Sputum Granulocytes Research Articles

Human Neutrophils Couple Nitric Oxide Production and Extracellular Trap Formation in Allergic Asthma.

Rationale: Nitric oxide (NO) is increased in the airways and serum of patients with allergic asthma, suggesting an important role in asthma. NO production has been widely attributed to the canonical inducible NO synthase. Much effort has been made to inhibit this enzyme, with two outcomes: no asthma improvement and partial NO reduction, suggesting the involvement of an inducible NO synthase-independent source. Objectives: Neutrophils produce NO under inflammatory conditions, and their role in asthma has been overlooked. The present study analyzes their possible role as sources of NO. Methods: Our hypothesis was tested in 99 allergic patients with intermittent bronchial asthma and 26 healthy donors. NO production by blood and sputum neutrophils in response to allergens, anti-IgE, and anti-IgE receptor antibodies was assessed by Griess reagent, flow cytometry, and confocal microscopy. The formation of extracellular traps (ETs) as a possible consequence of NO production was quantified by Western blot and confocal microscopy, and reactive oxygen species were assessed with luminol-enhanced chemiluminescence. Measurements and Main Results: Among blood and sputum granulocytes from patients with allergic asthma, only neutrophils produce NO by an IgE-dependent mechanism. This production is independent of NO synthase, but dependent on a reaction between L-arginine and reactive oxygen species from NOX2 (NADPH oxidase). NO and ETosis are induced in parallel, and NO amplifies ET formation, which is a key mediator in asthma. Conclusions: Our findings reveal a novel role of neutrophils as the unique allergen/IgE-dependent NO source in allergic asthma, enhancing ET formation. These results suggest that NO produced by neutrophils needs further consideration in the treatment of allergic asthma.

Exploring proteomic plasma biomarkers in eosinophilic and neutrophilic asthma.

Few biomarkers identify eosinophilic and neutrophilic asthma beyond cell concentrations in blood or sputum. Finding novel biomarkers for asthma endotypes could give insight about disease mechanisms and guide tailored treatment. Our aim was to investigate clinical characteristics and inflammation-related plasma proteins in relation to blood eosinophil and neutrophil concentrations in subjects with and without asthma. We included 24-26-year-old subjects (n=2063) from the Swedish population-based cohort BAMSE. Subjects with asthma (n=239) and without asthma (n=1824) were subdivided based on blood eosinophil and neutrophil concentrations (cut-offs 0.3 × 109 /L and 5.0 × 109 /L, respectively). We measured the levels of 92 plasma proteins using Olink Proseek Multiplex Inflammation Panel Assay. Group statistics tests were used to analyse the data, as well as adjusted multiple logistic regression models. Among subjects with asthma, 21.8% had eosinophilic asthma and 20.5% neutrophilic asthma. Eosinophilic asthma, but not neutrophilic asthma, was associated with a distinct clinical phenotype with, for example, higher proportions of eczema and sensitization. Most plasma proteins that associated with high eosinophil and/or neutrophil blood concentrations in subjects with asthma showed similar associations in subjects without asthma. However, out of these proteins, MMP10 levels were associated with eosinophilic asthma and were significantly higher as compared to controls with high eosinophilic concentration, while CCL4 levels associated with high neutrophil concentration only in subjects with asthma. Eosinophilic asthma was associated with a clear clinical phenotype. With our definitions, we identified MMP10 as a possible plasma biomarker for eosinophilic asthma and CCL4 was linked to neutrophilic asthma. These proteins should be evaluated further in clinical settings and using sputum granulocytes to define the asthma endotypes.

The role of eosinophil-mediated inflammation in the formation of respiratory remodeling in patients with bronchial asthma of school age children

Objective. To investigate the features of inflammatory changes in the airways, taking into account remodeling markers, determined directly in the sputum of school age children with bronchial asthma, and to optimize the management of this disease.Material and methods. To study the features of the formation of airway remodeling 75 children with persistent bronchial asthma were comprehensively examined on the basis of the Regional Communal Non-Commercial Enterprise "Chernivtsi Regional Children's Clinical Hospital". The children were divided into 2 clinical groups depending on the level of vasoendothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) in the sputum supernatant. The first (I) group included 37 schoolchildren whose VEGF concentration exceeded 80.0 ng / ml, and MMP-9 was more than 5.2 ng / ml. The second (II) comparison group was formed by 38 children in whom BA was not accompanied by accumulation of airway remodeling markers in the sputum supernatant above the indicated distribution point.Research results. The features of the formation of airway remodeling based on the accumulation of sputum markers such as VEGF> 80.0 ng / ml, MMP-9> 5.2 ng / ml were established. It was shown that bronchial remodeling is associated with a decrease in the group of patients with mild persistent asthma, loss of control over its symptoms, prevalence of family allergic history burdens according to both parental pedigrees and the negative effect of maternity smoking. The most sensitive marker in the verification of bronchial remodeling turned out to be a high marginal content of total IgE (> 750 IU / ml) in blood serum (test sensitivity - 95.8% (95% CI 89.7-98.8), which is advisable to combine in the diagnostic algorithm with more specific tests to optimize airway remodeling verification and develop personalized treatment strategies.Conclusion. The formation of bronchial remodeling in bronchial asthma in school age children is associated with the eosinophilic nature of bronchial inflammation, which is accompanied by an increase in the level of blood eosinophilia (> 10%) and sputum (on average 10.5 ± 1.0%) with an increase in myeloperoxidase activity and signs of degranulation of acidophilic granulocytes in sputum, as well as a high marginal concentration of total IgE (> 750 IU / ml) in blood serum in 95.8% of patients.

Особливості відповіді на базисну протизапальну терапію школярів з альтернативними запальними фенотипами бронхіальної астми

Purpose — to evaluate the effectiveness of long-term anti-inflammatory therapy with inhaled glucocorticosteroids in children with alternative inflammatory phenotypes of bronchial asthma (BA) for the development of individualized control treatment. Materials and methods. A comprehensive survey of 94 school-age children with BA was conducted. According to the results of cytological examination of sputum, 2 clinical observation groups were formed. The first group of patients was formed by 38 patients with non3eosinophilic (neutrophilic) nature of the inflammatory process of the bronchi (mean age — 11.1±2.9 years, the proportion of boys — 52.6±8.1%), and the second group — 56 children with eosinophilic type of airway inflammation (3% or more of eosinophilic granulocytes in sputum), ie eosinophilic asthma phenotype (mean age — 12.2±3.2 (P>0.05) years, the proportion of boys — 67.9±6.2%, P>0.05). According to the main characteristics of the observation group could be compared. All patients underwent a comprehensive clinical and paraclinical (spirographic) examination. Scoring control of BA symptoms was performed using a questionnaire at the beginning and at the end of the course of anti-inflammatory basic therapy. The questionnaire included the clinical signs of BA reflected in the scores, which were evaluated by patients and their parents, as well as the scale of instrumental studies according to the spirographic examination of patients. Results. The paper shows that the best effect of long-term courses of basic therapy with inhaled glucocorticosteroids (ICS) was observed in patients with eosinophilic airway inflammation. Thus, the share of patients with a relatively satisfactory level of clinical control of the disease and with close to normal spirographic indicators was in group II: before the course of treatment with inhaled corticosteroids 25.0% and 31.9%, and after treatment — 81.3% (P<0,01) and 69.0% (P<0.01), respectively. Thus, an increase of 69.3% in relative risk (IRR) and 56.3% in absolute risk (IAR) of BA control reflected a pronounced control effect of inhaled corticosteroids in the eosinophilic nature of airway inflammation. The minimum number of patients needed to treat (NNT) in order to obtain at least one positive result was 2. At the same time, in patients with neutrophilic inflammatory process, the proportion of patients with a relatively satisfactory level of clinical and paraclinical (according to spirography) disease control was: before the appointment of ICS — 30.8% and 30.8%, and after treatment — 50% (PFisher's exact test>0.05) and 76.9% (PFisher's exact test<0.01), respectively. This persistence of clinical manifestations of the disease indicated an insufficient level of control of non3eosinophilic BA in the treatment of inhaled corticosteroids and questioned the feasibility of monotherapy with this group of neutrophilic bronchitis. The insufficient effect of anti3inflammatory therapy with inhaled corticosteroids was evidenced by the fact that IRR of satisfactory level of clinical control was 38.4%, IAR — 19.2%, and NNT — 6 patients. Conclusions. In patients with the eosinophilic phenotype of BA, the use of long courses of inhaled glucocorticosteroids led to an increased chance of achieving disease control, while the chances of improving of the pulmonary function tests were also observed in the neutrophilic phenotype of the disease. The research was carried out in accordance with the principles of the Helsinki declaration. The study protocol was approved by the Local ethics committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interest was declared by the authors. Key words: bronchial asthma, children, basic anti-inflammatory therapy.

Hot Topic: Precision Medicine for Asthma-Has the Time Come?

Asthma is a common inflammatory airway disease, which affects more than 300 million people worldwide. Although conventional drugs are effective for most of the patients with mild-to-moderate asthma, they are less effective for patients with difficult-to-treat or severe asthma. Identification of asthma endotypes and biomarkers will lead to more precise approaches to treat asthma. Asthma subphenotypes and endotypes have been described based on clinical variables and sputum granulocytes. A recent asthma endotype study has been summarized based on the combination of T2 (FeNO) and non-T2 (IL-6) biomarkers. Discovery of potential biomarkers for asthma has been discussed in the context of omics approaches. Current biologic drugs for asthma have been summarized, and the future direction of precise treatment of asthma has been suggested. This review provides a concise overview of the current state of subphenotypes, endotypes, biomarkers, omics approaches, and biologic drugs in asthma.

Modulatory Effects of Aspergillus Colonization and Abpa on Blood and Sputum Granulocytes in CF

Modulatory Effects of Aspergillus Colonization and Abpa on Blood and Sputum Granulocytes in CF

Autophagy mechanisms in sputum and peripheral blood cells of patients with severe asthma: a new therapeutic target.

Autophagy and genetic predisposition have been suggested to potentially play roles in the development of asthma. However, little is known about the role of autophagy in the pathogenesis of severe asthma. We compared autophagy in the sputum granulocytes, peripheral blood cells (PBCs) and peripheral blood eosinophils (PBEs) between patients with severe asthma and those with non-severe asthma and investigated the functional effects of autophagy. We enrolled 36 patients with severe asthma, 14 with non-severe asthma and 23 normal healthy controls in this study. Sputum granulocytes, PBCs and PBEs were isolated from each subject. Autophagy was evaluated based on the expression of microtubule-associated protein light chain 3 (LC3) by Western blot, confocal microscopy, transmission electron microscopy and flow cytometry. IL-8 levels were measured by ELISA. To induce autophagy, HL-60 cells, human primary small airway epithelial cells (SAECs) and A549 cells were treated with IL-5, IL-1β and TNF-α. To inhibit autophagy, PI3K inhibitors (LY29400 and 3-methyladenine [3-MA]) and hydroxychloroquine (HCQ) were used. Knockdown of ATG5 and Beclin-1 was performed in A549 cells, and the therapeutic effects of dexamethasone were evaluated. Higher autophagy levels were noted in sputum granulocytes, PBCs and PBEs from patients with severe asthma than from patients with non-severe asthma and healthy controls (P < 0.05 for all). IL-5 increased autophagy levels in both PBCs and PBEs (P < 0.05). 3-MA attenuated the increased expression of LC3-II and eosinophil cationic protein in HL-60 cells induced by IL-5 (P = 0.034 for both). Dexamethasone did not affect autophagy levels in PBEs. IL-1β increased LC3-II expression and IL-8 production (P < 0.01) in SAECs, and this was attenuated by LY294002, 3-MA, HCQ and knockdown of ATG5 and Beclin-1 (in A549 cells) (P < 0.01). Autophagy could play a role in the pathogenesis of severe asthma. Autophagy modulation may be a novel therapeutic target for conventional therapy-resistant severe asthma.

Similar activation state of neutrophils in sputum of asthma patients irrespective of sputum eosinophilia.

Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13, <3%: n = 8). Eosinophils and neutrophils from blood and sputum were analysed for expression of activation and degranulation markers by flow cytometry. Data were analysed by both classical, non-parametric statistics and a multi-dimensional approach, using principal component analysis (PCA). Patients with sputum eosinophilia were characterized by increased asthma control questionnaire (ACQ) scores and blood eosinophil counts. Both sputum neutrophils and eosinophils displayed an activated and degranulated phenotype compared to cells obtained from blood. Specifically, degranulation of all granule types was detected in sputum cells, combined with an increased expression of the activation markers (activated) Mac-1 (CD11b), programmed death ligand 1 (PD-L1) (CD274) and a decreased expression of CD62L. CD69 expression was only increased on sputum eosinophils. Surface marker expression of neutrophils was similar in the presence or absence of eosinophilia, either by single or multi-dimensional analysis. Sputum neutrophils were highly activated and degranulated irrespective of sputum eosinophilia. Therefore, we conclude that differences in granulocyte activation in sputum and/or blood are not associated with clinical differences in the two groups of asthma patients. The finding of PD-L1 expression on sputum granulocytes suggests an immunomodulatory role of these cells in the tissue.

Similar activation of sputum granulocytes in eosinophilic and non‐eosinophilic asthma

Method We obtained peripheral blood and induced sputum from 21 asthma patients and defined two patient groups based on sputum eosinophilia (cut-off 3%). Eosinophils and neutrophils from blood and sputum were analysed by flow cytometry for expression of activation and degranulation markers. Data were analysed by both classical, non-parametric statistics and a more advanced multi-dimensional approach, using principal component analysis (PCA).

Systemic upregulation of neutrophil -defensins and serine proteases in neutrophilic asthma

The well-characterised airway inflammatory phenotypes of asthma include eosinophilic, neutrophilic, mixed eosinophilic/neutrophilic and paucigranulocytic asthma, identified based on the proportion of sputum granulocytes. Systemic inflammation is now recognised as an important part of some airway diseases, but the involvement of systemic inflammation in the pathogenesis of airway inflammatory phenotypes of asthma remains unknown. Induced sputum samples and peripheral blood were collected from participants with asthma (n=36). Airway inflammatory cell counts were performed from induced sputum and inflammatory phenotype assigned based on the airway eosinophil and neutrophil cut-offs of 3% and 61%, respectively. RNA was extracted from whole blood and gene expression profiles were generated (Illumina Humanref-8 V3) and analysed using GeneSpring GX11. There were six genes classified as differentially expressed between the four asthma phenotypes, including the α-defensins (DEFA) 1, 1B, 3 and 4 and neutrophil proteases cathepsin G (CTSG) and elastase (ELA2). Systemic expression of DEFA1,1B,3,4,CTSG and ELA2 was significantly higher in the neutrophilic asthma (NA) phenotype. Microarray results of the α-defensins and neutrophil proteases were successfully validated using real-time PCR. Plasma elastase was significantly increased in people with neutrophilic airway inflammation. There is systemic upregulation of α-defensin and neutrophil protease expression in NA, which may represent proinflammatory effects on the bone marrow and the release of immature neutrophils into the circulation. This demonstrates a systemic inflammatory component in NA that further differentiates this from other asthma phenotypes and indicates different mechanisms in NA.

Analyses of asthma severity phenotypes and inflammatory proteins in subjects stratified by sputum granulocytes

Patients with severe asthma have increased granulocytes in their sputum compared with patients with mild to moderate asthma. We hypothesized that inflammatory granulocytes in sputum may identify specific asthma severity phenotypes and are associated with different patterns of inflammatory proteins in sputum supernatants. This hypothesis was tested in 242 patients with asthma enrolled in the Severe Asthma Research Program who provided sputum samples for cell count, differential cell determinations, cell lysates for Western blot, and supernatant analyses by inflammatory protein microarrays and ELISAs. ANOVA and multiple linear regression models tested mediator associations. Stratified by sputum granulocytes, <2% or > or = 2% eosinophils and <40% or > or = 40% neutrophils, subjects with both increased eosinophils and neutrophils had the lowest lung function and increased symptoms and health care use. Subjects with elevated eosinophils with or without increased neutrophils had significantly increased fraction exhaled nitric oxide (FeNO) and serum eosinophils and greater frequency of daily beta-agonist use. Microarray data stratified by granulocytes revealed 25 to 28 inflammatory proteins increased >2-fold in sputa with > or = 40% neutrophils. Microarray analyses stratified by severity of asthma identified 6 to 9 proteins increased >2-fold in sputa in subjects with severe asthma compared with nonsevere asthma. ELISA data stratified by sputum granulocytes showed significant increases in brain-derived neurotrophic factor, IL-1beta, and macrophage inflammatory protein 3alpha/CCL20 for those with > or = 40% neutrophils; these mediators demonstrated positive associations with neutrophil counts. Combined increased sputum eosinophils and neutrophils identified patients with asthma with the lowest lung function, worse asthma control, and increased symptoms and health care requirements. Inflammatory protein analyses of sputum supernatants found novel mediators increased in patients with asthma, predominantly associated with increased sputum neutrophils.

Wood smoke exposure induces a pulmonary and systemic inflammatory response in firefighters

Epidemiological studies report an association between exposure to biomass smoke and cardiopulmonary morbidity. The mechanisms for this association are unclear. The aim of the present study was to characterise the acute pulmonary and systemic inflammatory effects of exposure to forest fire smoke. Seasonal forest firefighters (n = 52) were recruited before and/or after a day of fire-fighting. Exposure was assessed by questionnaires and measurement of carbon monoxide levels (used to estimate respirable particulate matter exposure). The pulmonary response was assessed by questionnaires, spirometry and sputum induction. Peripheral blood cell counts and inflammatory cytokines were measured to define the systemic response. Estimated respirable particulate matter exposure was high (peak levels >2 mg x m(-3)) during fire-fighting activities. Respiratory symptoms were reported by 65% of the firefighters. The percentage sputum granulocytes increased significantly from 6.5 to 10.9% following fire-fighting shifts, with concurrent increases in circulating white blood cells (5.55x10(9) to 7.06x10(9) cells x L(-1)) and band cells (0.11x10(9) to 0.16x10(9) cells x L(-1)). Serum interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 levels significantly increased following fire-fighting. There were no changes in band cells, IL-6, and IL-8 following strenuous physical exertion without fire-fighting. There was a significant association between changes in sputum macrophages containing phagocytosed particles and circulating band cells. In conclusion, acute exposure to air pollution from forest fire smoke elicits inflammation within the lungs, as well as a systemic inflammatory response.