Results were similar using sputum or blood eosinophil cut-offs. Sputum IL5 and IL13 were highly intercorrelated in severe asthma (r = 0.907, p < 0.001) and COPD (r = 0.824, p < 0.001), were moderately correlated with sputum eosinophils in severe asthma (IL5 r = 0.440, p < 0.001; IL13 r = 0.428, p < 0.001) and were weakly correlated in COPD (IL5 r = 0.245, p < 0.05; IL13 r = 0.317, p < 0.05). CONCLUSIONS: Molecular markers of type 2 airway inflammation do not differ between eosinophilic asthma and eosinophilic COPD; however, the relationship between eosinophilia and type 2 airway markers appears weaker in COPD than in severe asthma.

Neutrophilic asthma features increased airway classical monocytes.

BACKGROUND: Monocytes and macrophages are critical innate immune cells of the airways. Despite their differing functions, few clinical studies discriminate between them and little is known about their regulation in asthma. OBJECTIVE: We aimed to distinguish and quantify macrophages, monocytes and monocyte subsets in induced sputum and blood and examine their relationship with inflammatory and clinical features of asthma. METHODS: We applied flow cytometry to distinguish macrophages, monocytes and subsets in sputum and blood (n = 53; 45 asthma, 8 non-asthma) and a second asthma sputum cohort (n = 26). Monocyte subsets were identified by surface CD14/CD16 (CD14++ CD16- classical, CD14+ CD16+ intermediate and CD14+ CD16++ non-classical monocytes). Surface CD206, a marker of monocyte tissue differentiation, was measured in sputum. Relationship to airway inflammatory phenotype (neutrophilic n = 9, eosinophilic n = 14, paucigranulocytic n = 22) and asthma severity (severe n = 12, non-severe n = 33) was assessed. RESULTS: Flow cytometry- and microscope-quantified sputum differential cell proportions were significantly correlated. Sputum macrophage number was reduced (p = .036), while classical monocyte proportion was increased in asthma vs non-asthma (p = .032). Sputum classical monocyte number was significantly higher in neutrophilic vs paucigranulocytic asthma (p = .013). CD206- monocyte proportion and number were increased in neutrophilic vs eosinophilic asthma (p < .001, p = .013). Increased sputum classical and CD206- monocyte numbers in neutrophilic asthma were confirmed in the second cohort. Blood monocytes did not vary with airway inflammatory phenotype, but blood classical monocyte proportion and number were increased in severe vs non-severe asthma (p = .022, p = .011). CONCLUSION AND CLINICAL RELEVANCE: Flow cytometry allowed distinction of sputum macrophages, monocytes and subsets, revealing compartment-specific dysregulation of monocytes in asthma. We observed an increase in classical and CD206- monocytes in sputum in neutrophilic asthma, suggesting co-recruitment of monocytes and neutrophils to the airways in asthma. Our data suggest further investigation of how airway monocyte dysregulation impacts on asthma-related disease activity is merited.

Sensitization to Hymenoptera venom in pollen allergic patients: Frequency and involvement of cross-reacting carbohydrate determinants (CCD).

Sensitization to Hymenoptera venom in patients without a history of systemic allergic reactions to Hymenoptera stings is frequently found and can be due to the presence of specific IgE to cross-reactive carbohydrate determinants (CCD). This study investigates 105 pollen allergic subjects for the presence of specific IgE to honeybee or wasp venom, pollen, the MUXF3 carbohydrate epitope from bromelain and recombinant Hymenoptera venom components. In addition, in a subgroup of patients (n = 10) a basophil activation test (BAT) using bee and wasp venom was performed. Specific IgE to Hymenoptera venom was detected in 45.7% of the pollen allergic subjects and in 26.7% of the non-atopic controls, both without a history of systemic allergic reactions to Hymenoptera stings. The high sensitization rate in atopic patients could partially be explained by cross-sensitization between pollen and Hymenoptera venom due to specific IgE to CCDs. In our study population, only 20% showed a sensitization to CCDs. Primary sensitization due to sting exposure, high total IgE values or unspecific binding and detection of low affinity antibodies in the test procedure could be reasons. Thus, determination of specific IgE to Hymenoptera venom in patients without a history of systemic allergic reactions as screening test is not recommended.

A Sputum 6 Gene Expression Signature Predicts Inflammatory Phenotypes and Future Exacerbations of COPD.

Background: The 6 gene expression signature (6GS) predicts inflammatory phenotype, exacerbation risk, and corticosteroid responsiveness in asthma. In COPD, patterns of airway inflammation are similar, suggesting the 6GS may be useful. This study determines the diagnostic and prognostic ability of 6GS in predicting inflammatory phenotypes and exacerbation risk in COPD. Methods: We performed 2 studies: a cross-sectional phenotype prediction study in stable COPD (total N=132; n=34 eosinophilic (E)-COPD, n=42 neutrophilic (N)-COPD, n=39 paucigranulocytic (PG)-COPD, n=17 mixed-granulocytic (MG)-COPD) that assessed 6GS ability to discriminate phenotypes (eosinophilia≥3%; neutrophilia≥61%); and a prospective cohort study (total n=54, n=8 E-COPD; n=18 N-COPD; n=20 PG-COPD; n=8 MG-COPD, n=21 exacerbation prone (≥2/year)) that investigated phenotype and exacerbation prediction utility. 6GS was measured by qPCR and evaluated using multiple logistic regression and area under the curve (AUC). Short-term reproducibility (intra-class correlation) and phenotyping method agreement (κ statistic) were assessed. Results: In the phenotype prediction study, 6GS could accurately identify and discriminate patients with E-COPD from N-COPD (AUC=96.4%; p<0.0001), PG-COPD (AUC=88.2%; p<0.0001) or MG-COPD (AUC=86.2%; p=0.0001), as well as N-COPD from PG-COPD (AUC=83.6%; p<0.0001) or MG-COPD (AUC=87.4%; p<0.0001) and was reproducible. In the prospective cohort study, 6GS had substantial agreement for neutrophilic inflammation (82%, κ=0.63, p<0.001) and moderate agreement for eosinophilic inflammation (78%, κ=0.42, p<0.001). 6GS could significantly discriminate exacerbation prone patients (AUC=77.2%; p=0.034). Higher IL1B levels were associated with poorer lung function and increased COPD severity. Conclusion: 6GS can significantly and reproducibly discriminate COPD inflammatory phenotypes and predict exacerbation prone patients and may become a useful molecular diagnostic tool assisting COPD management.

Relationship of sputum mast cells with clinical and inflammatory characteristics of asthma.

BACKGROUND: Mast cells (MCs) are innate immune cells that regulate atopic and non-atopic inflammation in the airways. MCs play a critical role in the pathogenesis of asthma, yet their relationship to airway and systemic inflammation and clinical characteristics of asthma is poorly understood. OBJECTIVE: To quantify MCs in induced sputum samples and understand their relationship to airway and circulatory immune cells, and clinical variables in asthma. METHODS: We employed flow cytometry of sputum samples to quantify MCs, basophils and other immune cells in 51 participants (45 asthma and 6 non-asthma controls). Relationship of MCs to airway (n = 45) and blood (n = 19) immune cells, participant demographics, asthma history, spirometry and airways hyperresponsiveness (AHR) to hypertonic saline was determined by correlation and comparison of cut-off-based sputum MC high vs low participants. RESULTS: Mast cells, basophils and eosinophils were increased in asthma vs non-asthma control sputum. In asthma sputum, MCs, basophils and eosinophils were significantly intercorrelated, and MCs and basophils were elevated in participants with eosinophilic asthma. MCs and basophils, but not eosinophils, correlated with AHR. Sputum MC high asthma was characterized by an increased proportion of participants with uncontrolled asthma and reduced FEV1 and FVC. Trends towards similar clinical associations with elevated MCs were observed in a paucigranulocytic subpopulation (n = 15) lacking airway eosinophilia or neutrophilia. Receiver operator characteristic (ROC) analysis showed peripheral blood eosinophil (PBE) count predicted elevated sputum eosinophils and basophils, but not MCs. CONCLUSIONS AND CLINICAL RELEVANCE: Sputum MCs are elevated in asthma, and their measurement may be useful as they relate to key clinical features of asthma (spirometry, asthma control, AHR). PBE count did not predict airway MC status, suggesting direct measurement of airway MCs by sensitive methods such as flow cytometry should be further developed.

Management of allergy transfer upon solid organ transplantation.

Allergy transfer upon solid organ transplantation has been reported in the literature, although only few data are available as to the frequency, significance, and management of these cases. Based on a review of 577 consecutive deceased donors from the Swisstransplant Donor-Registry, 3 cases (0.5%) of fatal anaphylaxis were identified, 2 because of peanut and 1 of wasp allergy. The sera of all 3 donors and their 10 paired recipients, prospectively collected before and after transplantation for the Swiss Transplant Cohort Study, were retrospectively processed using a commercial protein microarray fluorescent test. As early as 5 days posttransplantation, newly acquired peanut-specific IgE were transiently detected from 1 donor to 3 recipients, of whom 1 liver and lung recipients developed grade III anaphylaxis. Yet, to define how allergy testing should be performed in transplant recipients and to better understand the impact of immunosuppressive therapy on IgE sensitization, we prospectively studied 5 atopic living-donor kidney recipients. All pollen-specific IgE and >90% of skin prick tests remained positive 7 days and 3 months after transplantation, indicating that early diagnosis of donor-derived IgE sensitization is possible. Importantly, we propose recommendations with respect to safety for recipients undergoing solid-organ transplantation from donors with a history of fatal anaphylaxis.

Sputum transcriptomics implicates increased p38 signalling activity in severe asthma.

BACKGROUND AND OBJECTIVE: Severe asthma is responsible for a disproportionate burden of illness and healthcare costs spent on asthma. This study analyses sputum transcriptomics to investigate the mechanisms and novel treatment targets of severe asthma. METHODS: Induced sputum samples were collected in a cross-sectional study from participants with severe asthma (n = 12, defined as per GINA criteria), non-severe uncontrolled (n = 21) and controlled asthma (n = 21) and healthy controls (n = 15). Sputum RNA was extracted and transcriptomic profiles were generated (Illumina HumanRef-8 V2) and analysed (GeneSpring). Sputum protein lysates were analysed for p38 activation in a validation study (n = 24 asthma, n = 8 healthy) by western blotting. RESULTS: There were 2166 genes differentially expressed between the four groups. In severe asthma, the expression of 1875, 1308 and 563 genes was altered compared to healthy controls, controlled and uncontrolled asthma, respectively. Of the 1875 genes significantly different to healthy controls, 123 were >2-fold change from which four networks were identified. Thirty genes (>2-fold change) were significantly different in severe asthma compared to both controlled asthma and healthy controls. There was enrichment of genes in the p38 signalling pathway that were associated with severe asthma. Phosphorylation of p38 was increased in a subset of severe asthma samples, correlating with neutrophilic airway inflammation. CONCLUSION: Severe asthma is associated with substantial differences in sputum gene expression that underlie unique cellular mechanisms. The p38 signalling pathway may be important in the pathogenesis of severe asthma, and future investigations into p38 inhibition are warranted as a 'non-Th2' therapeutic option.

Blood Neutrophils In COPD But Not Asthma Exhibit A Primed Phenotype With Downregulated CD62L Expression.

Purpose: To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression. Patients and methods: Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI). Results: Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p<0.017. Conclusion: Reduced CD62L expression suggests blood neutrophils have undergone priming in COPD but not in asthma, which may be the result of systemic inflammation. The increased shedding of CD62L receptor by COPD blood neutrophils suggests a high sensitivity for activation.