Bronchoalveolar Lavage Cell Counts Research Articles

Yes-associated Protein Mediates Acute Lung Injury in Adult but not Juvenile Mice

Objective: Adults with The Acute Respiratory Distress Syndrome (ARDS) have worse outcomes than children with Pediatric ARDS. This finding can be reproduced in animal models, suggesting a difference in Acute Lung Injury (ALI) mechanisms. Understanding these mechanistic differences could inform age-specific treatment. Hypothesis: Since Yes-associated protein (YAP) is important for lung development and reactive to mechanical stretch, we hypothesized that YAP is important for ALI mechanisms. Methods: To induce ALI, we intranasally instilled mice with Pseudomonas aeruginosa strain K (2.5 x 105 colony forming units (c.f.u.)) in juvenile (3-week-old) and adult (10-week-old) C57BL/6J mice. 24 h later, we collected bronchoalveolar lavage (BAL) fluid and measured protein, cell count, and c.f.u. To knockdown YAP expression, we injected siRNA 24 h before P. aeruginosa instillation. We removed the lungs and enriched for CD45+ and CD41+ fraction, and CD31+ fraction using Dynabeads sheep anti-rat IgG (Invitrogen). In separate experiments, lungs were separated into nuclear and cytosolic fractions for immunoblot analysis. For immunoprecipitation, the cytosolic fraction was added to beads coated with anti-YAP antibody (rabbit monoclonal). We washed the beads and eluted bound proteins, which were identified by label-free mass spectrometry. Data and results: While both juvenile and adult mice had significantly increased BAL cell counts and protein 24 h after P. aeruginosa instillation, adult mice had significantly greater cell counts and protein than juveniles. BAL c.f.u. were similar in both groups, suggesting equal instillation of P. aeruginosa. P. aeruginosa instillation increased whole lung YAP expression in both cytosolic and nuclear fractions in adult mice by >3-fold, but YAP expression was not increased in P. aeruginosa-treated juvenile mice. YAP protein increases occurred in CD31+ fraction (endothelial cells), but no YAP was quantified in CD45+/CD41+ fraction (leukocytes and platelets). Vascular knockdown of YAP expression by ~75% in adult mice blocked P. aeruginosa-induced BAL cell count increases. Proteomics analysis revealed 62 proteins that had significantly altered YAP binding upon P. aeruginosa treatment (47 with increased YAP binding). The complement factors H and I had a log2-fold-change for YAP binding of 4.56 and 2.26, respectively, upon P. aeruginosa treatment. Conclusions: Adult mice exhibited YAP-dependent Acute Lung Injury; juveniles did not. YAP-dependent mechanisms of injury may be secondary to YAP binding to the complement inhibitory proteins H and I. More work is needed to establish the cell-specificity of YAP-complement factor binding and to determine the location of complement signaling. HL148403, Parker B Francis, and Columbia University Department of Pediatrics Children’s Health Innovation Nucleation Fund to RFH; Ernest E Just Summer Scholarship to AMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dimethyl Sulfoxide as a Novel Therapy in a Murine Model of Acute Lung Injury.

The endothelial glycocalyx (EGX) on the luminal surface of endothelial cells contributes to the permeability barrier of the pulmonary vasculature. Dimethyl sulfoxide (DMSO) has a disordering effect on plasma membranes, which prevents the formation of ordered membrane domains important in the shedding of the EGX. We hypothesized that DMSO would protect against protein leak by preserving the EGX in a murine model of acute respiratory distress syndrome (ARDS). C57BL/6 mice were given ARDS via intra-tracheally administered lipopolysaccharide (LPS). DMSO (220 mg/kg) was administered intravenously for 4 days. Animals were sacrificed post-injury day 4 after bronchoalveolar lavage (BAL). BAL cell counts and protein content was quantified. Lung sections were stained with FITC-labelled wheat germ agglutinin (FITC-WGA) to quantify the EGX. Cultured endothelial cells (HUVECs) were exposed to LPS. EGX was measured using FITC-WGA, and co-immunoprecipitation was performed to measure interaction between sheddases and syndecan-1. DMSO treatment resulted in greater EGX staining intensity in the lung when compared to sham (9,641 vs. 36,659 A.U. p < 0.001). Total BAL cell counts were less for animals receiving DMSO (6.93 x 106 vs. 2.49 x 106 cells, p = 0.04). The treated group had less BAL macrophages (189.2 vs. 76.9 cells, p = 0.02) and lymphocytes (527.7 vs. 200.0 cells, p = 0.02). Interleukin-6 levels were lower in DMSO treated. Animals that received DMSO had less protein leak in BAL (1.48 vs. 1.08 ug/ul, p = 0.02). DMSO prevented LPS-induced EGX loss in HUVECs, and reduced the interaction between Matrix Metalloproteinase (MMP) 16 and syndecan-1. Systemically administered DMSO protects the EGX in the pulmonary vasculature, mitigating pulmonary capillary leak after acute lung injury. DMSO also results in decreased inflammatory response. DMSO reduced the interaction between MMP16 and Syndecan-1 and prevented LPS-induced glycocalyx damage in cultured endothelial cells. DMSO may be a novel therapeutic for ARDS. Not applicable (animal studies).

The efficacy of N-acetylcysteine in decreasing airway inflammation and mucus accumulation in horses with 18 hours of head confinement.

During transportation many horses develop post-transportation infection, which can be life-threatening and end their sport career. Preventing mucus accumulation and inflammation during transportation is vital, emphasizing the need for effective strategies to enhance overall horse health welfare. Assess the impact of N-acetylcysteine (NAC) on mucus accumulation and inflammation in horses subjected to 18 hours of head confinement. Six healthy crossbred horses, 5.3 ± 2.1 years of age and weighing 387 ± 30 kg. Prospective placebo-controlled cross-over design study. The horses' heads were restrained in their stalls for a period of 18 hours. They were studied under 4 conditions: Not confined (NC): before head confinement, placebo (P), and confined head (CH): 18 hours of head confinement without treatment, and N-Acetylcysteine (NAC): 18 hours of head confinement treated with NAC before confinement (15 mg/kg/day NAC PO for 3 days). Bronchoalveolar lavage (BAL) was performed in each condition. Mucus accumulation along the trachea was evaluated by endoscopy. Endoscopic scores were significantly different between CH and other conditions, whereas no significant differences were found among NC, P, and NAC. The BAL cell count (34 291 ± 2624 cells/μL), neutrophil and lymphocyte count (18 601 ± 3193 cells/μL and 3337.4 ± 593 cells/μL, respectively) in CH were significantly higher compared to NAC. Neutrophil percentage was significantly higher in CH (53.8 ± 8%) compared to horses that received NAC (20.08 ± 8%). Conversely, in comparison to NAC (66.33 ± 9%), the percentage of macrophages was significantly lower in CH (35.7 ± 10%). N-acetylcysteine was found to significantly decrease mucus accumulation and inflammatory cell counts in horses with head confinement.

Dimethyl malonate protects the lung in a murine model of acute respiratory distress syndrome.

Succinate is a proinflammatory citric acid cycle metabolite that accumulates in tissues during pathophysiological states. Oxidation of succinate after ischemia-reperfusion leads to reversal of the electron transport chain and generation of reactive oxygen species. Dimethyl malonate (DMM) is a competitive inhibitor of succinate dehydrogenase, which has been shown to reduce succinate accumulation. We hypothesized that DMM would protect against inflammation in a murine model of ARDS. C57BL/6 mice were given ARDS via 67.7 μg of intratracheally administered lipopolysaccharide. Dimethyl malonate (50 mg/kg) was administered via tail vein injection 30 minutes after injury, then daily for 3 days. The animals were sacrificed on day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts were performed to examine cellular influx. Supernatant protein was quantified via Bradford protein assay. Animals receiving DMM (n = 8) were compared with those receiving sham injection (n = 8). Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify the endothelial glycocalyx (EGX). Total cell counts in BAL was less for animals receiving DMM (6.93 × 10 6 vs. 2.46 × 10 6 , p = 0.04). The DMM group had less BAL macrophages (168.6 vs. 85.1, p = 0.04) and lymphocytes (527.7 vs. 248.3; p = 0.04). Dimethyl malonate-treated animals had less protein leak in BAL than sham treated (1.48 vs. 1.15 μg/μl, p = 0.03). Treatment with DMM resulted in greater staining intensity of the EGX in the lung when compared with sham (12,016 vs. 15,186 arbitrary units, p = 0.03). Untreated animals had a greater degree of weight loss than treated animals (3.7% vs. 1.1%, p = 0.04). Dimethyl malonate prevented the upregulation of monocyte chemoattractant protein-1 (1.66 vs. 0.92 RE, p = 0.02) and ICAM-1 (1.40 vs. 1.01 RE, p = 0.05). Dimethyl malonate reduces lung inflammation and capillary leak in ARDS. This may be mediated by protection of the EGX and inhibition of monocyte chemoattractant protein-1 and ICAM-1. Dimethyl malonate may be a novel therapeutic for ARDS.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

A methanolic extract of Eclipta prostrata (L.) L. decreases inflammation in a murine model of chronic allergic asthma via inhibition of the NF-kappa-B pathway

Ethnopharmacological relevanceEclipta prostrata (L.) L. is a medicinal plant used by many ethnic groups in Brazil to treat respiratory diseases, hepatitis and the bites of venomous animals. A methanolic extract of E. prostrata (MEEP), the major components of which are wedelolactone (WED) and demethylwedelolactone (DMW), exhibited anti-inflammatory activity in acute asthma models but the effects on lung inflammation and the mechanisms of action of MEEP in a chronic asthma model are not known. Aim of the studyTo study the effects of MEEP in vivo using a chronic ovalbumin (OVA)-induced allergic asthma model in mice. Materials and methodsThe identities of WED and DMW in MEEP were confirmed and the concentrations determined by liquid chromatography and tandem mass spectrometry. Male Balb/c mice were sensitized and challenged with OVA and experimental animals were treated with MEEP (100, 250 or 500 mg/kg) while control animals were treated with dexamethasone (2 mg/kg) or normal saline. Bronchial hyperresponsiveness, total and differential cell counts in bronchoalveolar lavage (BAL), and the production of Th2 cytokines in lung homogenates were assessed. Lung inflammation and mucus production were evaluated by histological analysis while nuclear factor kappa-B (NF-κB) activation was assessed immunohistochemically. ResultsConcentrations of WED and DMW in MEEP were 5.12% and 1.04%, respectively. Treatments with MEEP (250 or 500 mg/kg) significantly decreased bronchial hyperresponsiveness, reduced total cell and eosinophil counts in BAL and IL-4 concentrations in lung homogenate, and inhibited NF-κB activation. Treatment with MEEP at 500 mg/kg reduced the level of IL-5 in lung homogenates but did not decrease IL-13 concentration or mucus production. ConclusionsMEEP attenuated bronchial hyperresponsiveness and decreased lung and airway inflammation in a chronic asthma model in mice. The mechanism of action involves inhibition of NF-κB activation, most likely associated with the presence of the coumestans WED and DMW. These results support the ethnopharmacological evidence for the use of E. prostrata against asthma and other respiratory inflammatory diseases.

The impact of intravenous dodecafluoropentane on a murine model of acute lung injury

IntroductionIntravenous oxygen therapeutics present an appealing option for improving arterial oxygenation in patients with acute hypoxemic respiratory failure, while limiting iatrogenic injury from conventional respiratory management.MethodsWe used an established two-hit murine model of acute lung injury (ARDS/VILI) to evaluate the effect of intravenous dodecafluoropentane (DDFPe) on oxygen saturation and bronchoalveolar lavage cell counts and protein levels. Twenty hours after challenge with intratracheal lipopolysaccharide, mice were intubated and ventilated with high tidal volumes (4 h) to produce acute lung injury. DDFPe (0.6 mL/kg) or saline was administered by IV bolus injection at the initiation of mechanical ventilation and again at 2 h. Oxygen saturation was measured every 15 min. Bronchoalveolar lavage was performed at the conclusion of the experiment.ResultsThe two-hit ARDS/VILI model produced substantial inflammatory acute lung injury reflected by markedly increased bronchoalveolar lavage (BAL) cell counts compared to BAL cell counts in spontaneous breathing controls (5.29 ± 1.50 × 10–6 vs 0.74 ± 0.014 × 10–6 cells/mL) Similarly, BAL protein levels were markedly elevated in ARDS/VILI-challenged mice compared with spontaneous breathing controls (1109.27 ± 223.80 vs 129.6 ± 9.75 ng/mL). We fit a linear mixed effects model that showed a significant difference in oxygen saturation over time between DDFPe-treated mice and saline-treated mice, with separation starting after the 2-h injection. DDFPe-treated ARDS/VILI-challenged mice also exhibited significant reductions in BAL cell counts but not in BAL protein.ConclusionDDFPe improves oxygen saturation in a murine model of ARDS/VILI injury with the potential for serving as an intravenous oxygen therapeutic.

Optimising the yield from bronchoalveolar lavage on human participants in infectious disease immunology research

Bronchoalveolar lavage (BAL) is becoming a common procedure for research into infectious disease immunology. Little is known about the clinical factors which influence the main outcomes of the procedure. In research participants who underwent BAL according to guidelines, the BAL volume yield, and cell yield, concentration, viability, pellet colour and differential count were analysed for association with important participant characteristics such as active tuberculosis (TB) disease, TB exposure, HIV infection and recent SARS-CoV-2 infection. In 337 participants, BAL volume and BAL cell count were correlated in those with active TB disease, and current smokers. The right middle lobe yielded the highest volume. BAL cell and volume yields were lower in older participants, who also had more neutrophils. Current smokers yielded lower volumes and higher numbers of all cell types, and usually had a black pellet. Active TB disease was associated with higher cell yields, but this declined at the end of treatment. HIV infection was associated with more bloody pellets, and recent SARS-CoV-2 infection with a higher proportion of lymphocytes. These results allow researchers to optimise their participant and end assay selection for projects involving lung immune cells.

Bronchoalveolar Lavage Cell Count and Lymphocytosis Are the Important Discriminators between Fibrotic Hypersensitivity Pneumonitis and Idiopathic Pulmonary Fibrosis

Fibrotic hypersensitivity pneumonitis (fHP) shares many features with other fibrotic interstitial lung diseases (ILD), and as a result it can be misdiagnosed as idiopathic pulmonary fibrosis (IPF). We aimed to determine the value of bronchoalveolar lavage (BAL) total cell count (TCC) and lymphocytosis in distinguishing fHP and IPF and to evaluate the best cut-off points discriminating these two fibrotic ILD. A retrospective cohort study of fHP and IPF patients diagnosed between 2005 and 2018 was conducted. Logistic regression was used to evaluate the diagnostic utility of clinical parameters in differentiating between fHP and IPF. Based on the ROC analysis, BAL parameters were evaluated for their diagnostic performance, and optimal diagnostic cut-offs were established. A total of 136 patients (65 fHP and 71 IPF) were included (mean age 54.97 ± 10.87 vs. 64.00 ± 7.18 years, respectively). BAL TCC and the percentage of lymphocytes were significantly higher in fHP compared to IPF (p < 0.001). BAL lymphocytosis >30% was found in 60% of fHP patients and none of the patients with IPF. The logistic regression revealed that younger age, never smoker status, identified exposure, lower FEV1, higher BAL TCC and higher BAL lymphocytosis increased the probability of fibrotic HP diagnosis. The lymphocytosis >20% increased by 25 times the odds of fibrotic HP diagnosis. The optimal cut-off values to differentiate fibrotic HP from IPF were 15 × 106 for TCC and 21% for BAL lymphocytosis with AUC 0.69 and 0.84, respectively. Increased cellularity and lymphocytosis in BAL persist despite lung fibrosis in HP patients and may be used as important discriminators between IPF and fHP.

Supplementation of syringic acid-rich Phrynium pubinerve leaves imparts protection against allergic inflammatory responses by downregulating iNOS, COX-2, and NF-κB expressions

BackgroundThe present study was designed to characterize the role of ethanolic leaf extract of Phrynium pubinerve Blume (EPP) supplement in attenuating allergic inflammation, encouraged by the presence of syringic acid in it, as this phenolic acid is reportedly promising in suppressing serum immunoglobulin E (IgE) and inflammatory cytokine levels. Materials and methodsHPLC-DAD dereplication analysis was performed to determine the presence of the vital polyphenolic metabolites. The efficacy of EPP against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells was evaluated by measuring its inhibitory effects on NO and ROS/RNS production. The expressions of major inflammation-associated molecules (iNOS, COX-2, NF-κB, IL-6, and TNF-α) in RAW 264.7 cells were assessed through Western blot. Physiological and behavioral changes, BMI, and different biochemical parameters in mice blood serum were investigated in the toxicological assays. Formaldehyde-induced paw edema test in mice was conducted using established animal model. TDI-induced allergic model in mice was carried out to determine different allergy-like symptoms, and differential white blood cell (WBC) counts in blood and bronchoalveolar lavage (BAL) fluid. The intermolecular interaction analysis of the identified major metabolite of EPP with H1R and iNOS was studied by molecular docking. ResultsHPLC-DAD analysis showed the presence of syringic acid (89.19 mg/100 g EPP) and a few other compounds. LPS-induced NO generation was reduced by EPP in a concentration-dependent manner, showing IC50 of 28.20 ± 0.27 μg/mL. EPP exhibited a similar inhibitory effect on ROS/RNS production with IC50 of 29.47 ± 2.19 μg/mL. Western blotting revealed that EPP significantly downregulated the expressions of iNOS, COX-2, NF-κB, IL-6, and TNF-α in RAW 264.7 cells when challenged with LPS. The toxicological assays confirmed the dosage and organ-specific safety of EPP. In the formaldehyde-induced paw edema test, EPP caused a 66.41% reduction in mice paw volume at 500 mg/kg dose. It ameliorated TDI-induced allergy-like symptoms and decreased different inflammatory WBCs in mice's blood and BAL fluid in a dose-dependent manner. Finally, syringic acid demonstrated mentionable intermolecular binding affinity towards H1R (−6.6 Kcal/moL) and iNOS (−6.7 Kcal/moL). ConclusionsCollectively, considerable scientific reasoning was obtained in favor of the suppressive potential of EPP against allergic inflammatory responses that are proposed to be exerted via the downregulation of iNOS, COX-2, and NF-κB expressions, H1R antagonism and suppression of cytokines, such as IL-6, and TNF-α.

Chronic allergic lung inflammation negatively influences neurobehavioral outcomes in mice

BackgroundAsthma is a major public health problem worldwide. Emerging data from epidemiological studies show that allergies and allergic diseases may be linked to anxiety, depression and cognitive decline. However, little is known about the effect of asthma, an allergic lung inflammation, on cognitive decline/behavioral changes. Therefore, we investigated the hypothesis that allergic lung inflammation causes inflammation in the brain and leads to neurobehavioral changes in mice.MethodsWild-type C57BL/6J female mice were sensitized with nasal house dust mite (HDM) antigen or control PBS for 6 weeks to induce chronic allergic lung inflammation. A series of neurocognitive tests for anxiety and/or depression were performed before and after the intranasal HDM administration. After the behavior tests, tissues were harvested to measure inflammation in the lungs and the brains.ResultsHDM-treated mice exhibited significantly increased immobility times during tail suspension tests and significantly decreased sucrose preference compared with PBS controls, suggesting a more depressed and anhedonia phenotype. Spatial memory impairment was also observed in HDM-treated mice when assessed by the Y-maze novel arm tests. Development of lung inflammation after 6 weeks of HDM administration was confirmed by histology, bronchoalveolar lavage (BAL) cell count and lung cytokine measurements. Serum pro-inflammatory cytokines and Th2-related cytokines levels were elevated in HDM-sensitized mice. In the brain, the chemokine fractalkine was increased in the HDM group. The c-Fos protein, a marker for neuronal activity, Glial Fibrillary Acidic Protein (GFAP) and chymase, a serine protease from mast cells, were increased in the brains from mice in HDM group. Chymase expression in the brain was negatively correlated with the results of sucrose preference rate in individual mice.Conclusions6 weeks of intranasal HDM administration in mice to mimic the chronic status of lung inflammation in asthma, caused significant inflammatory histological changes in the lungs, and several behavioral changes consistent with depression and altered spatial memory. Chymase and c-Fos proteins were increased in the brain from HDM-treated mice, suggesting links between lung inflammation and brain mast cell activation, which could be responsible for depression-like behavior.

Role of TRPA1/TRPV1 in acute ozone exposure induced murine model of airway inflammation and bronchial hyperresponsiveness.

BackgroundTransient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) mediate the development of lung injury and inflammation. This study investigated the role and mechanism of the TRPA1/TRPV1 pathway in airway inflammation and bronchial hyperresponsiveness (BHR) induced by acute ozone exposure.MethodsC57BL/6 mice (8–10 weeks) were intraperitoneally injected with phosphate buffered saline (PBS), A967079 (TRPA1 inhibitor) or AMG9810 (TRPV1 inhibitor) 1 h before or after ozone exposure (2.5 ppm, 3 h). BHR, cell counts in bronchoalveolar lavage (BAL) fluid, oxidative stress biomarkers, inflammatory cytokines, TRPA1 and TPRV1 protein levels, mitochondrial dynamics- and mitophagy-related protein levels, and activities of mitochondrial respiratory chain (MRC) in lung were measured.ResultsThe preventive treatment effect was similar to the therapeutic treatment effect. Both A967079 and AMG9810 intervention suppressed BHR, inflammatory cytokines, total BAL fluid cells, malondialdehyde (MDA) levels and inflammatory cytokines mRNA including Substance P (SP), Keratinocyte-Derived Chemokine (KC), interleukin-18 (IL-18) and chemokine (C-X-C motif) ligand 8 (CXCL8) expression, and enhanced reduced glutathione (GSH)/oxidized glutathione (GSSG) levels compared with ozone-exposed mice. A967079 and AMG9810 intervention inhibited dynamin-related protein (DRP1), mitochondrial fission factor (MFF), Parkinson protein 2 E3 ubiquitin protein ligase (PARK2) and Sequestosome 1 (SQSTM1)/p62 expression, increased Optic atrophy 1 (OPA1), mitofusin 2 (MFN2) and PTEN-induced putative kinase 1 (PINK1) expression, and up-regulated the activities of MRC complex III and V in lung tissue.ConclusionsThe results show that both TRPA1 and TRPV1 pathways are involved in acute ozone exposure-induced airway inflammation and BHR and influence oxidative stress, mitochondrial quality control and MRC activity, which could be a potential target for clinical therapy of respiratory diseases.

NOX4 Mediates Epithelial Cell Death in Hyperoxic Acute Lung Injury Through Mitochondrial Reactive Oxygen Species.

Management of acute respiratory distress involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). NADPH oxidase (NOX) could be a major source of reactive oxygen species (ROS) in hyperoxia (HO). Epithelial cell death is a crucial step in the development of many lung diseases. Alveolar type II (AT2) cells are the metabolically active epithelial cells of alveoli that serve as a source of AT1 cells following lung injury. The aim of this study was to determine the possible role of AT2 epithelial cell NOX4 in epithelial cell death from HALI. Wild type (WT), Nox4 fl/fl (control), and Nox4 −/− Spc-Cre mice were exposed to room air (NO) or 95% O2 (HO) to investigate the structural and functional changes in the lung. C57BL/6J WT animals subjected to HO showed increased expression of lung NOX4 compared to NO. Significant HALI, increased bronchoalveolar lavage cell counts, increased protein levels, elevated proinflammatory cytokines and increased AT2 cell death seen in hyperoxic Nox4 fl/fl control mice were attenuated in HO-exposed Nox4 −/− Spc-Cre mice. HO-induced expression of NOX4 in MLE cells resulted in increased mitochondrial (mt) superoxide production and cell apoptosis, which was reduced in NOX4 siRNA silenced cells. This study demonstrates a novel role for epithelial cell NOX4 in accelerating lung epithelial cell apoptosis from HALI. Deletion of the Nox4 gene in AT2 cells or silencing NOX4 in lung epithelial cells protected the lungs from severe HALI with reduced apoptosis and decreased mt ROS production in HO. These results suggest NOX4 as a potential target for the treatment of HALI.

CD200 is required to control LPS-induced lung inflammation.

Abstract INTRODUCTION Acute respiratory distress syndrome (ARDS) is a severe lung inflammatory disease caused by a variety of precipitants, including SARS-CoV-2 (COVID-19). In addition to excessive inflammation, ARDS is characterised by the dysregulation of anti-inflammatory pathways, including CD200/CD200R pathway. OBJECTIVE To investigate the role of CD200/CD200R pathway in lung ARDS inflammatory response. METHODS LPS was administered intratracheally to induce ARDS in Sprague-Dawley CD200 KO and wild type (WT) rats. Inflammation was evaluated using bronchoalveolar lavage (BAL) cellularity. Lung injury was measured by total protein level in BAL fluid, and levels of proinflammatory cytokines (TNF, IL-6) and chemokines (CXCL2, CCL2) were determined in BAL supernatants. In a second experiment, recombinant CD200Fc was administered to KO rats to restore the anti-inflammatory response. RESULTS Although there was no difference in total BAL cell counts at 3 h, cell recruitment was greater in CD200 KO rats given the low cell number in naïve KO rats. BAL of KO rats had higher levels of TNF, IL-6, CXCL2, and CCL2 compared to WT rats. Total protein level in BAL was higher in CD200 KO rats, implying more pronounced pulmonary edema. CD200Fc administration in KO rats significantly decreased levels of TNF and CCL2 in BAL, suggesting an attenuation of the inflammatory response. CONCLUSION This study shows that ARDS inflammatory response is exacerbated in absence of CD200 in an experimental model of ARDS in rats and that CD200 supplementation alleviates this phenotype. Further analyses will be needed to better understand the contribution of different cell types expressing CD200 to control lung inflammatory response resulting from ARDS. Supported by grant by CIHR and IUCPQ fondation.

Inflammation on bronchoalveolar lavage cytology is associated with decreased chronic lung allograft dysfunction-free survival.

Lung transplant recipients undergo bronchoalveolar lavage (BAL) to detect antecedents of chronic lung allograft dysfunction (CLAD), but routine assessment of BAL cytology is controversial. We hypothesized that inflammation on BAL cytology would predict CLAD-free survival. In a single-center retrospective cohort, associations between cytology results and clinical characteristics were compared using generalized-estimating equation-adjusted regression. The association between BAL inflammation and CLAD or death risk was assessed using time-dependent Cox models. In 3365 cytology reports from 451 subjects, inflammation was the most common finding (6.2%, 210 cases), followed by fungal forms (5.3%, 178 cases, including 24 cases of suspectedAspergillus). Inflammation on BAL cytology was more common in procedures for symptoms (8.5%) versus surveillance (3.2%, p<.001). Inflammation on cytology was associated with automated neutrophil and lymphocyte counts, acute cellular rejection, infection, and portended a 2.2-fold hazard ratio (CI1.2-4.0, p=.007) for CLAD or death.However, inflammation by cytology did not inform CLAD-free survival risk beyond automated BAL cell counts (p=.57). Inflammation on BAL cytology is clinically significant, suggesting acute rejection or infection and increased risk of CLAD or death. However, other indicators of allograft inflammation can substitute for much of the information provided by BAL cytology.

Increased cytotoxic T-cells in the airways of adults with former bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) in preterm-born infants is a risk factor for chronic airway obstruction in adulthood. Cytotoxic T-cells are implicated in COPD, but their involvement in BPD is not known. To characterise the distribution of airway T-cell subsets in adults with a history of BPD. Young adults with former BPD (n=22; median age 19.6 years), age-matched adults born preterm (n=22), patients with allergic asthma born at term (n=22) and healthy control subjects born at term (n=24) underwent bronchoalveolar lavage (BAL). T-cell subsets in BAL were analysed using flow cytometry. The total number of cells and the differential cell counts in BAL were similar among the study groups. The percentage of CD3+CD8+ T-cells was higher (p=0.005) and the proportion of CD3+CD4+ T-cells was reduced (p=0.01) in the BPD group, resulting in a lower CD4/CD8 ratio (p=0.007) compared to the healthy controls (median 2.2 versus 5.3). In BPD and preterm-born study subjects, both CD3+CD4+ T-cells (rs=0.38, p=0.03) and CD4/CD8 ratio (rs=0.44, p=0.01) correlated positively with forced expiratory volume in 1 s (FEV1). Furthermore, CD3+CD8+ T-cells were negatively correlated with both FEV1 and FEV1/forced vital capacity (rs= -0.44, p=0.09 and rs= -0.41, p=0.01, respectively). Young adults with former BPD have a T-cell subset pattern in the airways resembling features of COPD. Our findings are compatible with the hypothesis that CD3+CD8+ T-cells are involved in mechanisms behind chronic airway obstruction in these patients.

A two-hit model of sepsis plus hyperoxia causes lung permeability and inflammation.

Mouse models of acute lung injury (ALI) have been instrumental for studies of the biological underpinnings of lung inflammation and permeability, but murine models of sepsis generate minimal lung injury. Our goal was to create a murine sepsis model of ALI that reflects the inflammation, lung edema, histological abnormalities, and physiological dysfunction that characterize ALI. Using a cecal slurry (CS) model of polymicrobial abdominal sepsis and exposure to hyperoxia (95%), we systematically varied the timing and dose of the CS injection, fluids and antibiotics, and dose of hyperoxia. We found that CS alone had a high mortality rate that was improved with the addition of antibiotics and fluids. Despite this, we did not see evidence of ALI as measured by bronchoalveolar lavage (BAL) cell count, total protein, C-X-C motif chemokine ligand 1 (CXCL-1) or by lung wet:dry weight ratio. Addition of hyperoxia [95% fraction of inspired oxygen ([Formula: see text])] to CS immediately after CS injection increased BAL cell counts, CXCL-1, and lung wet:dry weight ratio but was associated with 40% mortality. Splitting the hyperoxia treatment into two 12-h exposures (0-12 h and 24-36 h) after CS injection increased survival to 75% and caused significant lung injury compared with CS alone as measured by increased BAL total cell count (92,500 vs. 240,000, P = 0.0004), BAL protein (71 vs. 103 µg/mL, P = 0.0030), and lung wet:dry weight ratio (4.5 vs. 5.5, P = 0.0005), and compared with sham as measured by increased BAL CXCL-1 (20 vs. 2,372 pg/mL, P < 0.0001) and histological lung injury score (1.9 vs. 4.2, P = 0.0077). In addition, our final model showed evidence of lung epithelial [increased BAL and plasma receptor for advanced glycation end products (RAGE)] and endothelial (increased Syndecan-1 and sulfated glycosaminoglycans) injury. In conclusion, we have developed a clinically relevant mouse model of sepsis-induced ALI using intraperitoneal injection of CS, antibiotics and fluids, and hyperoxia. This clinically relevant model can be used for future studies of sepsis-induced ALI.

Hexarelin modulates lung mechanics, inflammation, and fibrosis in acute lung injury.

Introduction: Acute respiratory distress syndrome (ARDS) is an acute form of diffuse lung injury characterized by (i) an intense inflammatory response, (ii) increased pulmonary vascular permeability, and (iii) the loss of respiratory pulmonary tissue. In this article we explore the therapeutic potential of hexarelin, a synthetic hexapeptide growth hormone secretagogue (GHS), in an experimental model of ARDS. Hexarelin has anti-inflammatory properties and demonstrates cardiovascular-protective activities including the inhibition of cardiomyocyte apoptosis and cardiac fibrosis, both of which may involve the angiotensin-converting enzyme (ACE) system. Methods: In our experimental model, ARDS was induced by the instillation of 100 mM HCl into the right bronchus; these mice were treated with hexarelin (320 μg/kg, ip) before (Pre) or after (Post) HCl challenge, or with vehicle. Respiratory system compliance, blood gas analysis, and differential cell counts in a selective bronchoalveolar lavage (BAL) were determined 6 or 24 hours after HCl instillation. In an extended study, mice were observed for a subsequent 14 days in order to assess lung fibrosis. Results: Hexarelin induced a significant improvement in lung compliance and a reduction of the number of total immune cells in BAL 24 hours after HCl instillation, accompanied with a lower recruitment of neutrophils compared with the vehicle group. At day 14, hexarelin-treated mice presented with less pulmonary collagen deposition compared with vehicle-treated controls. Conclusions: Our data suggest that hexarelin can inhibit the early phase of the inflammatory response in a murine model of HCl-induced ARDS, thereby blunting lung remodeling processes and fibrotic development.

Benefits and risks of bronchoalveolar lavage in severe asthma in children.

BackgroundAlthough bronchoscopy can be part of the exploration of severe asthma in children, the benefit of bronchoalveolar lavage (BAL) is unknown. The present study aimed to decipher whether systematic BAL during a flexible bronchoscopy procedure could better specify the characteristics of severe asthma and improve asthma management.Material and methodsThe study took place in two departments of a university hospital in Paris. Children who underwent flexible bronchoscopy for the exploration of severe asthma between April 2017 and September 2019 were retrospectively included.ResultsIn total, 203 children were included, among whom 107 had a BAL. BAL cell count was normal in most cases, with an increasing number of eosinophils with age, independently from the atopic status of the patients. Compared with bronchial aspiration only, BAL increased the rate of identified bacterial infection by 1.5. Nonatopic patients had more bacterial infections (p<0.001). BAL induced a therapeutic modification only for azithromycin and omalizumab prescriptions. The practice of a BAL decreased bronchoscopy tolerance (p=0.037), especially in the presence of tracheobronchial malacia (p<0.01) and when performed in a symptomatic patient (p=0.019).Discussion and conclusionAlthough BAL may provide interesting information in characterising severe asthma, in most cases its impact on the patient's management remains limited. Moreover, BAL can be poorly tolerated and should be avoided in the case of tracheobronchial malacia or current asthma symptoms.

SNAP, CRACKLE, AND POP: A CASE OF PEMETREXED-INDUCED INTERSTITIAL PNEUMONITIS

TOPIC: Diffuse Lung Disease TYPE: Medical Student/Resident Case Reports INTRODUCTION: Pemetrexed is a folic acid inhibitor which is used for the treatment of non-small cell lung cancer (NSCLC) (1). Pemetrexed is generally well tolerated, but interstitial pneumonitis is an exceedingly rare, potentially fatal adverse effect (2). The diagnosis of drug induced interstitial pneumonitis is one of exclusion and based on nonspecific clinical and radiographic findings. Onset of symptoms must correlate clinically with timing of administration of the offending agent, and the symptoms improve with removal of the medication and initiation of corticosteroids (3). It is important for clinicians to have a high index of suspicion to initiate early treatment, reducing morbidity and mortality. Below is a case of a patient with lung adenocarcinoma who developed pemetrexed induced interstitial pneumonitis. CASE PRESENTATION: Our patient is an 85 year old man who was diagnosed with stage IVA lung adenocarcinoma. He received two cycles of Carboplatin and Pemetrexed with good radiologic response but experienced intolerable side effects. The decision was made to transition to Pemetrexed monotherapy. Two weeks after his second cycle, he developed dyspnea, low grade fevers and fatigue, requiring supplemental oxygen (Vapotherm 60% FiO2 at 40 L/min) and hospital admission. Physical exam was notable for diffuse crackles bilaterally. CTA Chest demonstrated bilateral ground glass opacities and was negative for pulmonary embolism. He was empirically started on broad spectrum antibiotics for multifocal pneumonia. COVID-19, respiratory viral test, blood and sputum cultures were all negative. Bronchoscopy with bronchoalveolar lavage (BAL) demonstrated normal airways. There was no evidence of alveolar hemorrhage. BAL cell count and differential was notable for a lymphocytic predominance. BAL cytology demonstrated pulmonary macrophages and mixed inflammatory cells but no atypical cells. High dose corticosteroids were started on hospital day five. Oxygen requirement steadily decreased, and he was discharged a few days later on 2 L NC and a prolonged corticosteroid taper. DISCUSSION: Pemetrexed is a widely used first-line treatment for NSCLC. Pemetrexed-induced interstitial pneumonitis has a range of clinical presentations, making it challenging to diagnose. Presentations vary from asymptomatic to life-threatening requiring mechanical ventilation. Moreover, it is a very rare side effect of Pemetrexed. This case highlights the importance of having strong clinical suspicion so that interstitial pneumonitis can be identified and treated early, mitigating morbidity and mortality. A thorough workup must be completed to rule out other causes, especially infection. CONCLUSIONS: Through early diagnosis, pemetrexed can be discontinued and corticosteroids can be initiated sooner, reducing risk to the patient and improving clinical outcomes (3). The patient should not be rechallenged with Pemetrexed, as this could be fatal. REFERENCE #1: Dhakal, B., Singh, V., Shrestha, A., Rao, A., & Choong, N. (2011). Pemetrexed induced pneumonitis. Clinics and Practice 1(4), 232-6. DOI: 10.4081/cp.2011.e106 REFERENCE #2: Kim, KH. et al. (2013). Interstitial pneumonitis after treatment with pemetrexed for non-small cell lung cancer. Cancer Research and Treatment 45(1), 74-7. DOI: 10.4143/crt.2013.45.1.74 REFERENCE #3: Skeoch, S. et al. (2018). Drug-induced interstitial lung disease: A systematic review. Journal of Clinical Medicine 7(10), 1-30. DOI: 10.3390/jcm7100356 DISCLOSURES: No relevant relationships by Brent Bagley, source=Web Response No relevant relationships by Ethan Tope, source=Web Response