Association between the Reduced Expression of RECK and Neutrophilic Inflammation in Chronic Obstructive Pulmonary Disease.

INTRODUCTION: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a recently discovered inhibitor of matrix metalloproteinase (MMP). There is a large number of chronic obstructive pulmonary disease (COPD) patients worldwide; however, the role of RECK on COPD has not been studied. This study explored the expression of RECK in COPD patients and its effect on neutrophil function to provide a new scientific basis for the prevention and treatment of COPD. METHOD: Fifty patients with acute exacerbation of COPD and fifty healthy controls were enrolled in the study. RECK was detected in lung tissue, sputum, and plasma of subjects as well as in BEAS-2B cells stimulated with cigarette smoke extract (CSE) by immunohistochemistry, ELISA, and qRT-PCR. Meanwhile, lung function (FEV1%pred) and inflammatory cytokines (IL-6 and IL-8) were examined, and correlation analysis was performed with RECK expression. The effect of RECK on proliferation, apoptosis, migration, and inflammatory cytokines and its potential mechanism was further quantified by neutrophil stimulated with recombinant human RECK protein (rhRECK) combined with CSE using CCK8, flow cytometry, Transwell assay, qRT-PCR, ELISA, and Western analysis. RESULTS: RECK was mainly expressed on airway epithelial cells in normal lung tissue and was significantly diminished in COPD patients. The levels of RECK in sputum and plasma were also significantly decreased in COPD patients. Pearson correlation analysis showed that RECK level in plasma was positively correlated with FEV1%pred (r = 0.458, p < 0.001) and negatively correlated with IL-6 and IL-8 (r = -0.386, -0.437; p = 0.006, 0.002) in COPD patients. The expression of RECK was decreased in BEAS-2B stimulated with CSE. The migration, inflammation, and MMP-9 expression of neutrophils were promoted by CSE, while inhibited by rhRECK. CONCLUSION: RECK is low expressed in COPD patients and negatively correlated with inflammation. It may inhibit the inflammation and migration of neutrophils by downregulating MMP-9.

Mapping atopic dermatitis and anti-IL-22 response signatures to type 2-low severe neutrophilic asthma.

BACKGROUND: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. OBJECTIVE: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti-IL-22 (fezakinumab [FZ]) is enriched in severe asthma. METHODS: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. RESULTS: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, TH2, and TH17/TH22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P < .05). The FZ-response signature (296 downregulated genes) was enriched in asthmatic blood (P < .05) and particularly in neutrophilic and mixed granulocytic sputum (P < .05). These data were confirmed in sputum of the Airway Disease Endotyping for Personalized Therapeutics cohort. IL-22 mRNA across tissues did not correlate with FZ-response enrichment scores, but this response signature correlated with TH22/IL-22 pathways. CONCLUSIONS: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.

Mometasone or Tiotropium in Mild Asthma with a Low Sputum Eosinophil Level.

BACKGROUND: In many patients with mild, persistent asthma, the percentage of eosinophils in sputum is less than 2% (low eosinophil level). The appropriate treatment for these patients is unknown. METHODS: In this 42-week, double-blind, crossover trial, we assigned 295 patients who were at least 12 years of age and who had mild, persistent asthma to receive mometasone (an inhaled glucocorticoid), tiotropium (a long-acting muscarinic antagonist), or placebo. The patients were categorized according to the sputum eosinophil level (<2% or ≥2%). The primary outcome was the response to mometasone as compared with placebo and to tiotropium as compared with placebo among patients with a low sputum eosinophil level who had a prespecified differential response to one of the trial agents. The response was determined according to a hierarchical composite outcome that incorporated treatment failure, asthma control days, and the forced expiratory volume in 1 second; a two-sided P value of less than 0.025 denoted statistical significance. A secondary outcome was a comparison of results in patients with a high sputum eosinophil level and those with a low level. RESULTS: A total of 73% of the patients had a low eosinophil level; of these patients, 59% had a differential response to a trial agent. However, there was no significant difference in the response to mometasone or tiotropium, as compared with placebo. Among the patients with a low eosinophil level who had a differential treatment response, 57% (95% confidence interval [CI], 48 to 66) had a better response to mometasone, and 43% (95% CI, 34 to 52) had a better response to placebo (P = 0.14). In contrast 60% (95% CI, 51 to 68) had a better response to tiotropium, whereas 40% (95% CI, 32 to 49) had a better response to placebo (P = 0.029). Among patients with a high eosinophil level, the response to mometasone was significantly better than the response to placebo (74% vs. 26%) but the response to tiotropium was not (57% vs. 43%). CONCLUSIONS: The majority of patients with mild, persistent asthma had a low sputum eosinophil level and had no significant difference in their response to either mometasone or tiotropium as compared with placebo. These data provide equipoise for a clinically directive trial to compare an inhaled glucocorticoid with other treatments in patients with a low eosinophil level. (Funded by the National Heart, Lung, and Blood Institute; SIENA ClinicalTrials.gov number, NCT02066298.).

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.

Assessment of bacterial exposure on phagocytic capability and surface marker expression of sputum macrophages and neutrophils in COPD patients.

Defective phagocytosis has been shown in chronic obstructive pulmonary disease (COPD) bronchoalveolar lavage and blood monocyte-derived macrophages. Phagocytic capabilities of sputum macrophages and neutrophils in COPD are unknown. We investigated phagocytosis in these cells from COPD patients and controls. Phagocytosis of Streptococcus pneumoniae or fluorescently labelled non-typeable Haemophilus influenzae (NTHi) by sputum macrophages and neutrophils was determined by gentamycin protection assay (COPD; n = 5) or flow cytometry in 14 COPD patients, 8 healthy smokers (HS) and 9 healthy never-smokers (HNS). Sputum macrophages and neutrophils were differentiated by adherence for the gentamycin protection assay or receptor expression (CD206 and CD66b, respectively), by flow cytometry. The effects of NTHi on macrophage expression of CD206 and CD14 and neutrophil expression of CD16 were determined by flow cytometry. There was greater uptake of S. pneumoniae [~10-fold more colony-forming units (CFU)/ml] by sputum neutrophils compared to macrophages in COPD patients. Flow cytometry showed greater NTHi uptake by neutrophils compared to macrophages in COPD (67 versus 38%, respectively) and HS (61 versus 31%, respectively). NTHi uptake by macrophages was lower in HS (31%, p = 0.019) and COPD patients (38%, p = 0.069) compared to HNS (57%). NTHi uptake by neutrophils was similar between groups. NTHi exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils. Sputum neutrophils showed more phagocytic activity than macrophages. There was some evidence that bacterial phagocytosis was impaired in HS sputum macrophages, but no impairment of neutrophils was observed in HS or COPD patients. These results highlight the relative contributions of neutrophils and macrophages to bacterial clearance in COPD.

Chronic bacterial pulmonary infections in advanced cystic fibrosis differently affect the level of sputum neutrophil elastase, IL-8 and IL-6.

Advanced cystic fibrosis (CF) lung disease is commonly characterized by a chronic Pseudomonas aeruginosa infection and destructive inflammation caused by neutrophils. However, the lack of convincing evidence from most informative biomarkers of severe lung dysfunction (SLD-CF) has hampered the formulation of a conclusive, targeted diagnosis of CF. The aim of this study was to determine whether SLD-CF is related to the high concentration of sputum inflammatory mediators and the presence of biofilm-forming bacterial strains. Forty-one patients with advanced CF lung disease were studied. The severity of pulmonary dysfunction was defined by forced expiratory volume in 1 second (FEV1) < 40%. C-reactive protein (CRP) and NLR (neutrophil-lymphocyte ratio) were examined as representative blood-based markers of inflammation. Expectorated sputum was collected and analysed for cytokines and neutrophil-derived defence proteins. Isolated sputum bacteria were identified and their biofilm-forming capacity was determined. There was no association between FEV1% and total number of sputum bacteria. However, in the high biofilm-forming group the median FEV1 was < 40%. Importantly, high density of sputum bacteria was associated with increased concentrations of neutrophil elastase and interleukin (IL)-8 and low concentrations of IL-6 and IL-10. The low concentration of sputum IL-6 is unique for CF and distinct from that observed in other chronic pulmonary inflammatory diseases. These findings strongly suggest that expectorated sputum is an informative source of pulmonary biomarkers representative for advanced CF and may replace more invasive bronchoalveolar lavage analysis to monitor the disease. We recommend to use of the following inflammatory biomarkers: blood CRP, NLR and sputum elastase, IL-6, IL-8 and IL-10.

Asthma in elderly is characterized by increased sputum neutrophils, lower airway caliber variability and air trapping.

BACKGROUND: Elderly asthmatics represent an important group that is often excluded from clinical studies. In this study we wanted to present characteristics of asthmatics older than 70 years old as compared to younger patients. METHODS: We conducted a retrospective analysis on a series of 758 asthmatics subdivided in three groups: lower than 40, between 40 and 70 and older than 70. All the patients who had a successful sputum induction were included in the study. RESULTS: Older patients had a higher Body Mass Index, had less active smokers and were more often treated with Long Acting anti-Muscarinic Agents. We found a significant increase in sputum neutrophil counts with ageing. There was no significant difference in blood inflammatory cell counts whatever the age group. Forced expiratory volume in one second (FEV1) and FEV1/FVC values were significantly lower in elderly who had lower bronchial hyperresponsiveness and signs of air trapping. We found a lower occurrence of the allergic component in advanced ages. Asthmatics older than 70 years old had later onset of the disease and a significant longer disease duration. CONCLUSION: Our study highlights that asthmatics older than 70 years old have higher bronchial neutrophilic inflammation, a poorer lung function, signs of air trapping and lower airway variability. The role of immunosenescence inducing chronic low-grade inflammation in this asthma subtype remains to be elucidated.

Spontaneous sputum discriminates inflammatory phenotypes in patients with asthma.

BACKGROUND: Eosinophils in induced sputum are not only a useful biomarker for diagnosing asthma but are also associated with severe asthma. However, little is known about the association between eosinophils in spontaneous sputum and asthma severity. OBJECTIVE: To investigate whether spontaneous sputum eosinophils are related to severe asthma in adult patients with asthma. METHODS: We conducted a retrospective cross-sectional study on 86 people with asthma whose spontaneous sputa were successfully collected. Patients were classified into 4 phenotypes according to the eosinophil and neutrophil levels in spontaneous sputum. We determined the association between inflammatory phenotypes and severe asthma. Moreover, we also compared asthma severity among the phenotypes classified according to blood eosinophils and spontaneous sputum eosinophils. RESULTS: Asthma phenotypes were as follows: paucigranulocytic, 30.2%; neutrophilic, 18.6%; eosinophilic, 32.6%; and mixed, 18.6%. People with eosinophilic asthma had the highest blood eosinophils, total immunoglobulin E (IgE), and fractional exhaled nitric oxide among the 4 phenotypes. Significant differences were observed in asthma severity between the phenotypes (P = .019). In particular, 57.2% and 56.2% of patients had severe eosinophilic asthma and mixed asthma, respectively. The logistic regression analysis revealed that spontaneous sputum eosinophilia represented the strongest association with severe asthma among the inflammatory variables. Finally, more patients with severe asthma were included in the phenotype with spontaneous sputum eosinophils greater than 3% and blood eosinophils less than or equal to 300/µL and in the phenotype with spontaneous sputum eosinophils greater than 3% and blood eosinophils greater than 300/µL. CONCLUSION: Spontaneous sputum can provide helpful information on airway inflammatory phenotyping in patients with asthma.

Airway hyperresponsiveness to mannitol improves in both type 2 high and type 2 low asthma after specialist management.

OBJECTIVES: Type 2 low (T2-low) asthma is reported to respond less to anti-inflammatory treatment compared with Type 2 high (T2-high) asthma. Airway hyperresponsiveness (AHR) to mannitol, a marker of airway mast cell activation, may be indicative of response to treatment in patients with T2-low disease. We investigated whether AHR to mannitol improves in patients with T2-low asthma after specialist management. METHODS: Patients with asthma or suspected asthma, referred to our specialist outpatient clinic, were enrolled consecutively and assessed with FeNO, asthma control, blood eosinophils, mannitol and methacholine tests and induced sputum. T2-low asthma was defined in patients with FeNO < 25ppb and sputum eosinophils < 3% and blood eosinophils < 300µl-1 at inclusion. Patients with asthma and AHR to mannitol (PD15 ≤ 635 mg) were followed and reassessed after 12 months of specialist management. RESULTS: Thirty-two patients (Females: 56%, age: 22 years (15-59)) were followed. Fourteen (44%) with T2-high and 18 (56%) with T2-low asthma. Baseline AHR to mannitol was comparable: Gmean PD15: 150 mg (95% CI 61-368) and 214 mg (95% CI 106-432) for T2-high and T2-low asthma respectively (P = 0.51). Both groups improved equally: Gmean PD15: 488 mg (95% CI 311-767) and 507 mg (95% CI 345-746); corresponding to a doubling-dose of: 3.00 (95% CI 1.58-5.74, P = 0.003) and 2.28 (95% CI 1.47-3.53, P = 0.001) respectively. There were no concomitant improvements in AHR to methacholine. CONCLUSION: Patients with asthma and AHR to mannitol improve similarly in responsiveness to mannitol after 12 months of specialist management regardless of Type 2 inflammatory biomarker levels. Mechanisms driving AHR in T2-low asthma need to be further elucidated.

Fractional Exhaled Nitric Oxide (FeNO) Integrating Airway Hyperresponsiveness (AHR) Examination Promotes Etiologic Diagnosis and Treatment for Children with Chronic Cough.

BACKGROUND Chronic cough is the main reason why parents seek medical treatment for their children. This study aimed to evaluate changes in airway function and inflammation levels and associated values in diagnosing and treating chronic cough. MATERIAL AND METHODS This study involved 118 children with chronic cough, including 45 cough-variant asthma (CVA) patients, 53 upper-airway cough syndrome (UACS) patients, and 20 post-infection cough (PIC) patients. Chronic cough was diagnosed as described by guidelines of the American College of Chest Physicians for evaluating chronic cough. Pulmonary ventilation function and airway hyperresponsiveness (AHR) were evaluated. Fractional exhaled nitric oxide (FeNO) levels and eosinophilic airway inflammation were measured. Eosinophil (EOS) count in sputum was also examined. CVA patients were treated with inhaled glucocorticoids, which have anti-inflammatory effects. RESULTS FeNO and sputum EOS levels were higher in CVA patients compared with UACS and PIC patients (P<0.05). CVA patients demonstrated significantly higher small airway indexes, including 25% forced expiratory flow (FEF), 50% FEF, and 75% FEF, compared with UACS and PIC patients (P<0.05). FeNO level was positively correlated with EOS in sputum (r=0.468, P=0.0001) and cough symptom scores (r=0.402, P<0.05). FeNO, EOS, and cough symptoms were significantly improved in CVA patients after glucocorticoid treatment. AHR was improved in all chronic cough patients after treatment. Cough-relief CVA patients demonstrated significantly higher FeNO levels compared with those without cough relief (P<0.05). CONCLUSIONS FeNO integrating pulmonary function and AHR examination can improve etiologic diagnosis and treatment for chronic cough in children.

Small airway dysfunction in patients with cough variant asthma: a retrospective cohort study.

BACKGROUND: Cough variant asthma (CVA) is one of the special populations of asthma. The aim of the study was to compare small airways, the degree of bronchial hyperresponsiveness (BHR) and airway inflammatory subtypes between CVA and classic asthma (CA), and investigate the relationship between these markers to determine the accuracy as indicators of CVA. METHODS: A total of 825 asthmatic patients participated in the study and 614 were included. 614 patients underwent spirometry and a bronchial challenge with methacholine and 459 patients performed induction sputum cell test. RESULTS: The number of CVA patients showed less small airway dysfunction than those of CA patients (p < 0.005). The degree of small airways dysfunction was higher in the CA group compared with the CVA group (p < 0.001). Small airways dysfunction was severer in the eosinophilic airway inflammatory subtype compared with other subtypes (p < 0.05).The area under curve of MMEF, FEF50 and FEF75 (% predicted) was 0.615, 0.621, 0.606, respectively. 0.17mcg of PD20 and 4.7% of sputum eosinophils was the best diagnostic value for CVA with an AUC of 0.582 and 0.575 (p = 0.001 and p = 0.005, respectively). CONCLUSIONS: The eosinophilic airway inflammatory subtype may be increased small airway dysfunction. The value of small airways, BHR and induction sputum cells in CVA prediction, which reflected significant, but not enough to be clinically useful.

Signal inhibitory receptor on leukocytes (SIRL)-1 and leukocyte- associated immunoglobulin-like receptor (LAIR)-1 regulate neutrophil function in infants.

During severe respiratory syncytial virus (RSV) bronchiolitis there is a massive influx of activated neutrophils to the lungs. An exaggerated immune response contributes to lung damage and disease severity during RSV infection. We have previously shown that normal adult neutrophil function can be modulated by agonists of SIRL-1. Here we aimed to measure the potential of two immune checkpoints: SIRL-1 and LAIR-1, to regulate the function of fresh blood and sputum neutrophils from infants with and without severe RSV bronchiolitis. We show a modest inhibition of the oxidative burst through SIRL-1 and LAIR-1, in control and RSV-infected infants. In addition, SIRL-1 and LAIR-1 inhibited neutrophil extracellular traps (NET) formation by sputum neutrophils of RSV patients. Altogether our data show that inhibitory receptors LAIR-1 and SIRL-1 can be used to regulate neutrophil function.

Mucosal-associated invariant T cells and Vδ2+ γδ T cells in community acquired pneumonia: association of abundance in sputum with clinical severity and outcome.

Mucosal-associated invariant T (MAIT) cells and Vδ2+ γδ T cells are anti-bacterial innate-like lymphocytes (ILLs) that are enriched in blood and mucosa. ILLs have been implicated in control of infection. However, the role of ILLs in community-acquired pneumonia (CAP) is unknown. Using sputum samples from a well-characterized CAP cohort, MAIT cell and Vδ2+ T cell abundance was determined by quantitative polymerase chain reaction (qPCR). Cytokine and chemokine concentrations in sputum were measured. The capacity of bacteria in sputum to produce activating ligands for MAIT cells and Vδ2+ T cells was inferred by 16S rRNA sequencing. MAIT cell abundance in sputum was higher in patients with less severe pneumonia; duration of hospital admission was inversely correlated with both MAIT and Vδ2+ T cell abundance. The abundance of both ILLs was higher in patients with a confirmed bacterial aetiology; however, there was no correlation with total bacterial load or the predicted capacity of bacteria to produce activating ligands. Sputum MAIT cell abundance was associated with interferon (IFN)-α, IFN-γ, and sputum neutrophil abundance, while Vδ2+ T cell abundance was associated with CXCL11 and IFN-γ. Therefore, MAIT and Vδ2+ T cells can be detected in sputum in CAP, where they may contribute to improved clinical outcome.

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.

The effect of deep inhalation on mannitol responsiveness.

BACKGROUND: Mannitol inhalation testing is specific for asthmatics with eosinophilic airway inflammation, a factor that has been negatively correlated with the development of deep inhalation bronchoprotection. OBJECTIVE: To evaluate the effect of deep inhalations on responsiveness to inhaled mannitol in correlation with the degree of airway inflammation. METHODS: Twenty participants with stable asthma completed this randomized, crossover study. A screening visit assessed responsiveness to methacholine and airway inflammation through fractional exhaled nitric oxide (FeNO) measures and sputum induction. Participants next completed two mannitol challenges, one with deep inhalations (standard method) and one with inhalations to half of total lung capacity, and two methacholine challenges, one with tidal breathing (standard method) and one with deep inhalations. Only the inhalation technique for dose administration differed between repeat mannitol or methacholine challenges. RESULTS: Deep inhalations did not significantly influence the provocative dose of mannitol causing a 15% fall in forced expiratory volume in 1 second ((P = .73; n = 7) or the mannitol dose-response slope (P = .26; n = 20). Deep inhalations produced significant bronchoprotection against methacholine (P = .03; n = 20). FeNO levels were significantly correlated to sputum eosinophilia (P = .02; n = 15), responsiveness to deep inhalation methacholine (P = .005; n = 20), the dose-response slopes from deep inhalation mannitol (P = .01; n = 20), and the dose-response slope from non-deep inhalation mannitol (P = .005; n = 20). CONCLUSIONS AND CLINICAL RELEVANCE: Deep inhalations did not produce significant bronchoprotection against inhaled mannitol. This result is in agreement with past findings linking airway inflammation with loss of deep inhalation bronchoprotection. CLINICAL TRIAL REGISTRATION: This study was prospectively registered on clinicaltrials.gov (NCT03505489).

Clinical relevance of Aspergillus fumigatus sensitization in cystic fibrosis.

RATIONALE: The clinical relevance of sensitization to Aspergillus (A) fumigatus in cystic fibrosis (CF) is unclear. Some researchers propose that specific A fumigatus IgE is an innocent bystander, whereas others describe it as the major cause of TH-2-driven asthma-like disease. OBJECTIVES: Lung function parameters in mild CF patients may be different in patients with and without A fumigatus sensitization. We aimed to ascertain whether allergen exposure to A fumigatus by bronchial allergen provocation (BAP) induces TH-2 inflammation comparable to an asthma-like disease. METHODS: A total of 35 patients, aged 14.8 ± 8.5 years, and 20 healthy controls were investigated prospectively. The patients were divided into two groups: group 1 (n = 18): specific (s)IgE negative, and group 2 (n = 17): sIgE positive (≥0.7 KU/L) for A fumigatus. Lung function, exhaled NO, and induced sputum were analysed. All sensitized patients with an FEV1 > 75% (n = 13) underwent BAP with A fumigatus, and cell counts, and the expression of IL-5, IL-13, INF-γ, and IL-8 as well as transcription factors T-bet, GATA-3, and FoxP3, were measured. RESULTS: Lung function parameters decreased significantly compared to controls, but not within the CF patient group. After BAP, 8 of 13 patients (61%) had a significant asthmatic response and increased eNO 24 hours later. In addition, marked TH-2-mediated inflammation involving eosinophils, IL-5, IL-13, and FoxP3 became apparent in induced sputum cells. CONCLUSION: Our study demonstrated the clinical relevance of A fumigatus for the majority of sensitized CF patients. A distinct IgE/TH-2-dominated inflammation was found in induced sputum after A fumigatus exposure.

The effect of BCG vaccination on alveolar macrophages obtained from induced sputum from healthy volunteers.

The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) is able to boost innate immune responses through a process called 'trained immunity'. It is hypothesized that BCG-induced trained immunity contributes to protection against Mycobacterium tuberculosis infection. Since alveolar macrophages are the first cell type to encounter M. tuberculosis upon infection, we aimed to investigate the immunomodulatory effects of BCG vaccination on alveolar macrophages. Searching for a less-invasive method than bronchoalveolar lavage, we optimized the isolation of alveolar macrophages from induced sputum of healthy volunteers. Viable alveolar macrophages could be successfully isolated from induced sputum and showed signs of activation already upon retrieval. Further flow cytometric analyses revealed that at baseline, higher expression levels of activation markers were observed on the alveolar macrophages of smokers compared to non-smokers. In addition, BCG vaccination resulted in decreased expression of the activation markers CD11b and HLA-DR on alveolar macrophages. Future studies should evaluate the functional consequences of this reduced activation of alveolar macrophages after BCG vaccination.

Bacteria and sputum inflammatory cell counts; a COPD cohort analysis.

BACKGROUND: There is evidence that bacterial colonisation in chronic obstructive pulmonary disease (COPD) is associated with increased neutrophilic airway inflammation. This study tested the hypothesis that different bacterial phyla and species cause different inflammatory profiles in COPD patients. METHODS: Sputum was analysed by quantitative polymerase chain reaction (qPCR) to quantify bacterial load and 16S rRNA gene sequencing to identify taxonomic composition. Sputum differential cell counts (DCC) and blood DCC were obtained at baseline and 6 months. Patients were categorised into five groups based on bacterial load defined by genome copies/ml of ≥ 1 × 104, no colonisation and colonisation by Haemophilus influenzae (H. influenzae), Moraxella catarrhalis (M. catarrhalis), Streptococcus pneumoniae (S. pneumoniae), or > 1 potentially pathogenic microorganism (PPM). RESULTS: We observed an increase in sputum neutrophil (%), blood neutrophil (%) and neutrophil-lymphocyte ratio (NLR) in patients colonised with H. influenzae (82.6, 67.1, and 3.29 respectively) compared to those without PPM colonisation at baseline (69.5, 63.51 and 2.56 respectively) (p < 0.05 for all analyses), with similar findings at 6 months. The bacterial load of H. influenzae and Haemophilus determined by qPCR and 16s rRNA gene sequencing respectively, and sputum neutrophil % were positively correlated between baseline and 6 months visits (p < 0.0001, 0.0150 and 0.0002 with r = 0.53, 0.33 and 0.44 respectively). CONCLUSIONS: These results demonstrate a subgroup of COPD patients with persistent H. influenzae colonisation that is associated with increased airway and systemic neutrophilic airway inflammation, and less eosinophilic airway inflammation.

Mechanisms and therapeutic strategies for non-T2 asthma.

Non-T2 asthma is traditionally defined as asthma without features of T2 asthma. The definition is arbitrary and is generally based on the presence of neutrophils in sputum, or the absence (or normal levels) of eosinophils or other T2 markers in sputum (paucigranulocytic), airway biopsies or in blood. This definition may be imprecise as we gain more knowledge from applying transcriptomics and proteomics to blood and airway samples. The prevalence of non-T2 asthma is also difficult to estimate as most studies are cross-sectional and influenced by concomitant treatment with glucocorticosteroids, and by the presence of recognized or unrecognized airway infections. No specific therapies have shown any clinical benefits in patients with asthma that is associated with a non-T2 inflammatory process. It remains to be seen if such an endotype truly exists and to identify treatments to target that endotype. Meanwhile, identifying intense airway neutrophilia as an indicator of airway infection and airway hyperresponsiveness as an indicator of smooth muscle dysfunction, and treating them appropriately, and not increasing glucocorticosteroids in patients who do not have obvious T2 inflammation, seem reasonable.

Different endotypes and phenotypes drive the heterogeneity in severe asthma.

The identification of sputum eosinophilia indicating corticosteroid responsiveness in subjects with severe asthma heralded the beginning of phenotyping asthmatic subjects based on airways inflammation. Since then, the heterogeneity of severe asthma has been explored and the importance of immunobiology has come sharply into focus with the identification of the key type-2 cytokine pathways driving eosinophilic inflammation. The development of molecules targeting these type-2 pathways has transformed severe asthma treatment, but necessitates robust clinical evaluation, biomarker profiling and assessment of comorbid factors to identify subjects most likely to benefit from these therapies. It has also become clear that targeting these pathways does not eradicate asthma symptoms and exacerbation risk; further work is needed to clarify underlying non-type-2 mechanisms in severe asthma pathways and possible therapeutic targets. This review addresses progress to date in clinical assessment and management of severe asthma and some of the challenges and unmet needs in severe asthma to achieve the goal of delivering individualized patient care.