Bronchoalveolar Lavage Neutrophils Research Articles

Bronchoscopic and histologic findings during lymphatic intervention for plastic bronchitis

Percutaneous lymphatic intervention (PCL) is a promising new therapy for plastic bronchitis (PB). We characterized bronchoalveolar lavage (BAL) and cast morphology in surgically repaired congenital heart disease (CHD) patients with PB during PCL. We quantified respiratory and bronchoscopic characteristics and correlated them with post-intervention respiratory outcomes. We retrospectively reviewed patients with PB and surgically repaired CHD undergoing PCL and bronchoscopy at our institution. Pre-intervention characteristics, bronchoscopy notes, BAL cell counts, virology, and cultures were collected. A pathologist blinded to clinical data reviewed cast specimens. Respiratory outcomes were evaluated through standardized telephone questionnaire. Sixty-two patients were included with a median follow-up of 20 months. No patients experienced airway bleeding, obstruction, or prolonged intubation related to bronchoscopy. Of BAL infectious studies, the positive results were 4 (8%) fungal, 6 (11%) bacterial, and 6 (14%) viral. Median BAL count per 100 cells for neutrophils, lymphocytes, and eosinophils were 13, 10, and 0, respectively. Of 23 bronchial casts analyzed, all contained lymphocytes, and 19 (83%) were proteinaceous, with 14 containing neutrophils and/or eosinophils. Median BAL neutrophil count was greater in patients with proteinaceous neutrophilic or eosinophilic casts compared to casts without neutrophils or lymphocytes (P = 0.030). Post-intervention, there was a significant reduction in respiratory medications and support and casting frequency. The predominance of neutrophilic proteinaceous casts and high percentage of positive BAL infectious studies support short-term fibrinolytic and anti-infective therapies in PB in select patients. Flexible bronchoscopy enables safe assessment of cast burden. PCL effectively treats PB and reduces respiratory therapies.

Seasonal Variation in Tracheal Mucous and Bronchoalveolar Lavage Cytology for Adult Clinically Healthy Stabled Horses

Seasonal changes affect the concentration of dust particles and aeroallergens in the equine stable environment and are hypothesized to change airway cytology in healthy stabled horses. The objective of the study was to evaluate if seasonal changes from May to November influenced the lower airway health cytological findings in clinically healthy stabled horses. Endoscopic examination, including tracheal aspiration (TA) and bronchoalveolar lavage (BAL) cytology was performed in November and May on 63 clinically healthy stabled Danish Warmblood geldings, aged 12.9 (±4.6) years. Tracheal aspiration and BAL cytology were evaluated via May-Grünwald-Giemsa stain after cytocentrifugation of samples. Five-hundred cells were identified microscopically as macrophages, lymphocytes, neutrophils, eosinophils, or mast cells. A significant difference in climatic conditions with lower temperatures (P < .001), fewer sunlight hours (P = .004), less precipitation (P = .006), and higher humidity (P < .001) was found in November compared to May. A significantly higher percentage of neutrophils (Median 6.10%, Quartiles 4.00–8.80, P < .001) and a higher tracheal mucus score (Median 3.50%, Quartiles 1.95–5.20, P = .010) were found during November compared to May period. Furthermore, a positive correlation between TA and BAL neutrophils was found for both sampling times. Mild winter weather conditions with lower temperatures, fewer minutes of sunlight, and a higher percent humidity were associated with significantly higher BAL neutrophil percentage and significantly higher mucus score in stabled clinical healthy horses. The clear seasonal influence on BAL neutrophilia and tracheal mucous findings in clinically normal horse needs to be included in the working diagnosis of asthma in horses.

Penehyclidine hydrochloride preconditioning provides pulmonary and systemic protection in a rat model of lung ischaemia reperfusion injury

Penehyclidine hydrochloride (PHC) is a new anticholinergic agent that provides protective effects in experimental models of heart and brain ischaemia as well as reperfusion (I/R) injury. In this study, we tested the hypothesis that PHC can alleviate lung ischaemia-reperfusion injury and improve pulmonary and systemic function in rats. PHC was administered intravenously at various doses (d= 0.1, 0.3, 1, 3 mg/kg) to I/R rats. We used six indicators, including lung function, histologic examination, pulmonary oedema, oxidative stress, inflammatory responses, and apoptosis staining to quantify the pulmonary and systemic protective effects of PHC. Haematoxylin and eosin staining was used for pulmonary histologic examination. The expression of Toll-like receptor (TLR) 4, phospho-inhibitor of NF-κB (p-IκB) and nuclear factor-kappa B (NF-κB) was analysed using western blotting. ELISA was conducted to detect inflammatory mediators. Oxidative stress markers as well as myeloperoxidase (MPO) were determined using an assay kit. PHC preconditioning (with concentrations ranging from 0.3 mg/kg to 3 mg/kg 30 min before the onset of I/R) significantly reduced lung histopathological changes, down regulated TLR4, p-IκB and NF-κB expression, and decreased inflammatory mediators as well as the total number of leukocytes and neutrophils in bronchoalveolar lavage (BAL) fluid and plasma. The lung tissue contents of reactive oxygen species (ROS), malondialdehyde (MDA), and MPO as well as pulmonary oedema formation decreased, while SOD (superoxide dismutase) activity was significantly upregulated. PHC preconditioning (with concentrations ranging from 1 mg/kg to 3 mg/kg) significantly improved the lung function and attenuated the apoptotic rate. The probable mechanism for this finding is the inhibition of proinflammatory mediators via the suppression of reactive oxygen species production and the TLR4/NF-κB signalling pathway.

Immunoproteasomes as a novel antiviral mechanism in rhinovirus-infected airways

Rhinovirus (RV) infection is involved in acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). RV primarily infects upper and lower airway epithelium. Immunoproteasomes (IP) are proteolytic machineries with multiple functions including the regulation of MHC class I antigen processing during viral infection. However, the role of IP in RV infection has not been explored. We sought to investigate the expression and function of IP during airway RV infection. Primary human tracheobronchial epithelial (HTBE) cells were cultured at air-liquid interface (ALI) and treated with RV16, RV1B, or interferon (IFN)-λ in the absence or presence of an IP inhibitor (ONX-0914). IP gene (i.e. LMP2) deficient mouse tracheal epithelial cells (mTECs) were cultured for the mechanistic studies. LMP2-deficient mouse model was used to define the in vivo role of IP in RV infection. IP subunits LMP2 and LMP7, antiviral genes MX1 and OAS1 and viral load were measured. Both RV16 and RV1B significantly increased the expression of LMP2 and LMP7 mRNA and proteins, and IFN-λ mRNA in HTBE cells. ONX-0914 down-regulated MX1 and OAS1, and increased RV16 load in HTBE cells. LMP2-deficient mTECs showed a significant increase in RV1B load compared with the wild-type (WT) cells. LMP2-deficient (compared with WT) mice increased viral load and neutrophils in bronchoalveolar lavage (BAL) fluid after 24 h of RV1B infection. Mechanistically, IFN-λ induction by RV infection contributed to LMP2 and LMP7 up-regulation in HTBE cells. Our data suggest that IP are induced during airway RV infection, which in turn may serve as an antiviral and anti-inflammatory mechanism.

Characterization of Siglec-8 Expression on Lavage Cells after Segmental Lung Allergen Challenge

Background: Siglec-8 is present at a high level on human blood eosinophils and low level on blood basophils. Engagement of Siglec-8 on blood eosinophils causes its internalization and results in death. Siglec-8 is a potential therapeutic target in eosinophilic asthma. Objectives: The aim of this study was to determine Siglec-8 levels on eosinophils and basophils recruited during lung inflammation. Method: We analyzed surface Siglec-8 by flow cytometry on cells obtained by bronchoalveolar lavage (BAL) 48 h after segmental lung allergen challenge of human subjects with mild allergic asthma and used confocal microscopy to compare Siglec-8 distribution on BAL and blood eosinophils. Results: Like their blood counterparts, BAL eosinophils had high unimodal surface Siglec-8, while BAL basophils had lower but detectable surface Siglec-8. BAL macrophages, monocytes, neutrophils, and plasmacytoid dendritic cells did not express surface Siglec-8. Microscopy of freshly isolated blood eosinophils demonstrated homogeneous Siglec-8 distribution over the cell surface. Upon incubation with IL-5, Siglec-8 on the surface of eosinophils became localized in patches both at the nucleopod tip and at the opposite cell pole. BAL eosinophils also had a patchy Siglec-8 distribution. Conclusions: We conclude that 48 h after segmental allergen challenge, overall levels of Siglec-8 expression on airway eosinophils resemble those on blood eosinophils, but with a patchier distribution, a pattern consistent with activation. Thus, therapeutic targeting of Siglec-8 has the potential to impact blood as well as lung eosinophils, which may be associated with an improved outcome in eosinophilic lung diseases.

Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants

To establish the reference values, diagnostic accuracy, and effect of various factors on cell count in intubated preterm neonates subjected to nonbronchoscopic bronchoalveolar lavage. This prospective, cross-sectional, blinded study included preterm neonates ventilated for any reason who underwent nonbronchoscopic bronchoalveolar lavage if they had not previously received postnatal antibiotics or steroids. Lavage was performed before surfactant replacement, if any. A gentle ventilation policy was applied. Pneumonia was diagnosed using clinical criteria, without considering cell count. Investigators performing cell counts were blinded to the clinical data. There were 276 neonates enrolled; 36 had congenital or ventilator-associated pneumonia. In the 240 noninfected babies, median neutrophil count increased significantly after the first 2 days of ventilation (day 1, 2 cells per field [IQR, 0.0-9.5 cells per field]; day 2, 2 cells per field [IQR, 0-15 cells per field]; day 3, 20 cells per field [IQR, 2-99 cells per field]; day 4, 15 cells per field [IQR, 2-96 cells per field]; P < .0001). No significant difference was seen over time in infected babies. Multivariate analysis indicated pneumonia (standardized β = 0.134; P = .033) and the time spent under mechanical ventilation before nonbronchoscopic bronchoalveolar lavage as factors significantly influencing neutrophil count (standardized β = 0.143; P = .027). Neutrophil count was correlated with the duration of ventilation (rho = 0.28; P <.001). Neutrophil counts were higher in infected (24 cells/field [IQR, 5-78] cells/field) than in noninfected babies (4 cells/field [IQR, 1-24 cells/field]; P <.001) and had an moderate reliability for pneumonia within the first 2 days of ventilation (area under the curve, 0.745; (95% CI, 0.672-0.810; P = .002). We provide reference values for airway neutrophil counts in ventilated preterm neonates. Bronchoalveolar lavage neutrophils significantly increase after 2 days of ventilation. Neutrophil count has moderate accuracy to diagnose pneumonia, but only within the first 2 days of ventilation.

Expression profile of microRNAs following bone marrow-derived mesenchymal stem cell treatment in lipopolysaccharide-induced acute lung injury.

Immunomodulatory or immunosuppressive properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) facilitate the treatment of acute respiratory distress syndrome and acute lung injury (ALI). Dysregulated miRNA (miRNA or miR) expression associated with the effects of BM-MSCs was assessed in a rat model of lipopolysaccharide (LPS)-induced ALI. The present study performed biochemical tests to assess five analytes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate, blood urea nitrogen (BUN), and creatinine (CREA). Total cell count was assessed and the percentage of bronchoalveolar lavage neutrophil content was also examined. The results Histopathological examination of rat upper lobe lung tissue was then used to estimate lung injury score (LIS). The levels of AST, lactate, BUN and creatinine (excluding ALT), released into the circulation upon injury, were significantly lower in ALI rats treated with BM-MSCs than in ALI rats alone (P<0.05). BM-MSC rats exhibited a significantly decreased bronchoalveolar lavage neutrophil percentage and LIS compared with that of LPS treated rats alone (P<0.05). In addition, the miRNA expression profile was determined following treatment with BM-MSCs via microarray analysis. A total of 95/690 miRNAs were differentially expressed following the treatment of BM-MSCs in rats with ALI. Among the 95 miRNAs, 66 were upregulated and 29 were downregulated; 9 miRNAs were significantly upregulated (miR-1843-3p, miR-323-3p, miR-183-5p, miR-182 and miR-196b-3p) or downregulated (miR-547-3p, miR-301b-5p, miR-503-3p and miR-142-3p). A total of 3 miRNAs were inversely expressed in ALI treated with BM-MSCs compared with untreated ALI. Of these 3 miRNAs, the expression of miR-142-3p and miR-503-3p was upregulated in the LPS groups and downregulated in the BM-MSC groups. miR-196b-3p was downregulated in the LPS group and upregulated in the BM-MSC groups. miRNAs have a role in cell proliferation, immune response, inflammation and apoptosis, which may be associated with the therapeutic effects of BM-MSCs in ALI. In summary, BM-MSCs improved multi-organ damage and attenuated lung injury. Different miRNA profiles were expressed following BM-MSC treatment of ALI. These dysregulated miRNAs participated in BM-MSC-mediated immunomodulation of ALI.

The clinical significance of oropharyngeal cultures in young children with cystic fibrosis.

In children with cystic fibrosis (CF) the associations between oropharyngeal swabs (OPSs) for detection of Pseudomonas and lung disease have not been evaluated.OPS and bronchoalveolar lavage (BAL) samples were obtained annually in children with CF from 2005 to 2017. OPS test characteristics were calculated using BAL as "gold standard". Results were related to lung inflammation (BAL neutrophil elastase and interleukin-8), structural lung disease (chest computed tomography PRAGMA-CF (Perth-Rotterdam Annotated Grid Morphometric Analysis for CF) scores), respiratory exacerbations and future detection of Pseudomonas on BAL.From 181 patients, 690 paired OPS-BAL cultures were obtained. Prevalence of Pseudomonas in BAL was 7.4%. OPS sensitivity was 23.0% and specificity was 91.4%, reducing the post-test probability for a positive BAL following a negative OPS to 6.3%. Pseudomonas on OPS was not associated with lung inflammation or respiratory exacerbations, but was weakly associated with current PRAGMA-CF %Disease score (p=0.043). Pseudomonas on BAL was associated with positive neutrophil elastase (OR 4.17, 95% CI 2.04-8.53; p<0.001), increased interleukin-8 (p<0.001), increased all baseline PRAGMA computed tomography scores (p<0.001), progression of PRAGMA computed tomography scores (p<0.05) and increased risk of respiratory exacerbations (incidence rate ratio 2.11, 95% CI 1.15-3.87; p=0.017).In children with CF OPSs only marginally change the probability of detecting lower airway Pseudomonas and are not associated with lung disease indices nor exacerbations risk.

Endothelial Colony-forming Cells Attenuate Ventilator-induced Lung Injury in Rats with Acute Respiratory Distress Syndrome

Mechanical ventilation (MV) can cause ventilator-induced lung injury (VILI). This study investigated whether endothelial colony-forming cells (ECFC) could inhibit VILI in a rat model of acute respiratory distress syndrome (ARDS). Male Wistar rats received the femoral artery and venous cannulation (sham group) or were injected intravenously with 500μg/kg lipopolysaccharide to induce ARDS. The ARDS rats were subjected to MV. Immediately after the MV, the rats were randomized and injected intravenously with vehicle (ARDS group) or ECFC (ECFC group, n=8 per group). The oxygen index, lung wet-to-dry weight (W/D) ratios, cytokine protein levels in serum or bronchoalveolar lavage fluid (BALF), neutrophil counts, neutrophil elastase and total protein levels in BALF, histology and cell apoptosis in the lung were detected. The protein levels of endothelin-1, inducible nitric oxide synthase (iNOS), endothelial NOS, matrix metalloproteinase (MMP)-9, Bax, Bcl-2, gelsolin, cleaved caspase-3, phosphorylated NF-κBp65 and myosin light chain (MLC) in the lung were analyzed. Compared with the ARDS group, treatment with ECFC significantly increased the oxygen index, and decreased the lung W/D ratios and injury, and the numbers of apoptotic cells in the lungs, neutrophils counts, total protein and elastase concentrations in BALF of rats. ECFC treatment significantly minimized the protein levels of pro-inflammatory cytokines in BALF and serum, but increased interleukin 10 in rats. Furthermore, ECFC treatment significantly reduced the protein levels of endothelin-1, iNOS, Bax, Gelsolin, MMP-9, cleaved caspase-3, phosphorylated NF-κBp65 and MLC, but enhanced eNOS and Bcl-2 in the lungs of rats. Therefore, ECFC attenuated inflammation, cell apoptosis and VILI in ARDS rats.

ALX receptor ligands define a biochemical endotype for severe asthma.

BACKGROUND. In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). METHODS. ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). RESULTS. Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. CONCLUSIONS. Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. TRIAL REGISTRATION. Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov {type:clinical-trial,attrs:{text:NCT01606826,term_id:NCT01606826}}NCT01606826) FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology.

Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.

Although a deficiency of surfactant protein B (SFTPB) has been associated with lung injury, SFTPB expression has not yet been linked with nicotinamide phosphoribosyltransferase (NAMPT), a potential biomarker of acute lung injury (ALI). The effects of Nampt in the pulmonary epithelial cell on both SFTPB expression and lung inflammation were investigated in a LPS-induced ALI mouse model. Pulmonary epithelial cell-specific knockdown of Nampt gene expression, achieved by the crossing of Nampt gene exon 2 floxed mice with mice expressing epithelial-specific transgene Cre or by the use of epithelial-specific expression of anti-Nampt antibody cDNA, significantly attenuated LPS-induced ALI. Knockdown of Nampt expression was accompanied by lower levels of bronchoalveolar lavage (BAL) neutrophil infiltrates, total protein and TNF-α levels, as well as lower lung injury scores. Notably, Nampt knockdown was also associated with significantly increased BAL SFTPB levels relative to the wild-type control mice. Down-regulation of NAMPT increased the expression of SFTPB and rescued TNF-α-induced inhibition of SFTPB, whereas overexpression of NAMPT inhibited SFTPB expression in both H441 and A549 cells. Inhibition of NAMPT up-regulated SFTPB expression by enhancing histone acetylation to increase its transcription. Additional data indicated that these effects were mainly mediated by NAMPT nonenzymatic function via the JNK pathway. This study shows that pulmonary epithelial cell-specific knockdown of NAMPT expression attenuated ALI, in part, via up-regulation of SFTPB expression. Thus, epithelial cell-specific knockdown of Nampt may be a potential new and viable therapeutic modality to ALI.-Bi, G., Wu, L., Huang, P., Islam, S., Heruth, D. P., Zhang, L. Q., Li, D.-Y., Sampath, V., Huang, W., Simon, B. A., Easley, R. B., Ye, S. Q. Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.

The peroxisome proliferator-activated receptor agonist pioglitazone and 5-lipoxygenase inhibitor zileuton have no effect on lung inflammation in healthy volunteers by positron emission tomography in a single-blind placebo-controlled cohort study.

BackgroundAnti-inflammatory drug development efforts for lung disease have been hampered in part by the lack of noninvasive inflammation biomarkers and the limited ability of animal models to predict efficacy in humans. We used 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in a human model of lung inflammation to assess whether pioglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, and zileuton, a 5-lipoxygenase inhibitor, reduce lung inflammation.MethodsFor this single center, single-blind, placebo-controlled cohort study, we enrolled healthy volunteers sequentially into the following treatment cohorts (N = 6 per cohort): pioglitazone plus placebo, zileuton plus placebo, or dual placebo prior to bronchoscopic endotoxin instillation. 18F-FDG uptake pre- and post-endotoxin was quantified as the Patlak graphical analysis-determined Ki (primary outcome measure). Secondary outcome measures included the mean standard uptake value (SUVmean), post-endotoxin bronchoalveolar lavage (BAL) cell counts and differentials and blood adiponectin and urinary leukotriene E4 (LTE4) levels, determined by enzyme-linked immunosorbent assay, to verify treatment compliance. One- or two-way analysis of variance assessed for differences among cohorts in the outcome measures (expressed as mean ± standard deviation).ResultsTen females and eight males (29±6 years of age) completed all study procedures except for one volunteer who did not complete the post-endotoxin BAL. Ki and SUVmean increased in all cohorts after endotoxin instillation (Ki increased by 0.0021±0.0019, 0.0023±0.0017, and 0.0024±0.0020 and SUVmean by 0.47±0.14, 0.55±0.15, and 0.54±0.38 in placebo, pioglitazone, and zileuton cohorts, respectively, p<0.001) with no differences among treatment cohorts (p = 0.933). Adiponectin levels increased as expected with pioglitazone treatment but not urinary LTE4 levels as expected with zileuton treatment. BAL cell counts (p = 0.442) and neutrophil percentage (p = 0.773) were similar among the treatment cohorts.ConclusionsEndotoxin-induced lung inflammation in humans is not responsive to pioglitazone or zileuton, highlighting the challenge in translating anti-inflammatory drug efficacy results from murine models to humans.Trial registrationClinicalTrials.gov NCT01174056.

Protective effect of SIRT3 on acute lung injury by increasing manganese superoxide dismutase-mediated antioxidation.

Prolonged exposure to hyperoxia results in acute lung injury (ALI). Pulmonary damage caused by oxygen toxicity occurs due to the generation of reactive oxygen species and subsequent formation of more potent oxidants. The present study demonstrated that sirtuin 3 (SIRT3) may attenuate hyperoxia‑induced ALI due to its potential antioxidative effect. In the present study, a hyperoxia‑induced acute lung injury mouse model, reverse transcription‑quantitative polymerase chain reaction, western blotting, retroviral mediated gene over‑expression and knockdown assays revealed that the expression of SIRT3 in the lung tissue of mice with hyperoxia‑induced ALI was decreased and overexpression of SIRT3 may significantly reduce hyperoxia‑induced ALI, as reflected by decreases in protein concentration, infiltrated neutrophils in bronchoalveolar lavage (BAL) fluid and wet/dry ratio of lung tissues. Furthermore, overexpression of SIRT3 increased the protein levels and enzymatic activity of manganese superoxide dismutase (MnSOD), and inhibited oxidative stress in the lungs of ALI mice. Additionally, the current study demonstrated that SIRT3 promoted the expression of MnSOD, and this regulation was crucial for the protective effect of SIRT3 on hyperoxia‑induced ALI. In summary, the results of the current study indicated that SIRT3 overexpression may effectively ameliorate hyperoxia‑induced ALI in mice, which indicates a potential application for SIRT3‑based gene therapy to treat clinical adult respiratory distress syndrome.

The comparison of heparan sulfate and its fragments on the protection against extracellular histones during the pathogenesis of acute respiratory distress syndrome

Objective: In order to explore the role of heparan sulfate (HS) during the pathogenesis of acute respiratory distress syndrome (ARDS) , the protective effect of HS and its fragments against extracellular histones was compared. Methods: Calf thymus histones (CTH) were injected via femoral vein to induce ARDS in rats. HS, HS fragments or saline was intraperitoneally injected (10mg/kg, Q6h, 24h) to test the protective effect against CTH. The ratio of wet/dry lung weight, protein content in bronchoalveolar lavage fluid (BALF) , total leukocyte and neutrophil count in BALF were measured. Results: After CTH injection, the ratio of wet/dry lung weight (5.7±0.95) was much higher than the saline control group (3.1±0.15). The protein content (0.47±0.086mg/ml) , total leukocyte[ (97.4±15.6l) ×10(4)/ml] and neutrophil (18±3.4/LPF) in BALF were obviously increased compared with the saline control group. The intervention of HS evidently decreased ratio of wet/dry lung weight (4.2±0.41) , protein content[ (0.26±0.019) mg/ml], leukocyte[ (61.3±5.74) ×10(4)/ml] and neutrophil (12±1.8/LPF) in BALF. HS fragments also decreased ratio of wet/dry lung weight, protein content, leukocyte and neutrophil count in BALF though the strength was much less than HS. Conclusion: HS and its fragments could provide protection against extracellular histones during the pathogenesis of ARDS. For the protective effect full length HS was much better than HS fragments.

Phloretin attenuates mucus hypersecretion and airway inflammation induced by cigarette smoke

BackgroundsCigarette smoke (CS)-induced airway mucus hypersecretion and inflammation are the prominent features of chronic obstructive pulmonary disease (COPD). As an anti-inflammatory flavonoid, phloretin was found to be involved in various inflammatory disorders such as sepsis. In this study, the effects of phloretin on CS-induced airway mucin secretion and inflammation were investigated in vivo and in vitro. MethodsPhloretin dissolved in 1% DMSO was daily injected intraperitoneally to mice, which were then exposed to CS for four weeks. Mouse lung histologic changes were evaluated, the expression of mucin 5ac (MUC5AC) was measured, bronchoalveolar lavage fluid (BALF) total cells, neutrophils, and macrophages were counted. BALF and lung levels of tumor necrosis factor-alpha and interleukin-1 beta (IL-1β) were quantified. Moreover, the effects of phloretin on cigarette smoke extract (CSE)-induced expression of MUC5AC and IL-1β were investigated in NCI-H292 cells. Then, to explore the potential mechanisms, the signaling molecules including epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) and P38 were evaluated. ResultsPhloretin pretreatment dramatically suppressed the mucins secretion, inflammatory cell infiltration and inflammatory cytokine release in mouse lungs induced by CS, and it also suppressed CSE-induced expression of MUC5AC and IL-1β in NCI-H292 bronchial epithelial cells. Furthermore, western blot showed that phloretin attenuated the activation of EGFR, ERK and P38 both in vivo and in vitro. ConclusionsThis study highlights the protective effect of phloretin on CS-related airway mucus hypersecretion and inflammation, where EGFR, ERK and P38 might be involved. These findings suggest that phloretin could be a potential therapeutic drug for COPD.

Effects of Macrolide and Corticosteroid in Neutrophilic Asthma Mouse Model.

BackgroundAsthma is a disease of chronic airway inflammation with heterogeneous features. Neutrophilic asthma is corticosteroid-insensitive asthma related to absence or suppression of TH2 process and increased TH1 and/or TH17 process. Macrolides are immunomodulatory drug that reduce airway inflammation, but their role in asthma is not fully known. The purpose of this study was to evaluate the role of macrolides in neutrophilic asthma and compare their effects with those of corticosteroids.MethodsC57BL/6 female mice were sensitized with ovalbumin (OVA) and lipopolysaccharides (LPS). Clarithromycin (CAM) and/or dexamethasone (DXM) were administered at days 14, 15, 21, 22, and 23. At day 24, the mice were sacrificed.ResultsAirway resistance in the OVA+LPS exposed mice was elevated but was more attenuated after treatment with CAM+DXM compared with the monotherapy group (p<0.05 and p<0.01). In bronchoalveolar lavage fluid study, total cells and neutrophil counts in OVA+LPS mice were elevated but decreased after CAM+DXM treatment. In hematoxylin and eosin stain, the CAM+DXM-treated group showed less inflammation additively than the monotherapy group. There was less total protein, interleukin 17 (IL-17), interferon γ, and tumor necrosis factor α in the CAM+DXM group than in the monotherapy group (p<0.001, p<0.05, and p<0.001). More histone deacetylase 2 (HDAC2) activity was recovered in the DXM and CAM+DXM challenged groups than in the control group (p<0.05).ConclusionDecreased IL-17 and recovered relative HDAC2 activity correlated with airway resistance and inflammation in a neutrophilic asthma mouse model. This result suggests macrolides as a potential corticosteroid-sparing agent in neutrophilic asthma.

Mechanistic Basis for Obesity-related Increases in Ozone-induced Airway Hyperresponsiveness in Mice.

Obesity is a risk factor for asthma, especially nonallergic asthma. Ozone, a common air pollutant, is a nonallergic asthma trigger. Importantly, ozone-induced decrements in lung function are greater in obese and overweight human subjects than in lean individuals. Obese mice also exhibit exaggerated pulmonary responses to ozone. Ozone causes greater increases in pulmonary resistance, in bronchoalveolar lavage neutrophils, and in airway hyperresponsiveness in obese than in lean mice. Our data indicate that IL-33 plays a role in mediating these events. Ozone causes greater release of IL-33 into bronchoalveolar lavage fluid in obese than in lean mice. Furthermore, an antibody blocking the IL-33 receptor, ST2, attenuates ozone-induced airway hyperresponsiveness in obese but not in lean mice. Our data also indicate a complex role for tumor necrosis factor (TNF)-α in obesity-related effects on the response to ozone. In obese mice, genetic deficiency in either TNF-α or TNF-α receptor 2 augments ozone-induced airway hyperresponsiveness, whereas TNF-α receptor 2 deficiency virtually abolishes ozone-induced airway hyperresponsiveness in lean mice. Finally, obesity is known to alter the gut microbiome. In female mice, antibiotics attenuate obesity-related increases in the effect of ozone on airway hyperresponsiveness, possibly by altering microbial production of short-chain fatty acids. Asthma control is often difficult to achieve in obese patients with asthma. Our data suggest that therapeutics directed against IL-33 may ultimately prove effective in these patients. The data also suggest that dietary manipulations and other strategies (prebiotics, probiotics) that alter the microbiome and/or its metabolic products may represent a new frontier for treating asthma in obese individuals.

Rhinovirus Infection of ORMDL3 Transgenic Mice Is Associated with Reduced Rhinovirus Viral Load and Airway Inflammation.

Orosomucoid like 3 (ORMDL3), a gene localized to chromosome 17q21, has been linked in epidemiologic studies to childhood asthma and rhinovirus (RV) infections. As the single nucleotide polymorphisms linking ORMDL3 to asthma are associated with increased expression of ORMDL3, we have used hORMDL3zp3-Cre mice (which have universal increased expression of human ORMDL3) to determine whether infection of these transgenic mice with RV influences levels of airway inflammation or RV viral load. RV infection of hORMDL3zp3-Cre mice resulted in reduced RV viral load assessed by quantitative real-time PCR (lung and airway epithelium), as well as reduced airway inflammation (total bronchoalveolar lavage cells, neutrophils, macrophages, and lymphocytes) compared with RV-infected wild-type mice. Levels of the antiviral pathways including IFNs (IFN-α, IFN-β, IFN-λ) and RNAse L were significantly increased in the lungs of RV-infected hORMDL3zp3-Cre mice. Levels of the antiviral mouse oligoadenylate synthetase (mOas)1g pathway and RNAse L were upregulated in the lungs of unchallenged hORMDL3zp3-Cre mice. In addition, levels of mOas2, but not mOas1 (mOas1a, mOas1b, mOas1g), or mOas3 pathways were significantly more upregulated by IFNs (IFN-α, IFN-β, IFN-λ) in epithelial cells from hORMDL3zp3-Cre mice compared with RV-infected wild-type mouse epithelial cells. RNAse L-deficient mice infected with RV had increased RV viral load. Overall, these studies suggest that increased levels of ORMDL3 contribute to antiviral defense to RV infection in mice through pathways that may include IFNs (IFN-α, IFN-β, IFN-λ), OAS, and RNAse L.

Serum amyloid A: an ozone-induced circulating factor with potentially important functions in the lung-brain axis.

Accumulating evidence suggests that O3 exposure may contribute to CNS dysfunction. Here, we posit that inflammatory and acute-phase proteins in the circulation increase after O3 exposure and systemically convey signals of O3 exposure to the CNS. To model acute O3 exposure, female Balb/c mice were exposed to 3 ppm O3 or forced air for 2 h and were studied after 6 or 24 h. Of 23 cytokines and chemokines, only KC/CXCL1 was increased in blood 6 h after O3 exposure. The acute-phase protein serum amyloid A (A-SAA) was significantly increased by 24 h, whereas C-reactive protein was unchanged. A-SAA in blood correlated with total leukocytes, macrophages, and neutrophils in bronchoalveolar lavage from O3-exposed mice. A-SAA mRNA and protein were increased in the liver. We found that both isoforms of A-SAA completely crossed the intact blood-brain barrier, although the rate of SAA2.1 influx was approximately 5 times faster than that of SAA1.1. Finally, A-SAA protein, but not mRNA, was increased in the CNS 24 h post-O3 exposure. Our findings suggest that A-SAA is functionally linked to pulmonary inflammation in our O3 exposure model and that A-SAA could be an important systemic signal of O3 exposure to the CNS.-Erickson, M. A., Jude, J., Zhao, H., Rhea, E. M., Salameh, T. S., Jester, W., Pu, S., Harrowitz, J., Nguyen, N., Banks, W. A., Panettieri, R. A., Jr., Jordan-Sciutto, K. L. Serum amyloid A: an ozone-induced circulating factor with potentially important functions in the lung-brain axis.

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse.

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.