Neutrophils In Bronchoalveolar Lavage Fluid Research Articles

Mechanism of chlorogenic acid reducing lipopolysaccharide-induced acute lung injury in mice by regulating miR-223/NLRP3 axis.

Chlorogenic acid has various physiological activities such as antibacterial, anti-inflammatory, and antiviral activities. Studies have shown that chlorogenic acid can alleviate the inflammatory response of mice with acute lung injury (ALI), but the specific mechanism is still unclear. This study aims to investigate whether chlorogenic acid attenuates lipopolysaccharide (LPS)-induced ALI in mice by regulating the microRNA-223 (miR-223)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) axis. SPF grade BALBc male mice were randomly divided into a control group, a model group, a chlorogenic acid group, a chlorogenic acid+miR-223 negative control (miR-223 NC) group, and a chlorogenic acid+miR-223 inhibitor (miR-223 antagomir) group, 10 mice in each group. Except the control group, the other groups were instilled with 4 mg/kg LPS through the airway to establish the ALI mouse model. After the modeling, the mice in the chlorogenic acid group were continuously given chlorogenic acid (100 mg/kg) by gavage for 7 d. The chlorogenic acid+miR-223 NC group and the chlorogenic acid+miR-223 antagomir group were given 100 mg/kg chlorogenic acid by gavage every day, and then were injected with 10 μL of miR-223 NC (0.5 nmol/μL) and miR-223 antagomir (0.5 nmol/μL) respectively for 7 consecutive days.The control group and the model group were replaced with normal saline. The lung tissues of mice were taken to measure the ratios of lung wet to dry weight (W/D). The bronchoalveolar lavage fluid of mice was collected to measure the levels of TNF-α, IL-6, and IL-1β by ELISA kit and to count the number of eosinophils (EOS), lymphocytes, neutrophils under light microscope. After HE staining, the pathological changes of lung tissues were observed and lung injury was scored. qRT-PCR method were used to determine the expression levels of miR-223 in lung tissues. Western blotting was used to determine the expression levels of NLRP3 protein in mouse lung tissues. Luciferase reporter assay was used to analyze the targeting relationship of miR-223 to NLRP3. Compared with the control group, the lung W/D value, the lung injury score and the level of inflammatory factors in the bronchoalveolar lavage fluid were significantly increased in the model group (all P<0.05); the infiltration of inflammatory cells in the lung tissue was severe; the alveolar space was significantly increased; the alveolar wall was significantly thickened; the number of EOS, lymphocytes, and neutrophils in the bronchoalveolar lavage fluid was significantly increased (all P<0.05); the expression levels of miR-223 in lung tissue were significantly decreased (P<0.05); and the protein expression levels of NLRP3 were significantly increased (P<0.05). Compared with the model group, the W/D value of lungs, lung injury score, and levels of inflammatory factors in bronchoalveolar lavage fluid were significantly decreased in the chlorogenic acid group, the chlorogenic acid+miR-223 NC group, and the chlorogenic acid+miR-223 antagomir group (all P<0.05); lung tissues damage was alleviated; the numbers of EOS, lymphocytes, and neutrophils in bronchoalveolar lavage fluid were significantly decreased (all P<0.05); the expression levels of miR-223 in lung tissues were significantly increased (P<0.05); and the expression levels of NLRP3 protein were significantly decreased (P<0.05). Compared with the chlorogenic acid group, the lung W/D value, lung injury score, and inflammatory factor levels in the bronchoalveolar lavage fluid were significantly increased in the chlorogenic acid+miR-223 antagomir group (all P<0.05); lung tissue damage was aggravated; the number of EOS, lymphocytes and neutrophils in bronchoalveolar lavage fluid significantly increased (all P<0.05); the expression levels of miR-223 in lung tissues were significantly decreased (P<0.05); and the expression levels of NLRP3 protein were significantly increased (P<0.05). The results of luciferase reporter assay showed that miR-223 had a targeting relationship with NLRP3. Chlorogenic acid may increase the level of miR-223, target the inhibition of NLRP3 expression, reduce LPS-induced inflammatory response in ALI mice, and alleviate pathological damage of lung tissues.

Knockout of GGPPS1 restrains rab37-mediated autophagy in response to ventilator-induced lung injury.

Mechanical ventilation may cause ventilator-induced lung injury (VILI) in patients requiring ventilator support. Inhibition of autophagy is an important approach to ameliorate VILI as it always enhances lung injury after exposure to various stress agents. This study aimed to further reveal the potential mechanisms underlying the effects of geranylgeranyl diphosphate synthase large subunit 1 (GGPPS1) knockout and autophagy in VILI using C57BL/6 mice with lung-specific GGPPS1 knockout that were subjected to mechanical ventilation. The results demonstrate that GGPPS1 knockout mice exhibit significantly attenuated VILI based on the histologic score, the lung wet-to-dry ratio, total protein levels, neutrophils in bronchoalveolar lavage fluid, and reduced levels of inflammatory cytokines. Importantly, the expression levels of autophagy markers were obviously decreased in GGPPS1 knockout mice compared with wild-type mice. The inhibitory effects of GGPPS1 knockout on autophagy were further confirmed by measuring the ultrastructural change of lung tissues under transmission electron microscopy. In addition, knockdown of GGPPS1 in RAW264.7 cells reduced cyclic stretch-induced inflammation and autophagy. The benefits of GGPPS1 knockout for VILI can be partially eliminated through treatment with rapamycin. Further analysis revealed that Rab37 was significantly downregulated in GGPPS1 knockout mice after mechanical ventilation, while it was highly expressed in the control group. Simultaneously, Rab37 overexpression significantly enhances autophagy in cells that are treated with cyclin stretch, including GGPPS1 knockout cells. Collectively, our results indicate that GGPPS1 knockout results in reduced expression of Rab37 proteins, further restraining autophagy and VILI.

Treatment with senicapoc, a KCa 3.1 channel blocker, alleviates hypoxaemia in a mouse model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by pulmonary oedema and severe hypoxaemia. We investigated whether genetic deficit or blockade of calcium-activated potassium (KCa 3.1) channels would counteract pulmonary oedema and hypoxaemia in ventilator-induced lung injury, an experimental model for ARDS. KCa 3.1 channel knockout (Kccn4-/- ) mice were exposed to ventilator-induced lung injury. Control mice exposed to ventilator-induced lung injury were treated with the KCa 3.1 channel inhibitor, senicapoc. The outcomes were oxygenation (PaO2 /FiO2 ratio), lung compliance, lung wet-to-dry weight and protein and cytokines in bronchoalveolar lavage fluid (BALF). Ventilator-induced lung injury resulted in lung oedema, decreased lung compliance, a severe drop in PaO2 /FiO2 ratio, increased protein, neutrophils and tumour necrosis factor-alpha (TNF-α) in BALF from wild-type mice compared with Kccn4-/- mice. Pretreatment with senicapoc (10-70 mg·kg-1 ) prevented the reduction in PaO2 /FiO2 ratio, decrease in lung compliance, increased protein and TNF-α. Senicapoc (30 mg·kg-1 ) reduced histopathological lung injury score and neutrophils in BALF. After injurious ventilation, administration of 30 mg·kg-1 senicapoc also improved the PaO2 /FiO2 ratio and reduced lung injury score and neutrophils in the BALF compared with vehicle-treated mice. In human lung epithelial cells, senicapoc decreased TNF-α-induced permeability. Genetic deficiency of KCa 3.1 channels and senicapoc improved the PaO2 /FiO2 ratio and decreased the cytokines after a ventilator-induced lung injury. Moreover, senicapoc directly affects lung epithelial cells and blocks neutrophil infiltration in the injured lung. These findings indicate that blocking KCa 3.1 channels is a potential treatment in ARDS-like disease.

Neutrophils in Bronchoalveolar Lavage Fluid Indicating the Severity and Relapse of Pulmonary Sarcoidosis.

BackgroundPulmonary sarcoidosis is a highly heterogeneous granulomatous disease without any specific symptoms and manifestations. Neutrophils in bronchoalveolar lavage fluid (BALF) have been proposed to indicate the severity and prognosis of pulmonary sarcoidosis, but this needs confirmation in patients from different populations due to the heterogeneity of the disease. This study aimed to determine the characteristics of patients with pulmonary sarcoidosis in northeastern China and to explore the relationship between neutrophils in BALF and the severity of pulmonary sarcoidosis.MethodsWe enrolled 432 patients who were diagnosed with pulmonary sarcoidosis in this retrospective study. The symptoms, extrapulmonary involvement, forced vital capacity percentage predicted (FVC % pred), and diffusing capacity of the lung for carbon monoxide percentage predicted (DLco % pred) were recorded. BAL was performed in 319 patients, and the results of a cellular examination of BALF were collected. A total of 123 patients who received corticosteroid treatment were followed up for at least 12 months, and the outcomes were recorded.ResultsCough was the most common symptom, and cutaneous involvement was the most common extrapulmonary manifestation in 304 (70.4%) and 82 (19.0%) patients, respectively. The percentages of patients with high neutrophil counts in BALF (>3%) were higher at Stages 2 and 3 compared with Stages 0 and 1 (33.2 vs. 19.4%, p = 0.007), although the percentages of neutrophils in BALF showed no difference between patients at Stages 0, 1, 2, and 3. Patients with high neutrophil counts in BALF had lower FVC % pred compared with the other patients (79.5 ± 18.2 vs. 84.9 ± 14.5%, p = 0.025) and were prone to relapse after corticosteroids were tapered. High neutrophil counts in BALF were independently associated with relapse after corticosteroids were tapered in a binary logistic regression analysis (p = 0.027).ConclusionsPatients with pulmonary sarcoidosis lacked specific symptoms and manifestations. The neutrophil count in BALF could indicate the severity and outcomes of pulmonary sarcoidosis.

Effects of forages, dust exposure and proresolving lipids on airway inflammation in horses.

To investigate the role of omega-3 polyunsaturated fatty acids (Ω-3)-derived proresolving lipid mediators (PRLM) in the resolution of mild airway inflammation in horses. 20 horses with mild airway inflammation. Horses previously eating hay were fed hay pellets (low Ω-3 content; n = 10) or haylage (high Ω-3 content; 9) for 6 weeks. Dust exposure was measured in the breathing zone with a real-time particulate monitor. Bronchoalveolar lavage (BAL) was performed at baseline, week 3, and week 6. The effect of PRLM on neutrophil apoptosis and efferocytosis was examined in vitro. BAL fluid inflammatory cell proportions, apoptosis of circulating neutrophils, efferocytosis displayed by alveolar macrophages, and plasma lipid concentrations were compared between groups fed low and high amounts of Ω-3 by use of repeated measures of generalized linear models. Dust exposure was significantly higher with hay feeding, compared to haylage and pellets, and equivalent between haylage and pellets. BAL fluid neutrophil proportions decreased significantly in horses fed haylage (baseline, 11.8 ± 2.4%; week 6, 2.5 ± 1.1%) but not pellets (baseline, 12.1 ± 2.3%; week 6, 8.5% ± 1.7%). At week 6, horses eating haylage had significantly lower BAL neutrophil proportions than those eating pellets, and a significantly lower concentration of stearic acid than at baseline. PRLM treatments did not affect neutrophil apoptosis or efferocytosis. Despite similar reduction in dust exposure, horses fed haylage displayed greater resolution of airway inflammation than those fed pellets. This improvement was not associated with increased plasma Ω-3 concentrations. Feeding haylage improves airway inflammation beyond that due to reduced dust exposure, though the mechanism remains unclear.

Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection.

ABSTRACTMycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited.IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

Implication of mitochondrial ROS-NLRP3 inflammasome axis during two-hit mediated acute lung injury in mice

Acute lung injury (ALI) caused by acid aspiration often accompanies bacterial components leading to exaggerated inflammation and can result in acute respiratory distress syndrome (ARDS), but the underlying mechanisms behind such an exacerbation remain unclear. NLRP3 inflammasome and mitochondrial ROS (mtROS) have been implicated in ALI but its role in injury caused through two hit i.e. Hydrochloric acid (HCl) + Lipopolysaccharide (LPS) is not known. Therefore, the present study is designed to elucidate the role of mtROS-NLPR3 inflammasome upon “two-hit” mediated ALI. Our data showed that “two-hit” induced ALI results in aggravated lung inflammation as compared to either of single hit(s) as reflected by a steep increase in inflammatory cells particularly neutrophils in bronchoalveolar lavage fluid (BALF). Further, enhanced inflammation was associated with increased mtROS as depicted by data on mean fluorescence intensity (MFI) of MitoSOX+ neutrophils and macrophages in BALF of two-hit simulated mice. Importantly, ALI results in activation of NLRP3 inflammasome as reflected by active caspase-1 protein expression and IL-1β levels. Interestingly, NLRP3 inflammasome inhibitor, MCC950 suppressed the lung inflammation remarkably. Further, Mito-tempo, a mitochondrial-targeted antioxidant, halted “two-hit” mediated NLRP3 inflammasome activation and IL-1β release followed by amelioration of lung inflammation. Suppression in MFI of MitoSOX+ stained neutrophils and macrophages by Mito-tempo was associated with down-regulation of phospho-p65-NF-κB and its dependent genes (IL-1β/TNF-α/IL-6). Overall, our data suggest that NLRP3 inflammasome activation by mtROS plays a critical role in pathogenesis of exaggerated inflammation and therefore targeting mtROS-NLRP3 inflammasome axis may be an attractive option for combating ALI/ARDS.

Effects of Bedding Material on Equine Lower Airway Inflammation: A Comparison of Two Peat Beddings, Wood Pellet, and Straw Pellet.

The effects of bedding material on air quality are important amongst horses worldwide. Respiratory diseases, especially equine asthma, are highly prevalent with air hygiene playing a major role on the pathophysiology of these diseases. The objective of our study was to investigate the effects of four bedding materials on the respiratory signs, tracheal mucus score, and tracheal wash (TW) and bronchoalveolar lavage fluid (BALF) cytology in healthy adult horses. The study design was a prospective controlled cross-over study, and the subjects were healthy adult riding school horses (n = 32) from a single stable. Wood pellet, straw pellet, and loosely stored peat (Peat 3) were compared to peat packed in plastic-covered bales (Peat 2). Lower airway endoscopy and sampling (TW and BALF) for cytological examination were performed after each 35-day bedding period. The tracheal mucus scores (P = 0.014) and respiratory rate (P = 0.026) were higher during the straw pellet period compared to the Peat 2 period. The respiratory rate was lower during the wood pellet period compared to the Peat 2 period (P = 0.004). The TW neutrophil percentage during the straw pellet period was higher compared to the Peat 2 period (P = 0.0003). The BALF neutrophil percentage was higher during the straw pellet period (P = 0.005) and during the Peat 3 period compared to the Peat 2 period (P = 0.04). We conclude that baled peat (Peat 2) caused lower neutrophil percentages in the airway samples compared to straw pellet and loosely stored peat (Peat 3). No difference was observed between Peat 2 and wood pellet. The information gained from this study may assist veterinarians and horse owners in selecting appropriate bedding materials, especially for horses with equine asthma.

Salidroside Attenuates Airway Inflammation and Remodeling via the miR-323-3p/SOCS5 Axis in Asthmatic Mice

Introduction: Salidroside (Sal) a bioactive component extracted from Rhodiola rosea is remarkable for its anti-asthmatic effects. The study aimed to explore the molecular mechanism of Sal in airway inflammation and remodeling in asthmatic mice and provide a novel theoretical basis for asthma treatment. Methods: An asthmatic mouse model was established via ovalbumin (OVA) treatment, followed by injection of Sal and transfection of miR-323-3p-mimic and sh- suppressor of cytokine signaling 5 (SOCS5). Expressions of miR-323-3p, SOCS5 mRNA, collagen (COL)-I, and COL-III were detected via reverse transcription quantitative polymerase chain reaction. SOCS5 protein level was detected via Western blot. Levels of IgE, IL-13, IL-4, and IL-5 were detected via enzyme-linked immunosorbent assay. Inflammatory cell infiltration was observed via hematoxylin-eosin staining. Collagen disposition was observed via Masson staining. Resistance index (RI) of airway hyperresponsiveness, and the number of total cells, inflammatory cells (eosinophil, macrophage, neutrophil, and lymphocyte) in bronchoalveolar lavage fluid (BALF) were observed. The binding relationship between miR-323-3p and SOCS5 was predicted through the RNA22 website and verified via dual-luciferase reporter assay. Results: miR-323-3p was highly expressed in OVA-treated mice. Sal treatment reduced inflammatory cell infiltration, COL disposition, miR-323-3p expression, and IgE, IL-13, IL-4, IL-5, COL-I, and COL-III levels, RI value, and the number of total cells and inflammatory cells in BALF. miR-323-3p inhibited SOCS5 transcription. miR-323-3p overexpression or SOCS5 downregulation reversed the protecting role of Sal in asthmatic mice. Conclusion: Sal inhibited miR-323-3p expression to promote SOCS5 transcription, thereby attenuating airway inflammation and remodeling in asthmatic mice.

Erythropoietin alleviates acute lung injury induced by ischemia-reperfusion through blocking p38 MAPK signaling

Erythropoietin (EPO) has antiapoptotic, antioxidative, and anti-inflammatory effects on ischemia tissues and protects against acute lung injury (ALI) induced by ischemia-reperfusion (I/R). p38 mitogen-activated protein kinases (p38 MAPK) signaling is involved in the processes of I/R-induced ALI. However, the interaction of EPO with p38 MAPK signaling in I/R-induced ALI has not been reported. To explore this issue, we constructed an I/R-induced ALI model in vivo and in vitro using Sprague Dawley rats and BEAS-2B cells. Some I/R rats and hypoxia-reoxygenation (H/R)-induced cells were treated with EPO, and the others were used as control groups. The injuries of lung tissues and cells were respectively assessed by inflammatory cytokine, morphologic changes, cell viability, apoptosis, and oxidative damage-related factors. Western blot determined key proteins in the p38 MAPK signaling. Results indicated that I/R induced the increase of inflammatory factors, lung weight, filtration coefficient, bronchoalveolar lavage fluid protein content, apoptosis, neutrophil, and lung peroxidation, and H/R caused cell growth inhibition, apoptosis, and oxidative damage-related factors' release. EPO attenuated I/R-induced injury in vivo and in vitro. Furthermore, the increase of p-p38, p-JNK, and p-ERK1/2 in lung tissues and cells induced by I/R was downregulated by EPO. Moreover, both EPO and an inhibitor of p38 MAPK (SB203580) alleviated H/R-induced cell injury. Erythropoietin along with SB203580 had more obvious protection effects than EPO alone. Collectively, EPO alleviated I/R-induced ALI by blocking p38 MAPK signaling. The interaction mechanism of EPO with p38 MAPK signaling contributes to understanding the processes of I/R-induced ALI and provides new insights for the disease treatment.

The Role of Thoracic Ultrasonography and Airway Endoscopy in the Diagnosis of Equine Asthma and Exercise-Induced Pulmonary Hemorrhage.

Mild-moderate (MEA), severe (SEA) equine asthma and exercise-induced pulmonary hemorrhage (EIPH) are common respiratory disorders in horses. The present retrospective study aims to evaluate the role of ultrasonography and endoscopy in the diagnosis of these conditions. Three hundred and three horses were included and divided into SEA, MEA and MEA + EIPH groups, on the basis of history, clinical examination and bronchoalveolar lavage fluid (BALf) cytology; scores were assigned to lung ultrasonography, pharyngeal lymphoid hyperplasia (PLH), tracheal mucus (TM) and tracheal bifurcation edema (TB). These scores were compared between groups, and their associations with age, BALf cytology, tracheal wash microbiology and between endoscopic and ultrasonographic scores were statistically analyzed. Ultrasonographic scores were higher in the SEA and MEA + EIPH groups and associated with increased BALf neutrophils and hemosiderophages. The PLH score was higher in younger horses affected by MEA and EIPH and associated with increased eosinophils and hemosiderophages. TM and TB scores were greater in older horses affected by SEA, associated with increased neutrophils and inversely correlated with hemosiderophages. Moreover, TM grade was negatively correlated with mast cells. Thoracic ultrasonography and airway endoscopy can provide useful information about the inflammatory status of upper and lower airways in the horse.

PM2.5 induces airway hyperresponsiveness and inflammation via the AhR pathway in a sensitized Guinea pig asthma-like model

Exposure to fine particulate matter (PM2.5) induces airway inflammation and hyperreactivity that lead to asthma. The mechanisms involved are still under investigation. We investigated the effect of resveratrol (3,4’,5-trihydroxystilbene) (RES) on airway hyperresponsiveness, inflammation and CYP1A1 protein expression (an aryl hydrocarbon receptor (AhR) target) induced by PM2.5 exposure in an allergic asthma experimental guinea pig model. The polyphenolic compound RES was used due to its antioxidant and anti-inflammatory properties and as an antagonist of the AhR; thus, providing mechanistic insights.Animals were sensitized with aluminum hydroxide and ovalbumin and exposed to filtered air or PM2.5. Exposure to PM2.5 was conducted using a whole-body chamber particle concentrator (5 h/day) for 15 days. Animals received saline solution or RES (10 mg/kg per day) orally for 21 days simultaneously to the OVA challenge or PM2.5 exposure.PM2.5 exposure (mean 433 ± 111 μg/m3 in the exposure chamber) in OVA challenged animals induced an asthma-like phenotype characterized by increased baseline lung resistance (Rrs) and central airway resistance (Rn) in response to acetylcholine (ACh) evaluated using a flexiVent system®. A parallel increase of pro-inflammatory cytokines (IL-6, IL-17, TNF-α and IFN-γ), inflammatory cells (eosinophils and neutrophils) in bronchoalveolar lavage fluid (BALF) and lung CYP1A1 increase also occurred. RES significantly inhibited airway hyperresponsiveness, inflammation, and CYP1A1 protein expression in the OVA-challenged PM2.5 exposed animals.In summary, with the use of RES we demonstrate that PM-induced airway hyperreactivity is modulated by the inflammatory response via the AhR pathway in an allergic asthma guinea pig model.

Interventional effect of IL-17A on chronic obstructive pulmonary disease and its mechanism

Objective: To investigate the intervention effects and mechanism of interleukin-17A (IL-17A) on chronic obstructive pulmonary disease (COPD). Methods: C57BL/6 mice were randomly divided into wild type blank control group, wild type COPD group and IL-7A knockout COPD group. Mice in wild type blank control group received no treatment, and mice in the other two groups were exposed to cigarette smoke to induce COPD (Cigarette: 1 cigarette / time, 4 times/day, 45 minutes/time; interval time: 1 hour; total intervention time: 90 days). Lung function of mice was assessed using animal pulmonary function machine. Bronchoalveolar lavage fluid (BALF) of mice was collected and BALF cell count and classification were determined. The lung tissue of mice was collected, the expression level of IL-17A in airway epithelium was determined by flow cytometry, and the levels of inflammatory factors in lung tissue were determined by enzyme-linked immunosorbent assay. The expression level of JNK/AP1 signaling pathway protein in mouse lung tissue was determined by Western blot. Results: Compared with the wild type blank control group mice, the wild type COPD group mice had significantly higher expression level of IL-17A, significantly lower peak inspiratory flow rate (PIF) and peak expiratory flow rate (PEF), significantly higher number of BALF neutrophils, eosinophils, lymphocytes and macrophage, significantly higher expression levels of CXC chemokine 1(CXCL1), CXC chemokine 2 (CXCL2), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and significantly higher phosphorylation level of JNK, cJun and cFos and AP1 expression levels (P＜0.05). Compared with COPD mice, IL-17A expression level in airway epithelium of mice in IL-7A knockout COPD group was significantly lower, PIF and PEF were higher, the number of BALF neutrophils, eosinophils, lymphocytes and macrophage was significantly lower, the expression levels of CXCL1, CXCL2, IL-1β and IL-6 in lung tissue were lower, and the phosphorylation levels of JNK, cJun and cFos and AP1 expression levels were significantly lower (P＜0.05). Conclusion: Cigarette smoke can induce the production of IL-17A and reduce (or inhibit) the production (or expression or secretion) of IL-17A in mouse airway epithelium, thus inhibiting the JNK/AP1 signaling pathway to reduce the airway inflammation and improve the lung function of COPD mice.

Azilsartan attenuates lipopolysaccharide-induced acute lung injury via the Nrf2/HO-1 signaling pathway.

Acute lung injury (ALI) is a severe complication of sepsis and hemorrhagic shock with high morbidity. In the present study, the protective effect of Azilsartan on lipopolysaccharide (LPS)-induced ALI in mice was investigated to explore the potential therapeutic property of Azilsartan for the treatment of ALI. LPS was used to induce an ALI model in mice. Hematoxylin-eosin (HE) staining sections were then evaluated for the pathological state of lung tissues. Bronchoalveolar lavage fluid (BALF) protein concentration, wet/dry weight ratios of lung tissues, and pulmonary myeloperoxidase (MPO) activity were detected to determine the degree of pulmonary injury. The number of total cells, macrophages, and neutrophils in BALF were counted using a hemocytometer to illustrate the inflammatory cell infiltration. The lung function was monitored using a spirometer. The concentrations of interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and interleukin-8 (IL-8) were determined using enzyme-linked immunosorbent assay (ELISA). Oxidative stress was evaluated by the superoxide dismutase (SOD) activity, glutathione (GSH), and malondialdehyde (MDA) concentrations in the lung tissue. The expressions of nuclear erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined using Western blot analysis. Azilsartan therapy alleviated LPS-induced lung tissue damage, increased BALF protein concentration, lung wet to dry weight ratio, MPO activity, and macrophage and neutrophils infiltration. Also, Azilsartan ameliorated the production of inflammatory factors (IL-1β, MCP-1, and IL-8). Azilsartan ameliorated LPS-impaired lung SOD activity, the GSH concentration, and the MDA concentration. Mechanistically, Azilsartan activated the LPS-impaired Nrf2/HO-1 signaling pathway. Azilsartan therapy attenuates LPS-induced ALI via the Nrf2/HO-1 signaling pathway.

Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury.

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.

Diesel exhaust particles increase nasal symptoms and IL-17A in house dust mite-induced allergic mice

Diesel exhaust particles (DEPs), traffic-related air pollutants, are considered environmental factors adversely affecting allergic diseases. However, the immunological basis for the adjuvant effects of DEP in allergic rhinitis (AR) remains unclear. Therefore, this study aimed to investigate the effect of DEP exposure on AR using a mouse model. BALB/c mice sensitized to house dust mite (HDM) were intranasally challenged with HDM in the presence and absence of DEP. Allergic symptom scores, serum total and HDM-specific immunoglobulins (Igs), eosinophil infiltration in the nasal mucosa, cytological profiles in bronchoalveolar lavage fluid (BALF), and cytokine levels in the nasal mucosa and spleen cell culture were analyzed. Mice co-exposed to HDM and DEP showed increased allergic symptom scores compared with mice exposed to HDM alone. Reduced total IgE and HDM-specific IgE and IgG1 levels, decreased eosinophil infiltration in the nasal mucosa, and increased proportion of neutrophils in BALF were found in mice co-exposed to HDM and DEP. Interleukin (IL)-17A level was found to be increased in the nasal mucosa of the co-exposure group compared with that in the HDM-exposed group. The levels of IL-4, IL-13, interferon-γ, IL-25, IL-33, and TSLP expression showed no difference between the groups with and without DEP treatment. Increased expression of IL-17A in the nasal mucosa may contribute to DEP-mediated exacerbation of AR in HDM-sensitized murine AR model.

Long-term endogenous acetylcholine deficiency potentiates pulmonary inflammation in a murine model of elastase-induced emphysema

Acetylcholine (ACh), the neurotransmitter of the cholinergic system, regulates inflammation in several diseases including pulmonary diseases. ACh is also involved in a non-neuronal mechanism that modulates the innate immune response. Because inflammation and release of pro-inflammatory cytokines are involved in pulmonary emphysema, we hypothesized that vesicular acetylcholine transport protein (VAChT) deficiency, which leads to reduction in ACh release, can modulate lung inflammation in an experimental model of emphysema. Mice with genetical reduced expression of VAChT (VAChT KDHOM 70%) and wild-type mice (WT) received nasal instillation of 50 uL of porcine pancreatic elastase (PPE) or saline on day 0. Twenty-eight days after, animals were evaluated. Elastase instilled VAChT KDHOM mice presented an increase in macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage fluid and MAC2-positive macrophages in lung tissue and peribronchovascular area that was comparable to that observed in WT mice. Conversely, elastase instilled VAChT KDHOM mice showed significantly larger number of NF-κB-positive cells and isoprostane staining in the peribronchovascular area when compared to elastase-instilled WT-mice. Moreover, elastase-instilled VAChT-deficient mice showed increased MCP-1 levels in the lungs. Other cytokines, extracellular matrix remodeling, alveolar enlargement, and lung function were not worse in elastase-instilled VAChT deficiency than in elastase-instilled WT-controls. These data suggest that decreased VAChT expression may contribute to the pathogenesis of emphysema, at least in part, through NF-κB activation, MCP-1, and oxidative stress pathways. This study highlights novel pathways involved in lung inflammation that may contribute to the development of chronic obstrutive lung disease (COPD) in cholinergic deficient individuals such as Alzheimer’s disease patients.

Electronic Cigarette Exposure Enhances Lung Inflammatory and Fibrotic Responses in COPD Mice.

Although a few studies show that the use of electronic nicotine delivery systems (ENDS) may ameliorate objective and subjective outcomes in COPD smokers who switched to electronic cigarettes, it is unclear whether e-cigarette exposure alters lung pathological features and inflammatory response in COPD. Here, we employed βENaC-overexpressing mice bearing COPD-like pulmonary abnormality, and exposed them to ENDS. We found that ENDS exposure aggravated airspace enlargement and mucus production in βENaC-overexpressing mice, which was associated with increased MMP12 and Muc5ac, respectively. ENDS exposure to mice significantly increased the numbers of macrophages, particularly in M2 macrophages in bronchoalveolar lavage (BAL) fluid, despite ENDS did not induce M2 macrophage polarization in a cultured murine macrophage cell line (RAW264.7). There were no changes in neutrophils in BAL fluid by ENDS exposure. Multiple cytokine productions were increased including M-CSF, IL-1r, IL-10, and TGF-β1, in BAL fluid from mice when exposed to ENDS. The Sirius Red staining and hydroxyproline assay showed ENDS-exposed mice displayed enhanced fibrotic phenotypes compared to control mice. In conclusion, ENDS exposure enhances airspace enlargement, mucus secretion, and fibrogenesis in COPD mice. This is associated with increased MMP12, inflammatory responses, and M2 macrophage phenotype. This study provides pre-clinical data implicating that electronic cigarette exposure is not safe in COPD patients who want to replace traditional cigarettes with ENDS.

Prognostic significance of bronchoalveolar lavage cellular analysis in patients with acute exacerbation of interstitial lung disease

BackgroundAcute exacerbation (AE) of interstitial lung disease (ILD) is an acute respiratory deterioration of unknown etiology, associated with high mortality. Currently, bronchoalveolar lavage (BAL) has been no longer required for the diagnosis of AE-ILD; however, the clinical utility of BAL fluid (BALF) cellular analysis in AE-ILD remains unclear. MethodsA retrospective study of 71 patients who underwent BAL at our institution between 2005 and 2019 and were diagnosed with AE-ILD was conducted. We performed BALF cellular analysis and evaluated its prognostic significance. ResultsThere were 26 patients with AE of idiopathic pulmonary fibrosis (IPF) and 45 with AE of non-IPF, including idiopathic interstitial pneumonias/non-IPF (n = 22), ILD associated with collagen tissue disease (n = 20) and fibrotic hypersensitivity pneumonia (n = 3). All patients were treated with high-dose corticosteroids, and the 90-day mortality after AE was 31%. Most patients showed a high percentage of lymphocytes and/or neutrophils in BALF regardless of the underlying ILD. There was a significant negative correlation between BALF neutrophils and the PaO2/FiO2 ratio, and patients with UIP pattern or diffuse AE pattern on HRCT had a significantly higher percentage of BALF neutrophils than those with other patterns. Multivariate analysis revealed that lower and higher percentage of lymphocytes and neutrophils, respectively, in BALF were independent poor prognostic factors for 90-day survival. BALF lymphocyte and neutrophil count ≥25% and <20%, respectively, predicted favorable survival after AE. ConclusionsCellular analysis of BALF in AE-ILD is a potential biomarker for predicting prognosis after AE.

Cangrelor ameliorates CLP-induced pulmonary injury in sepsis by inhibiting GPR17

BackgroundSepsis is a common complication of severe wound injury and infection, with a very high mortality rate. The P2Y12 receptor inhibitor, cangrelor, is an antagonist anti-platelet drug.MethodsIn our study, we investigated the protective mechanisms of cangrelor in CLP-induced pulmonary injury in sepsis, using C57BL/6 mouse models.ResultsTdT-mediated dUTP Nick-End Labeling (TUNEL) and Masson staining showed that apoptosis and fibrosis in lungs were alleviated by cangrelor treatment. Cangrelor significantly promoted surface expression of CD40L on platelets and inhibited CLP-induced neutrophils in Bronchoalveolar lavage fluid (BALF) (p < 0.001). We also found that cangrelor decreased the inflammatory response in the CLP mouse model and inhibited the expression of inflammatory cytokines, IL-1β (p < 0.01), IL-6 (p < 0.05), and TNF-α (p < 0.001). Western blotting and RT-PCR showed that cangrelor inhibited the increased levels of G-protein-coupled receptor 17 (GPR17) induced by CLP (p < 0.001).ConclusionOur study indicated that cangrelor repressed the levels of GPR17, followed by a decrease in the inflammatory response and a rise of neutrophils in BALF, potentially reversing CLP-mediated pulmonary injury during sepsis.