Cell-free Bronchoalveolar Lavage Fluid Research Articles

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFβ1 and IL-27

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFβ and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFβ1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFβ1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFβ1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Interplay between the Lung Microbiome, Pulmonary Immunity and Viral Reservoirs in People Living with HIV under Antiretroviral Therapy.

Pulmonary dysbiosis may predispose people living with HIV (PLWH) to chronic lung disease. Herein, we assessed whether intrapulmonary HIV reservoir size and immune disruption are associated with reduced bacterial lung diversity in PLWH. Bacterial DNA was extracted and PCR-amplified from cell-free bronchoalveolar lavage (BAL) fluid from 28 PLWH and 9 HIV-negative controls. Amplicon sequence variant (ASV) relative abundances and taxonomic identities were analyzed using joint species distribution modeling. HIV-DNA was quantified from blood and pulmonary CD4+ T-cells using ultra-sensitive qPCR. Immunophenotyping of BAL T-cells was performed using flow cytometry. Lung microbiome diversity was lower in smokers than non-smokers and microbiome composition was more variable in PLWH than HIV-negative individuals. Frequencies of effector memory BAL CD4+ and CD8+ T-cells positively correlated with abundance of several bacterial families while frequencies of BAL activated CD4+ T-cells negatively correlated with abundance of most lung bacterial families. Higher HIV-DNA levels in blood, but not in BAL, as well as frequencies of senescent CD4+ T-cells were associated with reduced bacterial diversity. These findings suggest that HIV infection may weaken the relationship between the lung microbiome and smoking status. Viral reservoir and immune activation levels may impact the lung microbiome, predisposing PLWH to pulmonary comorbidities.

TGF-\u03b21 increases viral burden and promotes HIV-1 latency in primary differentiated human bronchial epithelial cells

Combination antiretroviral therapy (cART) has increased the life expectancy of HIV patients. However, the incidence of non-AIDS associated lung comorbidities, such as COPD and asthma, and that of opportunistic lung infections have become more common among this population. HIV proteins secreted by the anatomical HIV reservoirs can have both autocrine and paracrine effects contributing to the HIV-associated comorbidities. HIV has been recovered from cell-free bronchoalveolar lavage fluid, alveolar macrophages, and intrapulmonary lymphocytes. We have recently shown that ex-vivo cultured primary bronchial epithelial cells and the bronchial brushings from human subjects express canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. Together these studies suggest that the lung tissue can serve as an important reservoir for HIV. In this report, we show that TGF-β1 promotes HIV latency by upregulating a transcriptional repressor BLIMP-1. Furthermore, we identify miR-9-5p as an important intermediate in TGF-β-mediated BLIMP-1 upregulation and consequent HIV latency. The transcriptionally suppressed HIV can be reactivated by common latency reactivating agents. Together our data suggest that in patients with chronic airway diseases, TGF-β can elevate the HIV viral reservoir load that could further exacerbate the HIV associated lung comorbidities.

Role of Mouse Cytochrome P450 Enzymes of the Cyp2abfgs Subfamilies in the Induction of Lung Inflammation by Cigarette Smoke Exposure.

Many constituents of tobacco smoke (TS) require bioactivation to exert toxic effects; however, few studies have examined the role of bioactivation enzymes in the adverse effects of TS exposure. This knowledge gap is a major source of uncertainty for risk assessment and chemoprevention efforts. Our aim is to test the hypothesis that cytochrome P450 (P450) enzyme-mediated bioactivation is essential to the development of TS exposure-induced lung toxicity, by determining the contributions of P450 enzymes in the mouse Cyp2abfgs gene subfamilies to environmental tobacco smoke (ETS)-induced lung inflammation. Adult female wildtype (WT) and Cyp2abfgs-null mice (both on C57BL/6J background) were exposed to filtered air or ETS, intermittently, for 1 or 2 weeks. Lung inflammation was assessed by quantification of inflammatory cells, cytokines, chemokines, and proteins in bronchoalveolar lavage fluid (BALF) and histopathological analysis. Glutathione (GSH) conjugates of 2 ETS constituents, naphthalene (NA), and 3-methylindole (3MI), were measured in mice exposed to ETS for 4 h. Persistent macrophagic and neutrophilic lung inflammation was observed in ETS-exposed WT mice; the extent of which was significantly reduced in ETS-exposed Cyp2abfgs-null mice. Levels of proinflammatory cytokines and chemokines, along with the total protein concentration, were increased in cell-free BALF from ETS-exposed WT mice, but not Cyp2abfgs-null mice. Additionally, GSH conjugates of NA and 3MI were detected in the lungs of WT, but not Cyp2abfgs-null, mice following ETS exposure. These results provide the first in vivo evidence that the mouse Cyp2abfgs gene cluster plays an important role in ETS-induced lung inflammation.

Modeling the pasture-associated severe equine asthma bronchoalveolar lavage fluid proteome identifies molecular events mediating neutrophilic airway inflammation.

Background: Pasture-associated severe equine asthma is a warm season, environmentally-induced respiratory disease characterized by reversible airway obstruction, persistent and non-specific airway hyper-responsiveness, and chronic neutrophilic airway inflammation. During seasonal exacerbation, signs vary from mild to life-threatening episodes of wheezing, coughing, and chronic debilitating labored breathing.Purpose: In human asthma, neutrophilic airway inflammation is associated with more severe and steroid-refractory asthma phenotypes, highlighting a need to decipher the mechanistic basis of this disease characteristic. We hypothesize that the collective biological activities of proteins in bronchoalveolar lavage fluid (BALF) of horses with pasture-associated severe asthma predict changes in neutrophil functions that contribute to airway neutrophilic inflammation.Methods: Using shotgun proteomics, we identified 1,003 unique proteins in cell-free BALF from six horses experiencing asthma exacerbation and six control herdmates. Contributions of each protein to ten neutrophil functions were modeled using manual biocuration to determine each protein’s net effect on the respective neutrophil functions.Results: A total of 417 proteins were unique to asthmatic horses, 472 proteins were unique to control horses (p<0.05), and 114 proteins were common in both groups. Proteins whose biological activities are responsible for increasing neutrophil migration, chemotaxis, cell spreading, transmigration, and infiltration, which would collectively bring neutrophils to airways, were over-represented in the BALF of asthmatic relative to control horses. By contrast, proteins whose biological activities support neutrophil activation, adhesion, phagocytosis, respiratory burst, and apoptosis, which would collectively shorten neutrophil lifespan, were under-represented in BALF of asthmatic relative to control horses. Interaction networks generated using Ingenuity® Pathways Analysis further support the results of our biocuration.Conclusion: Congruent with our hypothesis, the collective biological functions represented in differentially expressed proteins of BALF from horses with pasture-associated severe asthma support neutrophilic airway inflammation. This illustrates the utility of systems modeling to organize functional genomics data in a manner that characterizes complex molecular events associated with clinically relevant disease.

Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing.

Pathogens causing pneumonia utilize the complement regulator vitronectin to evade complement-mediated killing. Although vitronectin is associated with several chronic lung diseases, the role of bronchoalveolar vitronectin in pneumonia has not been studied. This study sought to reveal the involvement of vitronectin in the bronchoalveolar space during pneumonia, to assess the effect of outer membrane vesicles and endotoxin on vitronectin release, and to determine whether bacterial pathogens utilize pulmonary vitronectin for evasion. Vitronectin was analyzed in cell-free bronchoalveolar lavage fluid harvested from patients with pneumonia (n = 8) and from healthy volunteers after subsegmental endotoxin instillation (n = 13). Vitronectin binding by Pseudomonas aeruginosa and Haemophilus influenzae was analyzed, and subsequent complement evasion was assessed by serum challenge. The effects of outer membrane vesicles on vitronectin production in mouse lungs and human type II alveolar epithelial cells (A549) were determined. We detected increased vitronectin concentrations in lavage fluid during pneumonia (p = 0.0063) and after bronchial endotoxin challenge (p = 0.016). The capture of vitronectin by bacteria significantly reduced complement-mediated lysis. Following challenge with vesicles, vitronectin was detected in mouse bronchoalveolar space, and mouse alveolar epithelial cells in vivo as well as A549 cells in vitro contained increased levels of vitronectin. Taken together, outer membrane vesicles and endotoxin from Gram-negative bacteria induce vitronectin, which is released into the bronchoalveolar space, and used for evasion of complement-mediated clearance.

Endotoxin Exposure Increases LL-37 - but Not Calprotectin - in Healthy Human Airways

Rationale: The antimicrobial peptides (AMPs) LL-37 and calprotectin are important players in the innate immunity of human airways. In patients with diseases characterized by bacterial colonization, the airway concentrations of these AMPs are increased. Less is known about their presence and release patterns in healthy humans. Our aim was to determine whether LL-37 and calprotectin are released after the activation of the innate immune response in the peripheral airways. Methods: Healthy volunteers underwent exposure to endotoxin and vehicle in contralateral segment bronchi. After 12 or 24 h, samples of bronchoalveolar lavage fluid (BALf) were collected bilaterally from exposed segments. Cell and AMP concentrations were assessed, as were the pro-form and active form of LL-37. Results: Both LL-37 and calprotectin were detected in cell-free BALf from both endotoxin- and vehicle-exposed segments. The concentrations of precursor and active LL-37 and neutrophils were significantly higher in endotoxin-exposed segments after 12 and 24 h, and the concentrations of LL-37 and neutrophils correlated positively. The concentrations of calprotectin were not markedly affected by exposure to endotoxin. Conclusions: Local endotoxin exposure elicits the release and activation of LL-37 but not calprotectin in healthy human peripheral airways, suggesting an inducible involvement of LL-37 in the local innate immune response.

Impact of tobacco smoking on cytokine signaling via interleukin-17A in the peripheral airways.

There is excessive accumulation of neutrophils in the airways in chronic obstructive pulmonary disease (COPD) but the underlying mechanisms remain poorly understood. It is known that extracellular cytokine signaling via interleukin (IL)-17A contributes to neutrophil accumulation in the airways but nothing is known about the impact of tobacco smoking on extracellular signaling via IL-17A. Here, we characterized the impact of tobacco smoking on extracellular cytokine signaling via IL-17A in the peripheral airways in long-term smokers with and without COPD and in occasional smokers before and after short-term exposure to tobacco smoke. We quantified concentrations of IL-17A protein in cell-free bronchoalveolar lavage (BAL) fluid samples (Immuno-quantitative PCR) and cytotoxic T-cells (immunoreactivity for CD8+ and CD3+) in bronchial biopsies. Matrix metalloproteinase-8 and human beta defensin 2 proteins were also quantified (enzyme-linked immunosorbent assay) in the BAL samples. The concentrations of IL-17A in BAL fluid were higher in long-term smokers without COPD compared with nonsmoking healthy controls, whereas those with COPD did not differ significantly from either of the other groups. Short-term exposure to tobacco smoke did not induce sustained alterations in these concentrations in occasional smokers. Long-term smokers displayed higher concentrations of IL-17A than did occasional smokers. Moreover, these concentrations correlated with CD8+ and CD3+ cells in biopsies among long-term smokers with COPD. In healthy nonsmokers, BAL concentrations of matrix metalloproteinase-8 and IL-17A correlated, whereas this was not the case in the pooled group of long-term smokers with and without COPD. In contrast, BAL concentrations of human beta defensin 2 and IL-17A correlated in all study groups. This study implies that long-term but not short-term exposure to tobacco smoke increases extracellular cytokine signaling via IL-17A in the peripheral airways. In the smokers with COPD, this signaling may involve cytotoxic T-cells. Long-term exposure to tobacco smoke leads to a disturbed association of extracellular IL-17A signaling and matrix metalloproteinase-8, of potential importance for the coordination of antibacterial activity.

On the contribution of the phagocytosis and the solubilization to the iron oxide nanoparticles retention in and elimination from lungs under long-term inhalation exposure.

The aim of our study was to test a hypothesis according to which the pulmonary clearance vs. retention of metal oxide nanoparticles (NPs) is controlled not only by physiological mechanisms but also by their solubilization which in some cases may even prevail. Airborne Fe2O3 NPs with the mean diameter of 14±4nm produced by sparking from 99.99% pure iron rods were fed into a nose-only exposure tower. Rats were exposed to these NPs for 4h a day, 5days a week during 3, 6 or 10 months at the mean concentration of 1.14±0.01mg/m(3). NPs collected from the air exhausted from the exposure tower proved insoluble in water but dissolved markedly in the cell free broncho-alveolar lavage fluid supernatant and in the sterile bovine blood serum. The Fe2O3 content of the lungs and lung-associated lymph nodes was measured by the Electron Paramagnetic Resonance (EPR) spectroscopy. We found a relatively low but significant pulmonary accumulation of Fe2O3, gradually increasing with time. Besides, we obtained TEM-images of nanoparticles within alveolocytes and the myelin sheaths of brain fibers associated with ultrastructural damage. We have developed a multicompartmental system model describing the toxicokinetics of inhaled nanoparticles after their deposition in the lower airways as a process controlled by their (a) high ability to penetrate through the alveolar membrane; (b) active endocytosis; (c) in vivo dissolution. To conclude, both experimental data and the identification of the system model confirmed our initial hypothesis and demonstrated that, as concerns iron oxide NPs of the dimensions used, the dissolution-depending mechanisms proved to be dominant.

Extracellular cadmium in the bronchoalveolar space of long-term tobacco smokers with and without COPD and its association with inflammation.

Tobacco contains cadmium, and this metal has been attributed a causative role in pulmonary emphysema among smokers, although extracellular cadmium has not to date been quantified in the bronchoalveolar space of tobacco smokers with or without COPD. We determined whether cadmium is enhanced in the bronchoalveolar space of long-term tobacco smokers with or without COPD in vivo, its association with inflammation, and its effect on chemokine release in macrophage-like cells in vitro. Bronchoalveolar lavage (BAL), sputum, and blood samples were collected from current, long-term smokers with and without COPD and from healthy nonsmokers. Cadmium concentrations were determined in cell-free BAL fluid using inductively coupled plasma mass spectrometry. Blood monocyte-derived macrophages were exposed to cadmium chloride in vitro. Depending upon the type of sample, molecular markers of inflammation were quantified either as protein (enzyme-linked immunosorbent assay) or as mRNA (real-time polymerase chain reaction). Cadmium concentrations were markedly increased in cell-free BAL fluid of smokers compared to that of nonsmokers (n=19–29; P<0.001), irrespective of COPD. In these smokers, the measured cadmium displayed positive correlations with macrophage TNF-α mRNA in BAL, neutrophil and CD8+ cell concentrations in blood, and finally with IL-6, IL-8, and MMP-9 protein in sputum (n=10–20; P<0.05). The cadmium chloride exposure caused a concentration-dependent increase in extracellular IL-8 protein in monocyte-derived macrophages in vitro. In conclusion, extracellular cadmium is enhanced in the bronchoalveolar space of long-term smokers and displays pro-inflammatory features. Its pathogenic role in tobacco-induced disease deserves further evaluation.

New Insights into Molecular Mechanisms of Immune Complex-Induced Injury in Lung.

While the phlogistic activities of IgM or IgG immune complexes (ICs) have been well established as complement-activating agents and seem likely to play important roles in humans with vasculitis, certain types of glomerulonephritis as well as in a variety of autoimmune diseases, the predominant clinical strategies have involved the use of immunosuppressive or anti-inflammatory drugs. Over the past decade, new insights into molecular events developing during IC models in rodents have identified new phlogistic products that may be candidates for therapeutic blockade. Extracellular histones, located in the web-like structures of neutrophil extracellular traps, are released from complement-activated polymorphonuclear neutrophils (PMNs) downstream of IC deposition. Extracellular histones appear to be a new class of highly tissue-damaging products derived from complement-activated PMNs. Histones have also been discovered in cell-free broncho-alveolar lavage fluids from humans with acute respiratory distress syndrome (ARDS). Recent studies emphasize that in the setting of ARDS-like reactions in rodents, extracellular histones are released and are exceedingly proinflammatory, tissue damaging, and prothrombotic. Such studies suggest that in humans with ARDS, extracellular histones may represent therapeutic targets for blockade.

Metabolic hormones, apolipoproteins, adipokines, and cytokines in the alveolar lining fluid of healthy adults: compartmentalization and physiological correlates.

ObjectivesOur current understanding of hormone regulation in lung parenchyma is quite limited. We aimed to quantify a diverse array of biologically relevant protein mediators in alveolar lining fluid (ALF), compared to serum concentrations, and explore factors associated with protein compartmentalization on either side of the air-blood barrier.Research Design and MethodsParticipants were 24 healthy adult non-smoker volunteers without respiratory symptoms or significant medical conditions, with normal lung exams and office spirometry. Cell-free bronchoalveolar lavage fluid and serum were analyzed for 24 proteins (including enteric and metabolic hormones, apolipoproteins, adipokines, and cytokines) using a highly sensitive multiplex ELISA. Measurements were normalized to ALF concentrations. The ALF:serum concentration ratios were examined in relation to measures of protein size, hydrophobicity, charge, and to participant clinical and spirometric values.ResultsALF measurements from 24 individuals detected 19 proteins, including adiponectin, adipsin, apoA-I, apoA-II, apoB, apoC-II, apoC-III, apoE, C-reactive protein, ghrelin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon, insulin, leptin, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, resistin, and visfatin. C-peptide and serpin E1 were not detected in ALF for any individual, and IL-6, IL-10, and TNF-alpha were not detected in either ALF or serum for any individual. In general, ALF levels were similar or lower in concentration for most proteins compared to serum. However, ghrelin, resistin, insulin, visfatin and GLP-1 had ALF concentrations significantly higher compared to serum. Importantly, elevated ALF:serum ratios of ghrelin, visfatin and resistin correlated with protein net charge and isoelectric point, but not with molecular weight or hydrophobicity.ConclusionsBiologically relevant enteric and metabolic hormones, apolipoproteins, adipokines, and cytokines can be detected in the ALF of healthy individuals. For the proteins measured, charge may influence trafficking and compartmentalization to the alveolar airspace more than molecular weight or hydrophobicity. These data may have implications for homeostasis and drug delivery to the lung.

Pulmonary and Atherogenic Effects of Multi-Walled Carbon Nanotubes (MWCNT) in Apolipoprotein-E-Deficient Mice

Rapid growth in nanotechnology has raised concerns regarding adverse health effects due to human exposure to manufactured nanoparticles. Carbon nanotubes (CNT) are among the most extensively used nanoparticles. This study examined pulmonary and atherosclerotic effects of multiwalled CNT (MWCNT) in a mouse model of atherosclerosis. Female apolipoprotein E-deficient (apoE–/–) mice were exposed to 40 μg MWCNT, once each week for 16 consecutive weeks by pharyngeal aspiration. On d 1 after the last administration, tissues were extracted from half the group, while the remaining animals were sacrificed at d 7. Bronchoalveolar lavage (BAL) was performed to obtain BAL fluid. In addition, plasma, lung, and aortas were extracted to assess pulmonary inflammation and atherosclerotic lesion formation. Polymorphonuclear leukocytes and total BAL cell number increased significantly in MWCNT-exposed mice on d 1 and 7 postexposure. Cell-free BAL fluid obtained from MWCNT-exposed mice at d 1 and 7 postexposure contained significantly elevated levels of total protein, lactate dehydrogenase (LDH), surfactant protein-D, and mucin. Although MWCNT exposure increased pulmonary injury and inflammation, the aortic intimal surface covered by atherosclerotic lesions was not significantly different between control apoE–/– mice and apoE–/– MNCNT-treated animals. Total plasma cholesterol concentrations also were not markedly affected by MWCNT exposure. These results demonstrate that pulmonary exposure to MWCNT affects local airway inflammation but did not appear to augment progression of atherosclerosis in female apoE–/– mice.

Abstract 4801: Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner.

Abstract Subchronic dosing of BHT in mice is a well-known model used to discretely study the tumor promotion stage of lung adenocarcinoma. Using this model, we have previously shown that functional toll-like receptor 4 (TLR4) is protective against inflammatory sequelae and primary tumor formation in the mouse lung. Gap junctional intercellular communication (GJIC) has been shown to regulate the signal transduction pathways of cells, and inhibition of this communication by toxicant exposure has been linked to dysregulation and eventual carcinogenic endpoints. As part of our research on the protective nature of TLR4 during tumor promotion, we utilized the BHT subchronic model (4 weekly i.p. doses of BHT) to examine the early effects on BALF in both BALB/c (BALB; Tlr4 sufficient) and C.C3H-Tlr4Lps-d mice (BALBLps-d-d; Tlr4 mutant). Analysis of the BALF 24 hours after the final dose for protein content and cytology was indicative of an inflammatory profile in all mice treated with BHT. Magnitude of response to BHT was highest in the BALBLps-d mice; the BALB appeared resistant. In an effort to further characterize the response of the pulmonary epithelium, the BALF samples were used to treat cultured C10 cells, a murine immortalized line derived from BALB/c alveolar type II cells, which are a precursor cell type for adenocarcinoma. Using the scrape-load dye transfer assay, a well-defined method to measure GJIC, we demonstrated marked inhibition of cellular communication after a 4 hour exposure to cell-free BALF that is both treatment and strain dependent. The direction and magnitude of effect is in parallel with that of the direct analysis of the BALF, i.e. we observed more inhibition in BALF from BALBLps-d compared to BALB mice. In addition, quantitative PCR (QPCR) analysis of C10 cells exposed to cell-free BALF for 24 hours indicates alterations in the transcription patterns in genes of innate immunity that are also treatment and strain dependent. These novel findings suggest endocrine signaling from the inflammatory milieu are complicit in the inhibition of GJIC during the promotion phase of carcinogenesis. Furthermore, these data establish a link between active gap junctions, tumor promotion, and the protective nature of TLR4 in an ex vivo model which will facilitate a mechanistic examination of these protective effects at the molecular level. Funded by ACS RSG-10-162-01-LIB (AKB). Citation Format: Thomas Hill, Carla-Maria Alexander, Alison K. Bauer. Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2013-4801

NADPH Oxidase and Nrf2 Regulate Gastric Aspiration–Induced Inflammation and Acute Lung Injury

Recruitment of neutrophils and release of reactive oxygen species are considered to be major pathogenic components driving acute lung injury (ALI). However, NADPH oxidase, the major source of reactive oxygen species in activated phagocytes, can paradoxically limit inflammation and injury. We hypothesized that NADPH oxidase protects against ALI by limiting neutrophilic inflammation and activating Nrf2, a transcriptional factor that induces antioxidative and cytoprotective pathways. Our objective was to delineate the roles of NADPH oxidase and Nrf2 in modulating acute lung inflammation and injury in clinically relevant models of acute gastric aspiration injury, a major cause of ALI. Acid aspiration caused increased ALI (as assessed by bronchoalveolar lavage fluid albumin concentration) in both NADPH oxidase-deficient mice and Nrf2(-/-) mice compared with wild-type mice. NADPH oxidase reduced airway neutrophil accumulation, but Nrf2 decreased ALI without affecting neutrophil recovery. Acid injury resulted in a 120-fold increase in mitochondrial DNA, a proinflammatory and injurious product of cellular necrosis, in cell-free bronchoalveolar lavage fluid. Pharmacologic activation of Nrf2 by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl]imidazole limited aspiration-induced ALI in wild-type mice and reduced endothelial cell injury caused by mitochondrial extract-primed human neutrophils, leading to the conclusion that NADPH oxidase and Nrf2 have coordinated, but distinct, functions in modulating inflammation and injury. These results also point to Nrf2 as a therapeutic target to limit ALI by attenuating neutrophil-induced cellular injury.

Atherogenic and pulmonary responses of ApoE- and LDL receptor-deficient mice to sidestream cigarette smoke

Plasma lipoproteins play important roles in the development and progression of atherosclerosis. Two widely used mouse models of experimental atherosclerosis, apolipoprotein E-deficient (ApoE −/−) and LDL receptor-deficient (LDLr −/−) mice, have major differences in lipoprotein characteristics. These include differences in lipoprotein cholesterol distribution, lipoprotein compositions, apoliporoteins distribution, and susceptibility to oxidation. In the present study, we compared pulmonary and cardiovascular responses of ApoE −/− and LDLr −/− mice to sidestream cigarette smoke (SSCS) exposure to determine if strain differences influence their predisposition to SSCS-mediated promotion of atherosclerosis. Female ApoE −/− and LDLr −/− mice were maintained on a saturated fat enriched diet and exposed to SSCS in whole body exposure chambers for 15 weeks (4h/day, 5 days/week). At terminations, the levels of pulmonary injury markers in bronchoalveolar lavage (BAL) fluids from 6 mice per group and atherosclerotic lesion formation in 14 mice per group were analyzed. Total BAL cells and polymorphonuclear leukocytes were not significantly altered by SSCS exposure in both mouse models. Total protein, LDH, and cytokine concentrations in cell-free BAL fluids were also not significantly affected by chronic SSCS exposure in either mouse strain. SSCS significantly reduced surfactant protein D levels in both strains to a similar extent. However, SSCS exposure increased significantly the percent atherosclerotic lesion areas covering aortic intimal surfaces of ApoE −/− (control-25.3±1.52 vs. SSCS-31.9±2.02, p=0.012) as well as in LDLr −/− (control-30.97±1.1 vs. SSCS-36.61±1.7, p=0.028) mice. In contrast, the serum cholesterol concentrations of SSCS-exposed ApoE −/− mice were similar to that of controls (control-1255±85 vs. SSCS-1190±61mg/dl, p=0.552) but increased significantly in SSCS-exposed LDLr −/− mice (control-998±114 vs. SSCS-1577±142mg/dl, p=0.008). These results showing different effects of identical SSCS exposure on plasma cholesterol concentrations in these two mouse models suggest a role of multiple mechanisms in SSCS-induced atherosclerosis.

Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences

The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort.

S79 Potential diagnostic significance of neutrophil proteases in ventilator-associated pneumonia

Introduction and ObjectivesThe clinical diagnosis of ventilator-associated pneumonia (VAP) remains notoriously difficult, as several non-infective conditions mimic VAP. Microbiological confirmation of the diagnosis using conventional cultures typically takes 48–72 h....

Effects of tobacco smoke on IL-16 in CD8+cells from human airways and blood: a key role for oxygen free radicals?

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8(+) and CD4(+) cells as well as the CD4(+) chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8(+) cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8(+) cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8(+) cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a β-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8(+) cells. OFR may play a key role in this context.

Surfactant alterations in horses with recurrent airway obstruction at various clinical stages

To evaluate the phospholipid composition and function of surfactant in horses with recurrent airway obstruction (RAO) at various clinical stages and compare these properties with findings in horses without RAO. 7 horses with confirmed RAO and 7 without RAO (non-RAO horses). Pairs of RAO-affected and non-RAO horses were evaluated before, during, and after exposure to hay. Evaluations included clinical scoring, lung function testing, airway endoscopy, and bronchoalveolar lavage fluid (BALF) absolute and differential cell counts. Cell-free BALF was separated into crude surfactant pellet and supernatant by ultracentrifugation, and phospholipid and protein concentrations were determined. Phospholipid composition of crude surfactant pellets and surface tension were evaluated with high-performance liquid chromatography and a pulsating bubble surfactometer, respectively. Findings were compared statistically via mixed-effects, repeated-measures ANOVA. Total phospholipid concentration in BALF was lower in RAO-affected versus non-RAO horses at all sample collection times. In the RAO-affected group, total phospholipid concentration was lower during exposure to hay than before or after exposure. There were no significant differences in BALF protein concentration, percentages of phospholipid classes, or surface tension between or within groups of horses. All clinical stages of RAO-affected horses were characterized by low surfactant concentration in BALF. Exacerbation of RAO led to an additional decrease in surfactant concentration. Causes for low surfactant concentration in RAO-affected horses remain to be determined. Low phospholipid concentration may render RAO-affected horses more susceptible than unaffected horses to surfactant alterations and contribute to clinical disease status and progression.