Bronchoalveolar Lavage Fluid Protein Research Articles

Thalictrum minus L. ameliorates particulate matter-induced acute lung injury in mice

Ethnopharmacological relevanceThalictrum minus L., which is widespread across Eurasia, is utilized as a folk medicine for treating dysentery, bedsore, fungal infection and lung inflammation in China, Mongolia and Iran. Aim of the studyA Mongolian folk medicinal plant named Thalictrum minus L. (TML) has been extensively used for the treatment of lung inflammation, bacterial and fungal infection and tuberculosis. Our present study aims to investigate the effectiveness of TML against particulate matter (PM)-induced acute lung injury (ALI) and the potential underlying mechanisms. Materials and methodsInitially, HPLC-Q-TOF was applied for the qualitative analysis and HPLC was used for quantitative analysis of main components in TML. Then, the mice model of ALI was induced by PM via intratracheally instilled with 50 mg/kg body weight of Standard Reference Material1648a (SRM1648a), and TML (10, 20, 40 mg/kg) were administered orally 1 h prior to PM. The efficacy and molecular mechanisms in the presence or absence of TML were elucidated. ResultsEleven main ingredients were detected in TML and the contents of homoorientin and berberine were quantified. Additionally, the results demonstrated that TML profoundly inhibited weight loss in mice and ameliorated lung pathological injury induced by PM. Furthermore, we also found that TML significantly decreased the lung wet to dry weight (W/D) ratios, reduced total protein in bronchoalveolar lavage fluid (BALF), and effectively attenuated PM-induced increased leukocyte and macrophages in BALF. Meanwhile, TML could pronouncedly inhibited myeloperoxidase (MPO) activity in lung tissues, decreased the PM-induced inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), reduced nitric oxide (NO) and increased superoxide dismutase (SOD) in BALF. In addition, TML markedly facilitated the expression of p-AMPK-Nrf2 and suppressed the expression of KEAP, prohibited the activation of the MAPKs-NLRP3/caspase-1 and cyclooxygenase-2 (COX2), and inhibited apoptotic pathways. ConclusionThese findings indicated that TML attenuated PM-induced ALI through suppressing the release of inflammatory cytokines and alleviating oxidative damage correlated with the AMPK-Nrf2/KEAP signaling pathways, MAPKs-NLRP3/caspase-1 signaling pathways, as well as apoptotic pathways.

Reduced allergic lung inflammation and airway responsiveness in mice lacking the cytoskeletal protein gelsolin.

Airway smooth muscle hyperresponsiveness associated with chronic airway inflammation leads to the typical symptoms of asthma including bronchoconstriction and wheezing. Asthma severity is associated with airway inflammation; therefore, reducing airway inflammation is an important therapeutic target. Gelsolin is an actin capping and severing protein that has been reported to be involved in modulation of the inflammatory response. Using mice genetically lacking gelsolin, we evaluated the role of gelsolin in the establishment of house dust mite (HDM) antigen-induced allergic lung inflammation. The genetic absence of gelsolin was found to be protective against HDM sensitization, resulting in reduced lung inflammation, inflammatory cytokines, and Muc5AC protein in bronchoalveolar lavage (BAL) fluid. The number of eosinophils, lymphocytes, and interstitial macrophages in the BAL were increased after HDM sensitization in wild-type mice but were attenuated in gelsolin-null mice. The observed attenuation of inflammation may be partly due to delayed migration of immune cells, because the reduced eosinophils in the BALs from gelsolin-null mice compared with controls occurred despite similar amounts of the chemoattractant eotaxin. Splenic T cells demonstrated similar proliferation rates, but ex vivo alveolar macrophage migration was delayed in gelsolin-null mice. In vivo, the reduced lung inflammation after HDM sensitization in gelsolin-null mice was associated with significantly diminished airway resistance to inhaled methacholine compared with HDM-treated wild-type mice. Our results suggest that modulation of gelsolin expression or function in selective inflammatory cell types that modulate allergic lung inflammation could be a therapeutic approach for asthma.

Novel protein pathways in development and progression of pulmonary sarcoidosis

Pulmonary involvement occurs in up to 95% of sarcoidosis cases. In this pilot study, we examine lung compartment-specific protein expression to identify pathways linked to development and progression of pulmonary sarcoidosis. We characterized bronchoalveolar lavage (BAL) cells and fluid (BALF) proteins in recently diagnosed sarcoidosis cases. We identified 4,306 proteins in BAL cells, of which 272 proteins were differentially expressed in sarcoidosis compared to controls. These proteins map to novel pathways such as integrin-linked kinase and IL-8 signaling and previously implicated pathways in sarcoidosis, including phagosome maturation, clathrin-mediated endocytic signaling and redox balance. In the BALF, the differentially expressed proteins map to several pathways identified in the BAL cells. The differentially expressed BALF proteins also map to aryl hydrocarbon signaling, communication between innate and adaptive immune response, integrin, PTEN and phospholipase C signaling, serotonin and tryptophan metabolism, autophagy, and B cell receptor signaling. Additional pathways that were different between progressive and non-progressive sarcoidosis in the BALF included CD28 signaling and PFKFB4 signaling. Our studies demonstrate the power of contemporary proteomics to reveal novel mechanisms operational in sarcoidosis. Application of our workflows in well-phenotyped large cohorts maybe beneficial to identify biomarkers for diagnosis and prognosis and therapeutically tenable molecular mechanisms.

MCTR1 enhances the resolution of lipopolysaccharide-induced lung injury through STAT6-mediated resident M2 alveolar macrophage polarization in mice.

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro‐resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post‐treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS‐induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin‐1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS‐induced lung injury.

Protective effect of S-nitrosoglutathione pretreatment on acute lung injury in septic rats.

Objective(s):To investigate the protective effect of S-nitrosoglutathione (SNG) pretreatment on acute lung injury (ALI) in septic rats. Materials and Methods:We constructed a rat model of sepsis by cecal ligation and perforation (CLP), and randomly divided into Sham, CLP, and CLP+SNG (0.25 and 0.5 mg/kg) groups. We used H&E staining and lung wet/dry ratio to assess the severity of lung injury, detected the levels of protein and cells in bronchoalveolar lavage fluid (BALF) and the levels of TNF-α, IL-1β, TLR4 mRNA, and NF-κB p65 mRNA in the lung tissue, and assessed the levels of glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase in the lung tissue.Results:A rat model of sepsis was successfully constructed by CLP, and pretreatment of SNG significantly increased the survival of septic rats (P<0.001) and decreased the lung tissue injury scores (P<0.001) and lung wet/dry ratio (P<0.01) in a dose-dependent manner. Furtherly, SNG pretreatment significantly reduced the number of total cells, total protein, neutrophils, and lympholytes (all P<0.001) in BALF, and which also decreased the levels of TNF-α, IL-1β, TLR4 mRNA, and NF-κB p65 mRNA (all P<0.001) in the lungs of CLP-induced rats. Moreover, pretreatment of SNG significantly increased the levels of anti-oxidant enzymes GSH, SOD, GSH-Px, and catalase (all P<0.001) in the lung tissue of septic rats. Conclusion:SNG pretreatment has a protective effect on ALI in septic rats, and the specific mechanism may be related to anti-endotoxic, anti-inflammatory, and anti-oxidative properties.

Glucagon-like peptide-1 receptor mediates the beneficial effect of liraglutide in an acute lung injury mouse model involving the thioredoxin-interacting protein.

Repurposing clinically used drugs is among the important strategies in drug discovery. Glucagon-like peptide-1 (GLP-1) and its diabetes-based drugs, such as liraglutide, possess a spectrum of extra-pancreatic functions, while GLP-1 receptor (GLP-1R) is most abundantly expressed in the lung. Recent studies have suggested that GLP-1-based drugs exert beneficial effects in chronic, as well as acute, lung injury rodent models. Here, we show that liraglutide pretreatment reduced LPS induced acute lung injury in mice. It significantly reduced lung injury score, wet/dry lung weight ratio, bronchoalveolar lavage fluid immune cell count and protein concentration, and cell apoptosis in the lung, and it was associated with reduced lung inflammatory cytokine and chemokine gene expression. Importantly, these effects were virtually absent in GLP-1R−/− mice. A well-known function of GLP-1 and GLP-based drugs in pancreatic β-cells is the attenuation of high-glucose stimulated expression of thioredoxin-interacting protein (TxNIP), a key component of inflammasome. LPS-challenged lungs showed elevated TxNIP mRNA and protein expression, which was attenuated by liraglutide treatment in a GLP-1R-dependent manner. Hence, our observations suggest that GLP-1R is essential in mediating beneficial effects of liraglutide in acute lung injury, with the inflammasome component TxNIP as a potential target.

Circulating and tissue biomarkers as predictors of bromine gas inhalation.

The threat from deliberate or accidental exposure to halogen gases is increasing, as is their industrial applications and use as chemical warfare agents. Biomarkers that can identify halogen exposure, diagnose victims of exposure or predict injury severity, and enable appropriate treatment are lacking. We conducted these studies to determine and validate biomarkers of bromine (Br2 ) toxicity and correlate the symptoms and the extent of cardiopulmonary injuries. Unanesthetized rats were exposed to Br2 and monitored noninvasively for clinical scores and pulse oximetry. Animals were euthanized and grouped at various time intervals to assess brominated fatty acid (BFA) content in the plasma, lung, and heart using mass spectrometry. Bronchoalveolar lavage fluid (BALF) protein content was used to assess pulmonary injury. Cardiac troponin I (cTnI) was assessed in the plasma to evaluate cardiac injury. The blood, lung, and cardiac tissue BFA content significantly correlated with the clinical scores, tissue oxygenation, heart rate, and cardiopulmonary injury parameters. Total (free + esterified) bromostearic acid levels correlated with lung injury, as indicated by BALF protein content, and free bromostearic acid levels correlated with plasma cTnI levels. Thus, BFAs and cardiac injury biomarkers can identify Br2 exposure and predict the severity of organ damage.

P53 deficiency potentiates LPS-Induced acute lung injury in vivo.

Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) represent a significant cause of morbidity and mortality in critically ill hospitalized patients. Emerging evidence suggest that the expression levels of P53 in the lungs are associated with the supportive effects of heat shock protein 90 inhibitors and growth hormone releasing hormone antagonists in the endothelium. In the current study, we employed an in vivo model of intratracheal administration of lipopolysaccharides (LPS)-induced ALI to investigate the role of P53 in counteracting LPS-induced lung inflammatory responses. In wild type mice, LPS induced the expression of IL-1α, IL-1β, and TNFα in the lungs, increased bronchoalveolar lavage fluid protein concentration, and activated cofilin. Remarkably; those responses were more potent in P53 knockout mice, suggesting the crucial role of P53 in orchestrating rigorous endothelial defenses against inflammatory stimuli. The present study supports previous endeavors on the protective role of P53 against lung inflammatory disease, and enrich our knowledge on the development of medical countermeasures against ARDS.

Associations of soluble metals and lung and liver toxicity in mice induced by fine particulate matter originating from a petrochemical complex.

Adverse health effects have been observed in nearby residents due to exposure to petrochemical-derived chemicals. The objective of this study was to examine associations of soluble metals with lung and liver toxicity in fine particulate matter (PM2.5) in the vicinity of a petrochemical complex. PM2.5 was collected in the vicinity of a petrochemical complex of Mailiao Township (Yunlin County, Taiwan) to investigate lung and liver toxicity in BALB/c mice. The PM2.5 concentration was 30.2 ± 11.2μg/m3, and the PM2.5 was clustered in major local emissions (19.1μg/m3) and minor local emissions (14.1μg/m3) using a k-means clustering model. The PM2.5 (50 and 150μg/kg) and PM2.5-equivalent soluble nickel (Ni), vanadium (V), and lead (Pb) concentrations were intratracheally instilled into BALB/c mice. PM2.5 and V significantly decreased the tidal volume after exposure (p< 0.05). The peak expiratory flow (PEF) and peak inspiratory flow (PIF)/PEF ratio were significantly altered by 150μg/kgV (p< 0.05). V and Pb significantly increased total protein and lactate dehydrogenase (LDH) levels in bronchoalveolar lavage fluid (BALF) (p< 0.05). Interleukin (IL)-6 in BALF significantly increased after exposure to Pb (p< 0.05) accompanied by lung inflammatory infiltration. PM2.5 and Pb significantly increased levels of 8-isoprostane (p< 0.05). The level of caspase-3 activity significantly increased after exposure to Pb (p< 0.05). LDH in the liver was significantly increased by PM2.5 (p< 0.05). 8-Isoprostane in the liver was significantly increased by PM2.5 and Pb (p< 0.05). IL-6 in the liver was significantly increased by PM2.5, Ni, V, and Pb after exposure (p< 0.05), accompanied by liver inflammatory infiltration. Our results demonstrated that V in PM2.5 was associated with an increase in 8-isoprostane for all emissions and major local petrochemical emissions. In conclusion, V contributes to in vivo liver toxicity induced by PM2.5 in the vicinity of a petrochemical complex.

Iron overload causes a mild and transient increase in acute lung injury.

Recent studies have demonstrated a strong link between acute respiratory distress syndrome (ARDS) and the levels of iron and iron‐related proteins in the lungs. However, the role of iron overload in ARDS development has yet to be characterized. In this study, we compared the highly iron‐overloaded hepcidin knockout mice (HKO) to their iron‐sufficient wild‐type (WT) littermates in a model of sterile acute lung injury (ALI) induced by treatment with oropharyngeal (OP) LPS. There were no major differences in systemic inflammatory response or airway neutrophil infiltration between the two groups at the time of maximal injury (days 2 and 3) or during the recovery phase (day 7). Hepcidin knockout mice had transiently increased bronchoalveolar lavage fluid (BALF) protein and MPO activity in the lung and BALF on day 3, indicating worse vascular leakage and increased neutrophil activity, respectively. The increased ALI severity in iron‐overloaded mice may be a result of increased apoptosis of lung tissue, as evidenced by an increase in cleaved capsase‐3 protein in lung homogenates from HKO mice versus WT mice on day 3. Altogether, our data suggest that even severe iron overload has a relatively minor and transient effect in LPS‐induced ALI.

Pulmonary and systemic responses to aerosolized lysate of Staphylococcus aureus and Escherichia coli in calves

BackgroundConstitutive and inducible defenses protect the respiratory tract from bacterial infection. The objective of this study was to characterize the response to an aerosolized lysate of killed bacteria, as a basis for studying the regulation and in vivo effects of these inducible innate immune responses.ResultsBacterial lysate consisting of heat-killed and sonicated Staphylococcus aureus and Escherichia coli was aerosolized to 6 calves and systemic and pulmonary innate immune and inflammatory responses were measured in the first 24 h relative to baseline. Evaluated parameters included clinical parameters (body temperature and heart and respiratory rates), blood acute phase proteins and leukocyte counts, and leukocytes and proteins in bronchoalveolar lavage fluid. Mild clinical signs with increased heart rates and rectal temperatures developed following administration of the lysate, with resolution by 24 h. Serum haptoglobin and plasma fibrinogen concentrations were elevated at 24 h relative to baseline. Bronchoalveolar lavage fluid (BALF) had increased cellularity and increased proportion of neutrophils, as well as higher concentrations of interleukin (IL)-8, IL-10 and total protein at 24 h relative to baseline. Mass spectrometry identified 965 unique proteins in BALF: 19 proteins were increased and 26 proteins were decreased relative to baseline. The upregulated proteins included those involved in innate immunity including activation of complement, neutrophils and platelets. At postmortem examination, calves receiving higher doses of lysate had areas of lobular consolidation and interlobular edema. Histologically, neutrophils were present within bronchioles and to a lesser extent within alveoli. Calves receiving highest doses of lysate had patchy areas of neutrophils, hemorrhage and hyaline membranes within alveoli.ConclusionsAerosolization of bacterial lysate stimulated an innate immune response in lungs and airways, with alveolar damage observed at higher doses. Such a stimulus could be of value for investigating the effects of inducible innate immune responses on occurrence of disease, or for evaluating how stress, drugs or genetics affect these dynamic responses of the respiratory tract.

Cyclophilin A as a Novel Mediator in Ventilator‐Induced Lung Injury

RationaleCyclophilin A (CypA) is a ubiquitously distributed cytoplasmic protein. While focus has previously been on its intracellular roles, extracellular CypA and its receptor CD147 have emerged as a novel signalling axis in various inflammatory diseases. We explore the role of the CypA:CD147 axis in ventilator‐induced lung injury (VILI) and hypothesise that CypA is secreted into the alveolar space following epithelial insult where it promotes inflammation during acute lung injury (ALI).MethodsC57BL/6 mice received low‐stretch (LS; tidal volume: 8 mL/kg) or high‐stretch (HS: 40 mL/kg) ventilation for up to three hours (n=6–8/group). For inhibition experiments, mice received a 50 μL intratracheal dose of either a non‐permeable extracellular‐specific CypA inhibitor, MM‐284 (0.67 mM), or vehicle control, DMSO (10%), immediately prior to HS ventilation. Bronchoalveolar lavage fluid (BALF) CypA and soluble receptor for advanced glycation endproducts (sRAGE) were assessed by ELISA; cell activation status (surface ICAM‐1 expression) and intracellular CypA (iCypA) by flow cytometry; and matrix metalloproteinase‐2 (MMP‐2) by gelatin zymography.ResultsDuring VILI, there is a significant early upregulation of BALF CypA at two hours (LS: 663±268 vs HS: 2,586±1,054 pg/mL; p&lt;0.05), preceding signs of physiological deterioration, with further increase at three hours. In contrast, plasma CypA levels showed no significant changes across three hours regardless of ventilation strategy and were consistently below BALF levels. iCypA decreased in EpCAM‐/T1α+ and EpCAM+/T1α‐epithelial cells but not alveolar macrophages. MM‐284 mediated inhibition of extracellular CypA led to protection from the development of VILI (vehicle: 50% vs MM‐284: 100% survival; p&lt;0.05), attenuated lung wet:dry weight ratio (7.0±1.2 vs 5.0±0.2; p&lt;0.001), BALF protein (3.7±1.4 vs 1.3±0.2 mg/mL; p&lt;0.001), BALF sRAGE (40.3±11.2 vs 8.3±3.8 ng/mL; p&lt;0.0001), and deteriorations in elastance and oxygenation (interaction p&lt;0.01). Surprisingly, CypA inhibition appeared to have no impact on leukocyte recruitment into lung tissue but was associated with a reduction in stretch‐induced activation of alveolar macrophages (135.6±18.4 vs 21.8±45.4 ICAM‐1 MFI; p&lt;0.001) and both EpCAM‐/T1α+ (129.1±28.7 vs 91.9±6.1 ICAM‐1 MFI; p&lt;0.05) and EpCAM+/T1α‐(99.8±12.1 vs 76.5±15.2 ICAM‐1 MFI; p&lt;0.05) epithelial cells. Further, CypA inhibition also led to a decrease in active MMP‐2 in BALF (0.9±0.5 vs 0.4±0.2 relative band intensity; p&lt;0.05).ConclusionsWe have demonstrated the involvement of a novel mediator, CypA, in VILI. The early upregulation in BALF and corresponding decrease of iCypA in epithelial cells suggest that CypA is produced locally following epithelial injury. Inhibition of extracellular CypA promoted an impressive attenuation of VILI, particularly parameters related to alveolar‐capillary barrier integrity (e.g. BALF sRAGE, oedema formation, oxygenation, and MMP activity). Together, our findings support the CypA:CD147 axis as a novel mediator of epithelial cell dysfunction during the pathogenesis of VILI.Support or Funding InformationBritish Journal of Anaesthesia/Royal College of Anaesthetists

PPARγ Regulates the Inflammatory Response to Ozone‐Induced Lung Injury in Mice

Ozone is an urban air pollutant that is highly reactive causing tissue damage and inflammation in the lung. Long term exposure is linked to various health complications and increased mortality due to respiratory diseases. The inflammatory response to ozone is regulated by distinct populations of macrophages, which consist of M1/pro‐inflammatory and M2/anti‐inflammatory subsets; these cells are involved in the acute response to injury and long term resolution of tissue damage, respectively. Previous studies from our laboratory demonstrated that exposure of mice to ozone caused significant downregulation of PPARγ, a transcription factor important in anti‐inflammatory signaling in macrophages, phagocytosis of apoptotic neutrophils (efferocytosis), and the resolution of inflammation. We hypothesized that administration of a pharmacological agonist of PPARγ would reduce ozone‐induced lung injury by altering macrophage phenotype. Female C57BL/6J mice were exposed to air or ozone (0.8 ppm, 3 hr) and treated with vehicle control or rosiglitazone, a PPARγ agonist, daily via intraperitoneal injection beginning 24 hr prior to ozone exposure; mice were euthanized 24, 48, and 72 hr post ozone. Bronchoalveolar lavage fluid (BAL) was collected and analyzed for total protein and phospholipid content. BAL was enriched for alveolar macrophages by gentle lung massage and isolated cells analyzed by flow cytometry and qPCR. Ozone caused increases in total protein and phospholipid content, consistent with lung injury. In rosiglitazone‐treated animals, there was a trend towards reduced BAL protein levels at 48 hr and significant reduction in phospholipid content at 72 hr, suggesting accelerated injury resolution. Flow cytometric analysis showed increases in both pro‐ and anti‐inflammatory macrophages in the lungs of ozone‐exposed animals throughout the time‐course; a trend towards reduced numbers of these cells was observed in rosiglitazone‐treated animals at 72 hr. These results were consistent with reduced mRNA expression of pro‐ and anti‐inflammatory markers Ptgs2 and Arg1, respectively, at 72 hr. mRNA expression of Caveolin 1, a downstream target of PPARγ involved in lipid catabolism, was reduced in response to ozone but maintained in rosiglitazone‐treated animals. Collectively, these results suggest that PPARγ may modulate ozone‐induced lung injury by regulating inflammatory signaling and lipid metabolism in alveolar macrophages.Support or Funding InformationSupported by NIH Grants ES004738, ES005022, ES029254, ES007148, ES030984.

Comparative Pulmonary Protective Efficacy of Amifostine and it’s Analogue S-2(2-aminoethylamino)ethyl Phenyl Sulfide (DRDE-07) against Sulphur Mustard Induced Oxidative Stress and Inflammation in Female Mice

Aim: The present study was undertaken to investigate the comparative pulmonary protective efficacy of Amifostine (S-2[3-aminoprophylamino] ethyl phosphorothioate) and its analogues DRDE-07 (S-2(2-aminoethylamino) ethyl phenyl sulfide) against sulfur mustard toxicity in mice. Materials and Methods: Twenty female mice were divided into four groups: Control, SM group animals were percutaneously exposed to 16.2 mg/kg. The third and fourth group of animals received amifostine and DRDE-07 (210 and 250 mg/kg respectively) through the oral route, 30 min before SM exposure. The clinical symptoms and body weight changes were observed daily and sacrificed on 7th day. Bronchoalveolar lavage fluid (BALF) and lung tissuewere collected for biochemical and histopathological studies. The following biochemical endpoints were studied in BALF (total cell count, lactate dehydrogenase, protein content, β-glucuronidase activity, MMP-2, 9 activityand FSH) whereas reactive oxygen species (ROS), reduced glutathione (GSH), lipid peroxidation, superoxide dismutase, catalase and myeloperoxidase activity was measured in lung tissue. The above biochemical observations are also supported by histopathology studies. Results: Dermal exposure to SM significantly reduced body weight. The significant increase in BALF LDH leakage, protein content, cell number and MMPs activity in the SM exposed animals suggest disruption of endothelial barrier in the lung (p<0.05). Significant ROS generation (p<0.05) was observed in lung tissue of SM group which results in a significant decrease in SOD GSHand CAT and an increase in MDA (p<0.05). These alterations in BALF as well in lung tissue due to SM exposure was significantly prevented by the pretreatment of amifostine and DRDE-07 (p<0.05). The histopathological observations also support the above results. The above results indicate that the preventive efficacy of DRDE-07 is better than amifostine. Conclusion: The percutaneous SM exposure-induced pulmonary damages were significantly protected by DRDE-07 than amifostine in mice.

Glyburide attenuates ozone-induced pulmonary inflammation and injury by blocking the NLRP3 inflammasome.

Glyburide is a classic antidiabetic drug that is dominant in inflammation regulation, but its specific role in ozone-induced lung inflammation and injury remains unclear. In order to investigate whether glyburide prevents ozone-induced pulmonary inflammation and its mechanism, C57BL/6 mice were intratracheally pre-instilled with glyburide or the vehicle 1 hour before ozone (1 ppm, 3 hours) or filtered air exposure. After 24 hours, the total inflammatory cells and total protein in bronchoalveolar lavage fluid (BALF) were detected. The pathological alternations in lung tissues were evaluated by HE staining. The expression of NLRP3, interleukin-1β (IL-1β), and IL-18 protein in lung tissues was detected by immunohistochemistry. Western blotting was used to examine the levels of caspase-1 p10 and active IL-1β protein. Levels of IL-1β and IL-18 in BALF were measured using ELISA kits. Glyburide treatment decreased the total cells in BALF, the inflammatory score, and the mean linear intercept induced by ozone in lung tissues. In addition, glyburide inhibited the expression of NLRP3, IL-18, and IL-1β protein in lung tissues, and also suppressed NLRP3 inflammasome activation, including caspase-1 p10, active IL-1β protein in lung tissues, IL-1β, and IL-18 in BALF. These results demonstrate that glyburide effectively attenuates ozone-induced pulmonary inflammation and injury via blocking the NLRP3 inflammasome.

Extracellular nucleic acid scavenging rescues rats from sulfur mustard analog-induced lung injury and mortality.

Sulfur mustard (SM) is a highly toxic war chemical that causes significant morbidity and mortality and lacks any effective therapy. Rats exposed to aerosolized CEES (2-chloroethyl ethyl sulfide; 10% in ethanol), an analog of SM, developed acute respiratory distress syndrome (ARDS), which is characterized by increased inflammation, hypoxemia and impaired gas exchange. We observed elevated levels of extracellular nucleic acids (eNA) in the bronchoalveolar lavage fluid (BALF) of CEES-exposed animals. eNA can induce inflammation, coagulation and barrier dysfunction. Treatment with hexadimethrine bromide (HDMBr; 10mg/kg), an eNA neutralizing agent, 2h post-exposure, reduced lung injury, inhibited disruption of alveolar-capillary barrier, improved blood oxygenation (PaO2/FiO2 ratio), thus reversing ARDS symptoms. HDMBr treatment also reduced lung inflammation in the CEES-exposed animals by decreasing IL-6, IL-1A, CXCL-1 and CCL-2 mRNA levels in lung tissues and HMGB1 protein in BALF. Furthermore, HDMBr treatment also reduced levels of lung tissue factor and plasminogen activator inhibitor-1 indicating reduction in clot formation and increased fibrinolysis. Fibrin was reduced in BALF of the HDMBr-treated animals. This was further confirmed by histology that revealed diminished airway fibrin, epithelial sloughing and hyaline membrane in the lungs of HDMBr-treated animals. HDMBr completely rescued the CEES-associated mortality 12h post-exposure when the survival rate in CEES-only group was just 50%. Experimental eNA treatment of cells caused increased inflammation that was reversed by HDMBr. These results demonstrate a role of eNA in the pathogenesis of CEES/SM-induced injury and that its neutralization can serve as a potential therapeutic approach in treating SM toxicity.

The Involvement of Type 2 Innate Lymphoid Cells in Airway Inflammation of Asthma.

The airway inflammatory response is closely associated with asthma. The purpose of this article was to study the roles of innate lymphoid cells (ILCs) in the process of airway inflammatory response in asthma. We established the asthmatic mice model with intraperitoneal injected ovalbumin medium, then with the flow cytometry analysis, we detected the ILCs and their surface proteins in the mice blood samples, besides, we analyzed the amounts of inflammatory cytokines and secreted proteins in the mice bronchoalveolar lavage fluid and blood serum. Moreover, Western blot analyzed the proteins in the mice bronchial epithelial tissues. The ILC2 amounts were obviously increased in young asthmatic mice model. And, the proteins CD25 and CCR10 were highly expressed in the sorted ILC2s. Besides, the cytokines interleukin (IL)-5, IL-13, IL-33, CCL22, and CCL27 were abundant in the bronchoalveolar lavage fluid of asthmatic mice model. And, the secretion of IL-5, IL-13, IL-33, TSLP, and CCL22 in blood serum was much more in asthmatic mice model than in the normal control mice, whereas the secretion of PGD2 was suppressed in asthmatic mice bronchoalveolar lavage fluid and blood serum. Additionally, the guanine nucleotide-binding proteins Gα12 and Gα13 were upregulated in asthmatic mice bronchial tissues, and the protein SERCA2 was downregulated; moreover, the proteins NFAT, IRF4, and its downstream signal STAT6 were all upregulated in the asthmatic mice bronchial tissues. ILC2s were involved in the response of airway inflammation through secretion of proinflammatory cytokines and chemokines to dysregulate the Ca2+ homeostasis in airway in the process of asthma. [Figure: see text].

Pretreatment of nafithromycin attenuates inflammatory response in murine lipopolysaccharide induced acute lung injury

Acute respiratory distress syndrome following an acute lung injury (ALI) is a life threatening inflammatory condition predominantly characterized by vascular protein leakage, neutrophil recruitment and overexpression of proinflammatory cytokines. Pulmonary and systemic bacterial infections are the major cause of ALI wherein the bacterial cell components play a crucial role. Macrolide/ketolide antibiotics are reported to possess immunomodulatory activity; as a result improved survival has been noted in pneumonia patients. Hence immunomodulatory activity of nafithromycin, a novel lactone ketolide antibacterial agent was assessed in the murine LPS induced ALI model. Vehicle, nafithromycin (100 mg/kg), azithromycin (600 mg/kg) and dexamethasone (20 mg/kg) were administered orally, 1 h prior to LPS challenge and bronchoalveolar lavage (BAL) fluid was collected thereafter at 18, 24 and 48 h to determine the total cell count, total protein, myeloperoxidase (MPO), tumor necrosis factor (TNF)-α and interleukin (IL)-6. Results from the current study showed that pretreatment with nafithromycin significantly reduced the total cell count, total protein, MPO, TNF-α and IL-6 levels in BAL fluid compared to LPS control group. Histopathological evaluations also suggest significant reduction in neutrophil infiltration by nafithromycin. Dexamethasone, a positive reference standard as expected exhibited potent anti-inflammatory activity. The immunomodulatory effect of nafithromycin at dose of 100 mg/kg was comparable to azithromycin dosed at 600 mg/kg. As a result of immunomodulatory activity, nafithromycin is expected to provide additional clinical benefits by resolving the secondary complications associated with severe pneumonia and thereby improving survival in such patients.

An animal model of transfusion-related acute lung injury and the role of soluble CD40 ligand.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of transfusion and is one of leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear. Soluble CD40 ligand (sCD40L) is a proinflammatory cytokine that accumulates during blood component storage and is involved in transfusion reactions. The objective of this study was to establish a clinically relevant TRALI animal model and to evaluate the role of sCD40L in TRALI. Rats' red-blood-cell (RBC) suspensions were prepared, and the quality of RBC was evaluated. A trauma-haemorrhage-transfusion strategy was applied to build the animal model. Lung oedema was evaluated by histopathology examination, total bronchoalveolar lavage fluid (BALF) protein concentration, Evans blue dye (EBD) leakage and inflammatory cytokines. The sCD40L concentrations were measured. Storage lesions of RBCs gradually increased over time. Obvious histological evidence of lung injury of rats transfused with a 35-day RBC was observed. The total BALF protein concentration, EBD leakage, inflammatory cytokines concentration were increased significantly in the Day 35 group. The sCD40L concentration increased significantly in the storage RBC suspension over time but was slightly elevated in rat plasma. These findings indicated successful establishment of a TRALI animal model with trauma-haemorrhage-transfusion, in which sCD40L may play a minor role in the development of TRALI.

Vascular Endothelial Growth Factor-121 Administration Mitigates Halogen Inhalation-Induced Pulmonary Injury and Fetal Growth Restriction in Pregnant Mice.

BackgroundCirculating levels of sFLT‐1 (soluble fms‐like tyrosine kinase 1), the extracellular domain of vascular endothelial growth factor (VEGF) receptor 1, and its ratio to levels of placental growth factor are markers of the occurrence and severity of preeclampsia.Methods and ResultsC57BL/6 pregnant mice on embryonic day 14.5 (E14.5), male, and non‐pregnant female mice were exposed to air or to Br2 at 600 ppm for 30 minutes and were treated with vehicle or with VEGF‐121 (100 μg/kg, subcutaneously) daily, starting 48 hours post‐exposure. Plasma, bronchoalveolar lavage fluid, lungs, fetuses, and placentas were collected 120 hours post‐exposure. In Br2‐exposed pregnant mice, there was a time‐dependent and significant increase in plasma levels of sFLT‐1 which correlated with increases in mouse lung wet/dry weights and bronchoalveolar lavage fluid protein content. Supplementation of exogenous VEGF‐121 improved survival and weight gain, reduced lung wet/dry weights, decreased bronchoalveolar lavage fluid protein levels, enhanced placental development, and improved fetal growth in pregnant mice exposed to Br2. Exogenous VEGF‐121 administration had no effect in non‐pregnant mice.ConclusionsThese results implicate inhibition of VEGF signaling driven by sFLT‐1 overexpression as a mechanism of pregnancy‐specific injury leading to lung edema, maternal mortality, and fetal growth restriction after bromine gas exposure.