Bronchoalveolar Lavage Macrophages Research Articles

Structural modifications of the antimicrobial peptide ubiquicidin for pulmonary imaging of bacteria in the alveolar space

BackgroundThe direct visualisation of bacteria in the distal lung would increase the spatiotemporal understanding of pulmonary infection and be a powerful tool to stratify patients with suspected pneumonia. In critically ill patients, the diagnostic dilemma of pulmonary opacities leads to overprescribing of antimicrobial agents while waiting for culture results from bronchoalveolar lavage. Ubiquicidin (UBI) is an innate cytosolic antimicrobial peptide with a twelve aminoacid portion (UBI29–41) that specifically binds bacteria. We aimed to modify the chemical structure of UBI29–41 so that in-situ bacterial imaging with optical endomicroscopy (OEM) could be achieved. MethodsUBI29-41 compounds were labelled with the environmentally sensitive fluorophore NBD (NBD-UBI), with incorporation of synthetic aminoacids (NBD-UBInma) and alteration of the secondary structure of the native peptide on a dendrimeric scaffold (NBD-UBIdend). These compounds were assessed in vitro and delivered endobronchially in an ex-vivo sheep lung model and then OEM applied to allow alveolar imaging. The NBD-UBIdend–OEM platform was also evaluated in explanted whole cystic fibrosis lungs. FindingsNBD-UBI selectively labelled bacteria over mammalian cells but remained susceptible to proteolytic degradation and poor affinity. NBD-UBInma improved stability but not affinity. NBD-UBIdend remained structurally stable and exhibited high affinity for bacteria in vitro. It retained bacterial selectivity over mononuclear cells (p=0·0015), neutrophils (p=0·0034), bronchoalveolar lavage macrophages (p=0·0169), and labelled Escherichia coli (p=0·0035), Klebsiella pneumoniae (p=0·0003), Pseudomonas aeruginosa (p=0·0009), and meticillin-sensitive Staphylococcus aureus (p<0·0001) segments in situ in an ex-vivo sheep lung model (area under the curve 0·926, SE 0·056; p=0·000128) with a relevant limit of detection of 1 × 105 colony-forming units per mL on lavage. NBD-UBIdend also detected bacteria in situ in ex-vivo explanted human cystic fibrosis lungs (p=0·0027 compared with peptide and fluorophore control segments). InterpretationWe describe an OEM strategy that can immediately detect bacteria in size-relevant preclinical models, with crucial requirements for pulmonary molecular imaging of peptide stability and affinity. This method has the potential to stratify pulmonary opacities in the intensive care unit when pneumonia is suspected and offers the substantial advantage of real-time detection, therefore allowing immediate decision making about antimicrobial treatment. This imaging strategy is now undergoing first-in-man translation. FundingWellcome Trust, Department of Health, Engineering and Physical Sciences Research Council.

The MIF Antagonist ISO-1 Attenuates Corticosteroid-Insensitive Inflammation and Airways Hyperresponsiveness in an Ozone-Induced Model of COPD.

IntroductionMacrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.MethodsSputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.ResultsMIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.ConclusionMIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.

Kinin B1 receptor antagonist BI113823 reduces allergen-induced airway inflammation and mucus secretion in mice

Kinin B1 receptors are implicated in asthmatic airway inflammation. Here we tested this hypothesis by examining the anti-inflammatory effects of BI113823, a novel non-peptide orally active kinin B1 receptor antagonist in mice sensitized to ovalbumin (OVA). Male Balb-c mice were randomly assigned to four study groups: (1) control, (2) OVA+vehicle, (3) OVA+BI113823, (4) OVA+dexamethasone. Mice were sensitized intraperitoneally with 75μg ovalbumin on days 1 and 8. On days 15–17, mice were challenged intranasally with 50μg of ovalbumin. Mice received vehicle, BI113823, or dexamethasone (positive control) on days 16–18. On day 19, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immuno-histological analysis. Compared to controls treatment with BI113823 significantly reduced the numbers of BAL eosinophils, macrophages, neutrophils and lymphocytes by 58.3%, 61.1%, 66.4% and 56.0%, respectively. Mice treated with dexamethasone showed similar reductions in BAL cells. Treatment with BI113823 and dexamethasone also significantly reduced total protein content, IgE, TNF-α and IL-1β in lavage fluid, reduced myeloperoxidase activity, mucus secretion in lung tissues, and reduced the expression of B1 receptors, matrix metalloproteinase (MMP)-2 and cyclooxygenase (COX)-2 compared to vehicle-treated mice. Only BI113823 reduced MMP-9 and inducible nitric oxide synthase (iNOS). BI113823 effectively reduced OVA-induced inflammatory cell, mediator and signaling pathways equal to or greater than that seen with steroids in a mouse asthma model. BI113823 might be useful in modulating inflammation in asthma.

Protective Role of Proton-Sensing TDAG8 in Lipopolysaccharide-Induced Acute Lung Injury.

Acute lung injury is characterized by the infiltration of neutrophils into lungs and the subsequent impairment of lung function. Here we explored the role of TDAG8 in lung injury induced by lipopolysaccharide (LPS) administrated intratracheally. In this model, cytokines and chemokines released from resident macrophages are shown to cause neutrophilic inflammation in the lungs. We found that LPS treatment increased TDAG8 expression in the lungs and confirmed its expression in resident macrophages in bronchoalveolar lavage (BAL) fluids. LPS administration remarkably increased neutrophil accumulation without appreciable change in the resident macrophages, which was associated with increased penetration of blood proteins into BAL fluids, interstitial accumulation of inflammatory cells, and damage of the alveolar architecture. The LPS-induced neutrophil accumulation and the associated lung damage were enhanced in TDAG8-deficient mice as compared with those in wild-type mice. LPS also increased several mRNA and protein expressions of inflammatory cytokines and chemokines in the lungs or BAL fluids. Among these inflammatory mediators, mRNA and protein expression of KC (also known as CXCL1), a chemokine of neutrophils, were significantly enhanced by TDAG8 deficiency. We conclude that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production.

RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema.

BackgroundChondroitin sulfate proteoglycans are an important mediators in inflammation and leukocyte trafficking. However, their roles in pulmonary emphysema have not been explored.In a murine model of elastase-induced pulmonary emphysema, we found increased carbohydrate sulfotransferase 3 (CHST3), a specific enzyme that synthesizes chondroitin 6-sulfate proteoglycan (C6SPG). To elucidate the role of C6SPG, we investigated the effect of small interfering RNA (siRNA) targeting CHST3 that inhibits C6SPG-synthesis on the pathogenesis of pulmonary emphysema.MethodsMice were intraperitoneally injected with CHST3 siRNA or negative control siRNA on day0 and 7 after intratracheal instillation of elastase. Histology, respiratory function, glycosaminoglycans (GAGs) content, bronchoalveolar lavage (BAL), elastin staining and gene expressions of tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)-9 mRNA were evaluated on day7 and/or day21.ResultsCHST3 mRNA increased at day 7 and decreased thereafter in lung. CHST3 siRNA successfully inhibited the expression of CHST3 mRNA throughout the study and this was associated with significant reduction of GAGs and C6SPG. Airway destruction and respiratory function were improved by the treatment with CHST3 siRNA. CHST3 siRNA reduced the number of macrophages both in BAL and lung parenchyma and also suppressed the increased expressions of TNF-α and MMP-9 mRNA. Futhermore, CHST3 siRNA improved the reduction of the elastin in the alveolar walls.ConclusionsCHST3 siRNA diminishes accumulation of excessive macrophages and the mediators, leading to accelerate the functional recovery from airway damage by repair of the elastin network associated with pulmonary emphysema.

γδ T Cells Are Required for M2 Macrophage Polarization and Resolution of Ozone-Induced Pulmonary Inflammation in Mice.

We examined the role of γδ T cells in the induction of alternatively activated M2 macrophages and the resolution of inflammation after ozone exposure. Wildtype (WT) mice and mice deficient in γδ T cells (TCRδ-/- mice) were exposed to air or to ozone (0.3 ppm for up to 72h) and euthanized immediately or 1, 3, or 5 days after cessation of exposure. In WT mice, M2 macrophages accumulated in the lungs over the course of ozone exposure. Pulmonary mRNA abundance of the M2 genes, Arg1, Retnla, and Clec10a, also increased after ozone. In contrast, no evidence of M2 polarization was observed in TCRδ-/- mice. WT but not TCRδ-/- mice expressed the M2c polarizing cytokine, IL-17A, after ozone exposure and WT mice treated with an IL-17A neutralizing antibody exhibited attenuated ozone-induced M2 gene expression. In WT mice, ozone-induced increases in bronchoalveolar lavage neutrophils and macrophages resolved quickly after cessation of ozone exposure returning to air exposed levels within 3 days. However, lack of M2 macrophages in TCRδ-/- mice was associated with delayed clearance of inflammatory cells after cessation of ozone and increased accumulation of apoptotic macrophages in the lungs. Delayed restoration of normal lung architecture was also observed in TCRδ-/- mice. In summary, our data indicate that γδ T cells are required for the resolution of ozone-induced inflammation, likely because γδ T cells, through their secretion of IL-17A, contribute to changes in macrophage polarization that promote clearance of apoptotic cells.

Innate and ozone-induced airway hyperresponsiveness in obese mice: role of TNF-α.

Innate airway hyperresponsiveness (AHR) and augmented responses to ozone, an asthma trigger, are characteristics of obese mice. Systemic inflammation, a condition of increased circulating concentrations of inflammatory moieties, occurs in obesity. We hypothesized that TNF-α, via its effects as a master effector of this systemic inflammation, regulates innate AHR and augmented responses to ozone in obese mice. Therefore, we examined pulmonary inflammation and airway responsiveness in unexposed or ozone-exposed (2 ppm for 3 h) lean wild-type and obese Cpe(fat) mice that were TNF-α sufficient or deficient. Cpe(fat) mice lack carboxypeptidase E, which regulates satiety. Compared with wild type, Cpe(fat) mice had elevated serum IL-17A, G-CSF, KC, MCP-1, IL-9, MIG, and leptin, indicating systemic inflammation. Despite reductions in most of these moieties in TNF-α-deficient vs. -sufficient Cpe(fat) mice, we observed no substantial difference in airway responsiveness in these two groups of mice. Ozone-induced increases in bronchoalveolar lavage (BAL) neutrophils and macrophages were lower, but ozone-induced AHR and increases in BAL hyaluronan, osteopontin, IL-13, and protein carbonyls, a marker of oxidative stress, were augmented in TNF-α-deficient vs. -sufficient Cpe(fat) mice. Our data indicate that TNF-α has an important role in promoting the systemic inflammation but not the innate AHR of obesity, suggesting that the systemic inflammation of obesity is not the major driver of this AHR. TNF-α is required for the augmented effects of acute ozone exposure on pulmonary inflammatory cell recruitment in obese mice, whereas TNF-α protects against ozone-induced AHR in obese mice, possibly by suppressing ozone-induced oxidative stress.

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

Regulation of ozone-induced lung inflammation and injury by the β-galactoside-binding lectin galectin-3

Macrophages play a dual role in ozone toxicity, contributing to both pro- and anti-inflammatory processes. Galectin-3 (Gal-3) is a lectin known to regulate macrophage activity. Herein, we analyzed the role of Gal-3 in the response of lung macrophages to ozone. Bronchoalveolar lavage (BAL) and lung tissue were collected 24–72h after exposure (3h) of WT and Gal-3-/- mice to air or 0.8ppm ozone. In WT mice, ozone inhalation resulted in increased numbers of proinflammatory (Gal-3+, iNOS+) and anti-inflammatory (MR-1+) macrophages in the lungs. While accumulation of iNOS+ macrophages was attenuated in Gal-3-/- mice, increased numbers of enlarged MR-1+ macrophages were noted. This correlated with increased numbers of macrophages in BAL. Flow cytometric analysis showed that these cells were CD11b+ and consisted mainly (>97%) of mature (F4/80+CD11c+) proinflammatory (Ly6GLy6Chi) and anti-inflammatory (Ly6GLy6Clo) macrophages. Increases in both macrophage subpopulations were observed following ozone inhalation. Loss of Gal-3 resulted in a decrease in Ly6Chi macrophages, with no effect on Ly6Clo macrophages. CD11b+Ly6G+Ly6C+ granulocytic (G) and monocytic (M) myeloid derived suppressor cells (MDSC) were also identified in the lung after ozone. In Gal-3-/- mice, the response of G-MDSC to ozone was attenuated, while the response of M-MDSC was heightened. Changes in inflammatory cell populations in the lung of ozone treated Gal-3-/- mice were correlated with reduced tissue injury as measured by cytochrome b5 expression. These data demonstrate that Gal-3 plays a role in promoting proinflammatory macrophage accumulation and toxicity in the lung following ozone exposure.

Airway mucus obstruction triggers macrophage activation and matrix metalloproteinase 12-dependent emphysema.

Whereas cigarette smoking remains the main risk factor for emphysema, recent studies in β-epithelial Na(+) channel-transgenic (βENaC-Tg) mice demonstrated that airway surface dehydration, a key pathophysiological mechanism in cystic fibrosis (CF), caused emphysema in the absence of cigarette smoke exposure. However, the underlying mechanisms remain unknown. The aim of this study was to elucidate mechanisms of emphysema formation triggered by airway surface dehydration. We therefore used expression profiling, genetic and pharmacological inhibition, Foerster resonance energy transfer (FRET)-based activity assays, and genetic association studies to identify and validate emphysema candidate genes in βENaC-Tg mice and patients with CF. We identified matrix metalloproteinase 12 (Mmp12) as a highly up-regulated gene in lungs from βENaC-Tg mice, and demonstrate that elevated Mmp12 expression was associated with progressive emphysema formation, which was reduced by genetic deletion and pharmacological inhibition of MMP12 in vivo. By using FRET reporters, we show that MMP12 activity was elevated on the surface of airway macrophages in bronchoalveolar lavage from βENaC-Tg mice and patients with CF. Furthermore, we demonstrate that a functional polymorphism in MMP12 (rs2276109) was associated with severity of lung disease in CF. Our results suggest that MMP12 released by macrophages activated on dehydrated airway surfaces may play an important role in emphysema formation in the absence of cigarette smoke exposure, and may serve as a therapeutic target in CF and potentially other chronic lung diseases associated with airway mucus dehydration and obstruction.

Novosphingobium and its potential role in chronic obstructive pulmonary diseases: insights from microbiome studies.

Bacterial infection of lung airways underlies some of the main complications of COPD, significantly impacting disease progression and outcome. Colonization by bacteria may further synergize, amplify, or trigger pathways of tissue damage started by cigarette smoke, contributing to the characteristic airway inflammation and alveolar destruction of COPD. We sought to elucidate the presence and types of lung bacterial populations in different stages of COPD, aimed at revealing important insights into the pathobiology of the disease. Sequencing of the bacterial small subunit ribosomal RNA gene in 55 well-characterized clinical lung samples, revealed the presence of Novosphingobium spp. (>2% abundance) in lungs of patients with GOLD 3-GOLD 4 COPD, cystic fibrosis and a subset of control individuals. Novosphingobium-specific quantitative PCR was concordant with the sequence data and high levels of Novosphingobium spp. were quantifiable in advanced COPD, but not from other disease stages. Using a mouse model of subacute lung injury due to inhalation of cigarette smoke, bronchoalveolar lavage neutrophil and macrophage counts were significantly higher in mice challenged intratracheally with N. panipatense compared to control mice (p<0.01). Frequencies of neutrophils and macrophages in lung tissue were increased in mice challenged with N. panipatense at room air compared to controls. However, we did not observe an interaction between N. panipatense and subacute cigarette smoke exposure in the mouse. In conclusion, Novosphingobium spp. are present in more severe COPD disease, and increase inflammation in a mouse model of smoke exposure.

Alveolar and intraparenchymal proteasome in sarcoidosis

In sarcoidosis, an antigen specific immune response is a putative mechanism, resulting in granulomatous inflammation. Since the proteasome is involved in antigen presentation, alterations in the alveolar and parenchymal proteasomal system may be a feature of sarcoidosis. To explore the role of proteasomes and immunoproteasomes in sarcoidosis. Total proteasome concentration and activity was assessed in bronchoalveolar lavage (BAL) supernatant obtained from sarcoidosis patients (n=67) and healthy controls (n=18) using ELISA and cleavage of specific fluorogenic substrates (±epoxomicin), respectively. Immunohistochemistry of lung tissue sections and immunocytochemistry of BAL macrophages for immunoproteasome was performed in sarcoidosis patients and controls. Proteasome was present in BAL supernatants of all sarcoidosis patients. In sarcoidosis, abundant immunoproteasome staining was seen in pneumocytes type II and granulomas. Total proteasome concentration was greater in active sarcoidosis, stages II (101ng/ml±79; p=0.009) and III (119ng/ml±66; p=0.012), than in inactive sarcoidosis or in healthy controls (35ng/ml±34). In the absence of epoxomicin, all fluorogenic substrates were hydrolyzed by BAL supernatant of sarcoidosis patients and controls. Patients with active sarcoidosis but not healthy controls demonstrate immunoproteasome in the lung tissue and in granulomas. Thus, the putative immune response in sarcoidosis may be mediated or sustained by the proteasomal system.

Effects of acute inhalation of aerosols generated during resistance spot welding with mild-steel on pulmonary, vascular and immune responses in rats

Spot welding is used in the automotive and aircraft industries, where high-speed, repetitive welding is needed to join thin sections of metal. Epoxy adhesives are applied as sealers to the metal seams. Pulmonary function abnormalities and airway irritation have been reported in spot welders, but no animal toxicology studies exist. Therefore, the goal of this study was to investigate vascular, immune and lung toxicity measures after exposure to these metal fumes in an animal model. Male Sprague-Dawley rats were exposed by inhalation to 25 mg/m3 to either mild-steel spot welding aerosols with sparking (high metal, HM) or without sparking (low metal, LM) for 4 h/d for 3, 8 and 13 d. Shams were exposed to filtered air. Bronchoalveolar lavage (BAL), lung gene expression and ex vivo BAL cell challenge were performed to assess lung toxicity. Lung resistance (RL) was evaluated before and after challenge with inhaled methacholine (MCh). Functional assessment of the vascular endothelium in isolated rat tail arteries and leukocyte differentiation in the spleen and lymph nodes via flow cytometry was also done. Immediately after exposure, baseline RL was significantly elevated in the LM spot welding aerosols, but returned to control level by 24 h postexposure. Airway reactivity to MCh was unaffected. Lung inflammation and cytotoxicity were mild and transient. Lung epithelial permeability was significantly increased after 3 and 8 d, but not after 13 d of exposure to the HM aerosol. HM aerosols also caused vascular endothelial dysfunction and increased CD4+, CD8+ and B cells in the spleen. Only LM aerosols caused increased IL-6 and MCP-1 levels compared with sham after ex vivo LPS stimulation in BAL macrophages. Acute inhalation of mild-steel spot welding fumes at occupationally relevant concentrations may act as an irritant as evidenced by the increased RL and result in endothelial dysfunction, but otherwise had minor effects on the lung.

Nanoparticle uptake by airway phagocytes after fungal spore challenge in murine allergic asthma and chronic bronchitis.

BackgroundIn healthy lungs, deposited micrometer-sized particles are efficiently phagocytosed by macrophages present on airway surfaces; however, uptake of nanoparticles (NP) by macrophages appears less effective and is largely unstudied in lung disease. Using mouse models of allergic asthma and chronic obstructive pulmonary disease (COPD), we investigated NP uptake after challenge with common biogenic ambient air microparticles.MethodsBronchoalveolar lavage (BAL) cells from diseased mice (allergic asthma: ovalbumin [OVA] sensitized and COPD: Scnn1b-transgenic [Tg]) and their respective healthy controls were exposed ex vivo first to 3-μm fungal spores of Calvatia excipuliformis and then to 20-nm gold (Au) NP. Electron microscopic imaging was performed and NP uptake was assessed by quantitative morphometry.ResultsMacrophages from diseased mice were significantly larger compared to controls in OVA-allergic versus sham controls and in Scnn1b-Tg versus wild type (WT) mice. The percentage of macrophages containing AuNP tended to be lower in Scnn1b-Tg than in WT mice. In all animal groups, fungal spores were localized in macrophage phagosomes, the membrane tightly surrounding the spore, whilst AuNP were found in vesicles largely exceeding NP size, co-localized in spore phagosomes and occasionally, in the cytoplasm. AuNP in vesicles were located close to the membrane. In BAL from OVA-allergic mice, 13.9 ± 8.3% of all eosinophils contained AuNP in vesicles exceeding NP size and close to the membrane.ConclusionsOverall, AuNP uptake by BAL macrophages occurred mainly by co-uptake together with other material, including micrometer-sized ambient air particles like fungal spores. The lower percentage of NP containing macrophages in BAL from Scnn1b-Tg mice points to a change in the macrophage population from a highly to a less phagocytic phenotype. This likely contributes to inefficient macrophage clearance of NP in lung disease. Finally, the AuNP containing eosinophils in OVA-allergic mice show that other inflammatory cells present on airway surfaces may substantially contribute to NP uptake.

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A+ CD45+ cells in the lungs and these effects were abolished in TCRδ−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A+ CD45+ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A+ γδ T cells. Il17a mRNA and IL-17A+ γδ T cells were also lower in obese Cpefat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A+ γδ T cells to the lung.

Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages

Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages

The Effects of Airway Microbiome on Corticosteroid Responsiveness in Asthma

The role of airway microbiome in corticosteroid response in asthma is unknown. To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids. 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation. Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-β-associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids. A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce corticosteroid resistance. TAK1 inhibition restored cellular sensitivity to corticosteroids.

The Bronchial Microbiome and Asthma Phenotypes

The Bronchial Microbiome and Asthma Phenotypes

S112 HDAC activity in macrophages in experimental rhinovirus infection in COPD

Introduction and ObjectivesAcute exacerbations are a major cause of morbidity and mortality in COPD and current treatments are not very effective. Histone deacetylase 2 (HDAC2) is deficient in stable COPD...

1,8-Cineol Attenuates LPS-Induced Acute Pulmonary Inflammation in Mice

Eucalyptol, also known as 1,8-cineol, is a monoterpene and has been shown to exert anti-inflammatory and antioxidant effect. It is traditionally used to treat respiratory disorders due to its secretolytic properties. In the present study, we evaluated the effect of 1,8-cineol on pulmonary inflammation in a mouse model of acute lung injury. We found that 1,8-cineol significantly decreased the level of TNF-α and IL-1β, and increased the level of IL-10 in lung tissues after acute lung injury induced by lipopolysaccharide (LPS). It also reduced the expression of nuclear factor kappa B (NF-κB) p65 and toll-like receptor 4 (TLR4), and myeloperoxidase activity in lung tissues. In addition, 1,8-cineol reduced the amounts of inflammatory cells in bronchoalveolar lavage fluid (BALF), including neutrophils and macrophages, and significantly decreased the protein content in BALF and the lung wet/dry weight (W/D) ratio. Its effect on LPS-induced pulmonary inflammation was associated with suppression of TLR4 and NF-κB expressions. Our results provide evidence that 1,8-cineol inhibits acute pulmonary inflammation, indicating its potential for the treatment of acute lung injury.