Neutrophilic Airway Research Articles

Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.

Trif-dependent induction of Th17 immunity by lung dendritic cells.

Allergic asthma is thought to stem largely from maladaptive T helper (Th)2 responses to inhaled allergens, which in turn lead to airway eosinophilia and airway hyperresponsiveness. However, many individuals with asthma have airway inflammation that is predominantly neutrophilic and resistant to treatment with inhaled glucocorticoids. An improved understanding of the molecular basis of this form of asthma might lead to improved strategies for its treatment. Here, we identify novel roles of the adaptor protein, TRIF, in neutrophilic responses to inhaled allergens. In different mouse models of asthma, Trif-deficient animals had marked reductions in interleukin (IL)-17, airway neutrophils, and airway hyperresponsiveness compared with WT mice, whereas airway eosinophils were generally similar in these two strains. Compared with lung dendritic cells (DCs) from WT mice, lung DCs from Trif-deficient mice displayed impaired LPS-induced migration to regional lymph nodes, lower levels of the costimulatory molecule, CD40, and produced smaller amounts of the Th17-promoting cytokines, IL-6 and IL-1β. When cultured with allergen-specific, naïve T cells, Trif-deficient lung DCs stimulated robust Th2 cell differentiation, but very weak Th1 and Th17 cell differentiation. Together, these findings reveal a TRIF-CD40-Th17 axis in the development of IL-17-associated, neutrophilic asthma.

Allergic airway inflammation decreases lung bacterial burden following acute Klebsiella pneumoniae infection in a neutrophil- and CCL8-dependent manner.

The Th17 cytokines interleukin-17A (IL-17A), IL-17F, and IL-22 are critical for the lung immune response to a variety of bacterial pathogens, including Klebsiella pneumoniae. Th2 cytokine expression in the airways is a characteristic feature of asthma and allergic airway inflammation. The Th2 cytokines IL-4 and IL-13 diminish ex vivo and in vivo IL-17A protein expression by Th17 cells. To determine the effect of IL-4 and IL-13 on IL-17-dependent lung immune responses to acute bacterial infection, we developed a combined model in which allergic airway inflammation and lung IL-4 and IL-13 expression were induced by ovalbumin sensitization and challenge prior to acute lung infection with K. pneumoniae. We hypothesized that preexisting allergic airway inflammation decreases lung IL-17A expression and airway neutrophil recruitment in response to acute K. pneumoniae infection and thereby increases the lung K. pneumoniae burden. As hypothesized, we found that allergic airway inflammation decreased the number of K. pneumoniae-induced airway neutrophils and lung IL-17A, IL-17F, and IL-22 expression. Despite the marked reduction in postinfection airway neutrophilia and lung expression of Th17 cytokines, allergic airway inflammation significantly decreased the lung K. pneumoniae burden and postinfection mortality. We showed that the decreased lung K. pneumoniae burden was independent of IL-4, IL-5, and IL-17A and partially dependent on IL-13 and STAT6. Additionally, we demonstrated that the decreased lung K. pneumoniae burden associated with allergic airway inflammation was both neutrophil and CCL8 dependent. These findings suggest a novel role for CCL8 in lung antibacterial immunity against K. pneumoniae and suggest new mechanisms of orchestrating lung antibacterial immunity.

CXC chemokines and antimicrobial peptides in rhinovirus‐induced experimental asthma exacerbations

SummaryRationaleRhinoviruses (RVs) are the major triggers of asthma exacerbations. We have shown previously that lower respiratory tract symptoms, airflow obstruction, and neutrophilic airway inflammation were increased in experimental RV‐induced asthma exacerbations.ObjectivesWe hypothesized that neutrophil‐related CXC chemokines and antimicrobial peptides are increased and related to clinical, virologic, and pathologic outcomes in RV‐induced exacerbations of asthma.MethodsProtein levels of antimicrobial peptides (SLPI, HNP 1–3, elafin, and LL‐37) and neutrophil chemokines (CXCL1/GRO‐α, CXCL2/GRO‐β, CXCL5/ENA‐78, CXCL6/GCP‐2, CXCL7/NAP‐2, and CXCL8/IL‐8) were determined in bronchoalveolar lavage (BAL) fluid of 10 asthmatics and 15 normal controls taken before, at day four during and 6 weeks post‐experimental infection.Results BAL HNP 1–3 and Elafin were higher, CXCL7/NAP‐2 was lower in asthmatics compared with controls at day 4 (P = 0.035, P = 0.048, and P = 0.025, respectively). BAL HNP 1–3 and CXCL8/IL‐8 were increased during infection (P = 0.003 and P = 0.011, respectively). There was a trend to increased BAL neutrophils at day 4 compared with baseline (P = 0.076). BAL HNP 1–3 was positively correlated with BAL neutrophil numbers at day 4. There were no correlations between clinical parameters and HNP1–3 or IL‐8 levels.ConclusionsWe propose that RV infection in asthma leads to increased release of CXCL8/IL‐8, attracting neutrophils into the airways where they release HNP 1–3, which further enhances airway neutrophilia. Strategies to inhibit CXCL8/IL‐8 may be useful in treatment of virus‐induced asthma exacerbations.

Mechanistic differences between phenotypes of chronic lung allograft dysfunction after lung transplantation.

Distinct phenotypes of chronic lung allograft dysfunction (CLAD) after lung transplantation are emerging with lymphocytic bronchiolitis (LB)/azithromycin reversible allograft dysfunction (ARAD), classical or fibrotic bronchiolitis obliterans syndrome (BOS), and restrictive allograft syndrome (RAS) proposed as separate entities. We have additionally identified lung transplant recipients with prior LB, demonstrating persistent airway neutrophilia (PAN) despite azithromycin treatment. The aim of this study was to evaluate differences in the airway microenvironment in different phenotypes of CLAD. Bronchoalveolar lavage (BAL) from recipients identified as stable (control), LB/ARAD, PAN, BOS, and RAS were evaluated for differential cell counts and concentrations of IL-1α, IL-1β, IL-6, IL-8, and TNF-α. Primary human bronchial epithelial cells were exposed to BAL supernatants from different phenotypes and their viability measured. BOS and RAS showed increased BAL neutrophilia but no change in cytokine concentrations compared with prediagnosis. In both LB/ARAD and PAN, significant increases in IL-1α, IL-1β, and IL-8 were present. BAL IL-6 and TNF-α concentrations were increased in PAN and only this phenotype demonstrated decreased epithelial cell viability after exposure to BAL fluid. This study demonstrates clear differences in the airway microenvironment between different CLAD phenotypes. Systematic phenotyping of CLAD may help the development of more personalized approaches to treatment.

Preceding human metapneumovirus infection increases adherence of Streptococcus pneumoniae and severity of murine pneumococcal pneumonia.

Coinfection with respiratory virus and Streptococcus pneumoniae has been frequently reported in several epidemiologic studies. The aim of this study was to explore the effect of preceding human metapneumovirus (hMPV) inoculation on subsequent pneumococcal infection. Hep-2 and A549 cells were infected with hMPV then inoculated with S. pneumoniae. Bacterial adhesion was measured using colony forming unit and cytometric-fluorescence assays. In vivo bacterial adhesion was examined in hMPV-infected mice after inoculation of fluorescence-conjugated S. pneumoniae. Pulmonary inflammation (bacterial titers, cytokine levels, and histopathology) of hMPV-infected mice was investigated after inoculation with S. pneumoniae. In vitro results of bacterial infection with S. pneumoniae on A549 and Hep-2 monolayer cells showed that even though cellular adherence was variable among different serotypes, there was significantly enhanced bacterial adherence in A549 cells with preceding hMPV infection. In addition, in vivo study of hMPV-infected mice showed increased adhesion of S. pneumoniae on the bronchial epithelium with delayed bacterial clearance and exacerbated histopathology. Furthermore, mice with preceding hMPV infection showed repressed recruitment of airway neutrophils with decreased expression of neutrophil chemoattractants during pneumococcal infection. These results suggest that hMPV-infected airway cells, especially the lower airway epithelium, express increased adherence with S. pneumoniae. Furthermore, hMPV-infected mice showed impaired recruitment of airway neutrophils, possibly leading to delayed bacterial clearance and exacerbated pulmonary inflammation, after secondary infection with pneumococcal isolates.

Airway Inflammation and Illness Severity in Response to Experimental Rhinovirus Infection in Asthma

Background:The nature of bronchial mucosal inflammation and its physiologic and clinical significance in rhinovirus-induced asthma exacerbations is unclear. We investigated bronchial mucosal inflammatory response and its association with physiologic and clinical outcomes in an experimental model of rhinovirus-induced asthma exacerbations.Methods:We used immunohistochemistry methods to detect phenotypes of inflammatory cells infiltrating the bronchial mucosa before and after experimental rhinovirus infection in 10 subjects with asthma and 15 normal subjects.Results:Compared with baseline, rhinovirus infection significantly increased the number of epithelial (P = .005) and subepithelial (P = .017) neutrophils in subjects with asthma only and subepithelial CD68+ macrophages in both subjects with asthma (P = .009) and normal subjects (P = .018) but more so in those with asthma (P = .021). Numbers of CD45+, CD68+, and CD20+ cells; neutrophils; and eosinophils at day 4 postinfection were positively associated with virus load (r = 0.50-0.72, P = .016-0.03). At acute infection in subjects with asthma, CD4+ cells correlated with chest symptom scores (r = 0.69, P = .029), the fall in the 10% fall in FEV1 (PC10) correlated with neutrophils (r = −0.89, P = .029), the PC10 correlated inversely with CD4+ (r = −0.67, P = .023) and CD8+ cells (r = −0.65, P = .03), the 20% fall in FEV1 was inversely associated with CD20+ cells (r = −0.65, P = .03), and higher epithelial CD8+ cell counts were significantly associated with a greater maximum fall in FEV1 (r = −0.72, P = .03), whereas higher subepithelial mast cell counts were significantly associated with a lower maximum percent fall in peak expiratory flow (r = 0.8, P = .024).Conclusions:In subjects with asthma, rhinovirus infection induces bronchial mucosal neutrophilia and more severe monocyte/macrophage infiltration than in normal subjects. Airway neutrophils, eosinophils, and T and B lymphocytes during infection are related to virus load and physiologic and clinical severity, whereas mast cells are related to greater lung function.

Prospective Characterization of Protracted Bacterial Bronchitis in Children

BackgroundPrior studies on protracted bacterial bronchitis (PBB) in children have been retrospective or based on small cohorts. As PBB shares common features with other pediatric conditions, further characterization is needed to improve diagnostic accuracy among clinicians. In this study, we aim to further delineate the clinical and laboratory features of PBB in a larger cohort, with a specific focus on concurrent viral detection.MethodsChildren with and without PBB (control subjects) undergoing flexible bronchoscopy were prospectively recruited. Basic immune function testing and lymphocyte subset analyses were performed. BAL specimens were processed for cellularity and microbiology. Viruses were identified using polymerase chain reaction (PCR) and bacteria were identified via culture.ResultsThe median age of the 104 children (69% male) with PBB was 19 months (interquartile range [IQR], 12-30 mo). Compared with control subjects, children with PBB were more likely to have attended childcare (OR, 8.43; 95% CI, 2.34-30.46). High rates of wheeze were present in both groups, and tracheobronchomalacia was common. Children with PBB had significantly elevated percentages of neutrophils in the lower airways compared with control subjects, and adenovirus was more likely to be detected in BAL specimens in those with PBB (OR, 6.69; 95% CI, 1.50-29.80). Median CD56 and CD16 natural killer (NK) cell levels in blood were elevated for age in children with PBB (0.7 × 109/L; IQR, 0.5-0.9 cells/L).ConclusionsChildren with PBB are, typically, very young boys with prolonged wet cough and parent-reported wheeze who have attended childcare. Coupled with elevated NK-cell levels, the association between adenovirus and PBB suggests a likely role of viruses in PBB pathogenesis.

Comparison of Bronchoalveolar Lavage and Tracheal Aspirate Results in 64 Non‐Thoroughbred Horses Presenting with Respiratory Signs or Exercise Intolerance

IntroductionIt has been debated which airway sampling method, bronchoalveolar lavage (BAL) or tracheal aspirate (TA), is superior in evaluating equine respiratory disease. Most studies suggest BAL is the best sampling method due to better correlation with lung histopathology. In this study we compared BAL and TA results of athletic non‐Thoroughbred horses with chronic respiratory signs or exercise intolerance during 2009–2013.Methods64 horses, 2–25 years of age (16 Coldbloods, 30 Warmbloods, 11 Standardbreds, 7 ponies) with recurrent or chronic (duration &gt;1 month) respiratory signs or exercise intolerance that were subject to both BAL and TA sampling at rest were included. Neutrophil counts &gt;5% in BAL and &gt;20% in TA were considered abnormal. Statistical analysis was performed with Spearman's correlation coefficient.ResultsBoth BAL and TA were abnormal in 29 horses and normal in 26 horses. 7 horses had only abnormal TA and 2 horses only abnormal BAL. Correlation was found between the amount of mucus in the trachea and both BAL (P = 0.002) and TA (P&lt;0.001) neutrophil percentage. Neutrophil (P&lt;0.001), eosinophil (P&lt;0.001), lymphocyte (P = 0.008) and macrophage (P = 0.004) percentages correlated between BAL and TA.ConclusionsIn our university hospital population, mucus in the trachea was a good indicator of airway neutrophilia. Unlike previous studies, in only 14% of the horses the interpretation of BAL and TA result was different with TA being slightly more sensitive at detecting neutrophilia. However, a gold standard for detecting inflammation in the equine airways is still lacking and the type and severity of the disease may greatly influence the best choice of sampling method.Ethical Animal ResearchRetrospective analysis of case records. Sources of funding: none. Competing interests: none.

Ethyl pyruvate decreases airway neutrophil infiltration partly through a high mobility group box 1-dependent mechanism in a chemical-induced murine asthma model.

Diisocyanates are one of the leading causes of occupational asthma, which is dominated by granulocytic inflammation in the airway. In this study, we intended to explore the role of ethyl pyruvate (EP) on neutrophil infiltration in a toluene-2,4-diisocyanate (TDI)-induced murine asthma model. The experimental mice were first dermally sensitized and then challenged with TDI via oropharyngeal aspiration. The mice were treated intraperitoneally with 100, 50 or 10mg/kg EP 1h before each challenge. One day after the last challenge, airway reactivity to methacholine was measured by a barometric plethysmographic chamber. Total and differential cell counts, along with levels of macrophage inflammatory protein-2 (MIP-2), TNF-α in bronchoalveolar lavage (BAL) fluid and mRNA expression of CXCR2 in the lung were assessed. To depict neutrophils, a naphthol AS-D chloroacetate esterase kit was used. High mobility group box 1 (HMGB1) was determined by western blot and immunohistochemistry. Treatment with EP dramatically decreased airway hyperresponsiveness in TDI-challenged mice, as well as numbers of neutrophils in BAL fluid and peribronchovascular regions. Both the TDI-induced raised protein level and abnormal distribution of HMGB1 were significantly recovered by EP in a dose-dependent manner. The concentration of MIP-2 in TDI-induced asthma mice was significantly higher than that of the control ones, while EP had few effects on MIP-2. The mRNA expression of CXCR2 didn't change significantly, and TNF-α was not detected in BAL fluids. EP reduces airway neutrophil infiltration partly through downregulating HMGB1 in a chemical-induced murine asthma model.

Oxidative stress-induced pulmonary neutrophil recruitment is independent of conventional dendritic cells (INC5P.325)

Abstract Oxidative stress and airway neutrophilia are key features of some asthma phenotypes. Ozone (O3), a potent oxidant, induces pulmonary neutrophil recruitment which requires IL-17A+ γδ T cells. IL-23 is required for IL-17A production by T cells and conventional dendritic cells (cDC) are a potent source of IL-23. We therefore hypothesized that oxidative stress-induced IL-17A+ γδ T cells require IL-23 production from cDC. Wild type (WT), Tcrd-/- (lacking γδ T cells), and flt3l-/- (deficient in cDC) mice were exposed to air or 0.3 ppm O3 for 72 h. Bronchoalveolar lavage (BAL) was collected for cell differentials and lungs were processed for qPCR analysis. Lungs from mice which were not lavaged were used for flow cytometric analysis. In WT mice, O3 caused a 4 fold increase in the percentage of lung CD11c+/MHCII+/Siglec-F+ dendritic cells. IL-23 mRNA was also increased after O3 exposure. Flt3l-/- and WT mice had similar amounts of IL-23 and IL-17A mRNA and BAL neutrophils, thus indicating that cDC are not the source of IL-23 after such oxidative stress. However, IL-23 mRNA was reduced in Tcrd-/- mice which had fewer CD11c-/ CD11b+/F4/80+ myeloid cells. These cells are thus the potential source of IL-23. Altogether these data suggest oxidative stress induces IL-23 production by non-dendritic myeloid cells leading to IL-17A+ γδ T cell driven neutrophil recruitment.

The IL-17-independent role of IL-23 in severe asthma (HYP7P.300)

Abstract Asthma is a common respiratory disease affecting approximately 300 million people worldwide with no current preventions or cures. Th17 cells are known to be influential in asthma pathogenesis, especially in asthmatics with severe disease that fail to respond to glucocorticoid therapy. Interleukin-23 is critical for the maintenance and full acquisition of Th17 cells, but very little is known about its direct effects outside of IL-17 signaling. This study seeks to investigate novel mechanisms by which IL-23 regulates allergic airway disease. To do this, BALB/c SCID and Rag2-/-γc-/- mice were treated with adenoviral IL-23 or control and airway disease was assessed 3 days later. Preliminary data show that exogenous IL-23 induced airway neutrophilia and pulmonary expression of genes related to asthma pathogenesis and Th17-related cytokines and chemokines, while it increased airway resistance in response to methacholine and decreased lung compliance in BALB/c SCID mice. Further, IL-23 induced airspace inflammation and production of Th17-related cytokines and chemokines, while not impacting lung function in Rag2-/-γc-/- mice. This work suggests a novel role for IL-23 in airway disease as IL-23 itself could induce inflammation, which was predominately neutrophilic, in mice regardless of immune status. More significantly, IL-23 was also shown to modulate pulmonary expression of extracellular matrix-related proteins in these mice, which is currently under investigation.

Lack of Neutrophil Elastase Reduces Inflammation, Mucus Hypersecretion, and Emphysema, but Not Mucus Obstruction, in Mice with Cystic Fibrosis–like Lung Disease

Recent evidence from clinical studies suggests that neutrophil elastase (NE) released in neutrophilic airway inflammation is a key risk factor for the onset and progression of lung disease in young children with cystic fibrosis (CF). However, the role of NE in the complex in vivo pathogenesis of CF lung disease remains poorly understood. To elucidate the role of NE in the development of key features of CF lung disease including airway inflammation, mucus hypersecretion, goblet cell metaplasia, bacterial infection, and structural lung damage in vivo. We used the Scnn1b-Tg mouse as a model of CF lung disease and determined effects of genetic deletion of NE (NE(-/-)) on the pulmonary phenotype. Furthermore, we used novel Foerster resonance energy transfer (FRET)-based NE reporter assays to assess NE activity in bronchoalveolar lavage from Scnn1b-Tg mice and sputum from patients with CF. Lack of NE significantly reduced airway neutrophilia, elevated mucin expression, goblet cell metaplasia, and distal airspace enlargement, but had no effect on airway mucus plugging, bacterial infection, or pulmonary mortality in Scnn1b-Tg mice. By using FRET reporters, we show that NE activity was elevated on the surface of airway neutrophils from Scnn1b-Tg mice and patients with CF. Our results suggest that NE plays an important role in the in vivo pathogenesis and may serve as a therapeutic target for inflammation, mucus hypersecretion, and structural lung damage and indicate that additional rehydration strategies may be required for effective treatment of airway mucus obstruction in CF.

ATF3 is a novel regulator of mouse neutrophil migration

AbstractExpression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3−/− neutrophil recruitment to wild-type lungs. In vitro, ATF3−/− neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3−/− neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3−/− and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.

Autocrine ligands of the epithelial growth factor receptor mediate inflammatory responses to diesel exhaust particles

BackgroundDiesel exhaust is associated with cardiovascular and respiratory mortality and morbidity. Acute exposure leads to increased IL-8 expression and airway neutrophilia, however the mechanism of this response is unknown. Objectives: As cigarette smoke-induced IL-8 expression by epithelial cells involves transactivation of the epidermal growth factor receptor (EGFR), we studied the effects of diesel exhaust particles (DEP) on IL-8 release and the role of the EGFR.MethodsPrimary bronchial epithelial cells (PBEC) were exposed to DEPs or carbon black. IL-8 and EGFR ligand expression (transforming growth factor alpha (TGFα), heparin-binding EGF-like growth factor, and amphiregulin (AR)) were assessed by quantitative RT-PCR and ELISA.ResultsDEP, but not carbon black, caused a dose-dependent increase in mitogen-activated protein kinase (MAPK) activation and IL-8 expression, however above 50 μg/ml there was an increase in cytotoxicity. At 50 μg/ml, DEPs stimulated transcription and release of IL-8 and EGFR ligands. IL-8 release was blocked by EGFR neutralizing antibodies, an EGFR-selective tyrosine kinase inhibitor and by the metalloprotease inhibitor, GM6001, which blocks EGFR ligand shedding. Neutralizing antibodies to AR, TGFα and heparin-binding (HB)-EGF reduced DEP-induced IL-8 by >50%. Conclusion Expression of IL-8 in response to DEPs is dependent on EGFR activation and that autocrine production of EGFR ligands makes a substantial contribution to this response. Capsule Summary: This study identifies a mechanism whereby diesel particles stimulates IL-8 release from bronchial epithelial cells. This mechanism may help to explain the recruitment of neutrophils into the airways of people exposed to particulate air pollution.

Predictors of neutrophilic airway inflammation in young smokers with asthma

Introduction: Asthma is one of the most widespread chronic diseases worldwide. In spite of numerous detrimental effects on asthma, smoking is common among asthma patients. These smoking-induced aggravations of asthma may be attributed to changes in airway inflammation, which is characterized by a higher degree of neutrophilic inflammation than in non-smokers. A state of neutrophilic inflammation may lead to increased steroid resistance and an accelerated loss of lung function owing to tissue destruction. The aim of this study was to elucidate predictors of neutrophilic inflammation in young asthmatic smokers not on steroid treatment, including analysis of tobacco history and bacterial colonization. Methods: In a cross-sectional study, 52 steroid-free, current smokers with asthma were examined with induced sputum, fractional exhaled nitric oxide (FeNO), lung function, ACQ6 score, mannitol and methacholine challenge. A sample from the sputum induction was taken for bacterial analysis using 16S gene PCR technique and sequencing. Results: Using one-way analysis of variance and binary and linear regression models, only age and ACQ6 score were found to be significant predictors for airway neutrophilia. The investigation also included analysis for effect of pack years, current tobacco consumption, body mass index, lung function, FeNO; methacholine and mannitol responsiveness, atopy, gender, asthma history and presence of bacteria. The most common potentially pathogenic bacteria found were Streptococcus spp., Haemophilus spp. and Mycoplasma spp. Conclusion: In this study, no tobacco-related predictors of airway neutrophilia were found, indicating that in the younger years of asthma patients who smoke, the amount of tobacco smoked in life does not influence the degree of neutrophilia. Conversely, for asthmatic smokers, neutrophilia may be induced when a certain threshold of tobacco consumption is reached.

A role for mitogen kinase kinase 3 in pulmonary inflammation validated from a proteomic approach

Proteomics is a powerful tool to ascertain which proteins are differentially expressed in the context of disease. We have used this approach on inflammatory cells obtained from patients with asthma to ascertain whether novel drugs targets could be illuminated and to investigate the role of any such target in a range of in vitro and in vivo models of inflammation.A proteomic study was undertaken using peripheral blood mononuclear cells from mild asthmatic subjects compared with healthy subjects. The analysis revealed an increased expression of the intracellular kinase, mitogen activated protein kinase (MKK3), and the function of this protein was investigated further in preclinical models of inflammation using MKK3 knockout mice.We describe a 3.65 fold increase in the expression of MKK3 in CD8+ T lymphocytes obtained from subjects with asthma compared with healthy subjects using a proteomic approach which we have confirmed in CD8+, but not in CD4+ T lymphocytes or human bronchial epithelial cells from asthmatic patients using a Western blot technique. In wild type mice, bacterial lipopolysaccharide (LPS) caused a significant increase in MKK3 expression and significantly reduced airway neutrophilia in MKK3−/− mice (median, 25, 75% percentile; wild/LPS; 5.3 (0.7–9.9) × 105 cells/mL vs MKK3−/−/LPS; 0 (0–1.9) × 105 cells/mL, P < 0.05). In contrast, eosinophilia in sensitized wild type mice challenged with allergen (0.5 (0.16–0.65) × 105 cells/mL) was significantly increased in MKK3−/− mice (2.2 (0.9–3.5) × 105 cells/mL, P < 0.05).Our results suggest that asthma is associated with MKK3 over-expression in CD8+ cells. We have also demonstrated that MKK3 may be critical for airway neutrophilia, but not eosinophilia, suggesting that this may be a target worthy of further consideration in the context of diseases associated with neutrophil activation such as severe asthma and COPD.

Impact Of BMI On Ozone-Induced IL-1β In The Airways Of Human Volunteers

Ozone is a ubiquitous environmental pollutant that induces an airway inflammatory response characterized by decrements in spirometry, airway neutrophilia, and pro-inflammatory cytokine production. Increased BMI has been shown to influence ozone-induced decrements in spirometry in otherwise healthy human volunteers. We sought to investigate if BMI influences ozone-induced cytokine production in the airways of human volunteers.

Supplementation with γ-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats

Supplementation with γ-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats

Altered sputum granzyme B and granzyme B/proteinase inhibitor‐9 in patients with non‐eosinophilic asthma

The non-eosinophilic phenotype of asthma (NEA) is associated with chronic airway inflammation and airway neutrophilia. An accumulation of apoptotic airway epithelial cells, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, with the potential for inflammation of surrounding tissues. Apoptosis may occur via the T cell granzyme B pathway. The role of granzyme B in NEA is not known. The aim of this study was to investigate production of granzyme B and its inhibitor proteinase inhibitor (PI)-9 by T cells from induced sputum and compare expression between eosinophilic, NEA and healthy controls. We investigated T cell intracellular granzyme B and its inhibitor, PI-9, in sputum from healthy control subjects (n = 10), and patients with NEA (n = 22) or eosinophilic asthma (EA) (n = 15) using flow cytometry. Granzyme B expression and the ratio of granzyme B to PI-9 positive cells were highest in those with NEA for both CD3+ and CD4+ T cells. The expression of granzyme B was not statistically different between patients with NEA and EA; however, the ratio of granzyme B to PI-9 positive cells for CD3+ T cells was significantly higher in those with NEA compared with EA. Induced sputum provides a non-invasive tool for investigating T cell cytotoxic mediators in the various asthma subtypes. Granzyme B expression is increased in NEA and the contribution of granzyme B to chronic inflammation requires further study.