Lung Homogenates Research Articles

Pharmacological Activation Of Aldehyde Dehydrogenase 2 Protects Against Heatstroke-Induced Acute Lung Injury by Modulating Oxidative Stress and Endothelial Dysfunction.

Heatstroke (HS) can cause acute lung injury (ALI). Heat stress induces inflammation and apoptosis via reactive oxygen species (ROS) and endogenous reactive aldehydes. Endothelial dysfunction also plays a crucial role in HS-induced ALI. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that detoxifies aldehydes such as 4-hydroxy-2-nonenal (4-HNE) protein adducts. A single point mutation in ALDH2 at E487K (ALDH2*2) intrinsically lowers the activity of ALDH2. Alda-1, an ALDH2 activator, attenuates the formation of 4-HNE protein adducts and ROS in several disease models. We hypothesized that ALDH2 can protect against heat stress-induced vascular inflammation and the accumulation of ROS and toxic aldehydes. Homozygous ALDH2*2 knock-in (KI) mice on a C57BL/6J background and C57BL/6J mice were used for the animal experiments. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro experiment. The mice were directly subjected to whole-body heating (WBH, 42°C) for 1 h at 80% relative humidity. Alda-1 (16 mg/kg) was administered intraperitoneally prior to WBH. The severity of ALI was assessed by analyzing the protein levels and cell counts in the bronchoalveolar lavage fluid, the wet/dry ratio and histology. ALDH2*2 KI mice were susceptible to HS-induced ALI in vivo. Silencing ALDH2 induced 4-HNE and ROS accumulation in HUVECs subjected to heat stress. Alda-1 attenuated the heat stress-induced activation of inflammatory pathways, senescence and apoptosis in HUVECs. The lung homogenates of mice pretreated with Alda-1 exhibited significantly elevated ALDH2 activity and decreased ROS accumulation after WBH. Alda-1 significantly decreased the WBH-induced accumulation of 4-HNE and p65 and p38 activation. Here, we demonstrated the crucial roles of ALDH2 in protecting against heat stress-induced ROS production and vascular inflammation and preserving the viability of ECs. The activation of ALDH2 by Alda-1 attenuates WBH-induced ALI in vivo.

Arsenic-induced lung inflammation and fibrosis in a rat model: Contribution of the HMGB1/RAGE, PI3K/AKT, and TGF-β1/SMAD pathways

An increasing number of studies have shown that arsenic exposure increases the risk of lung cancer as well as a variety of non-malignant respiratory diseases, including bronchitis and tracheobronchitis. HMGB1 is widely expressed in a variety of tissues and cells and is involved in the pathological processes of many lung diseases through binding to the corresponding receptors and activating the downstream signaling pathways. However, the exact role of HMGB1/RAGE in arsenic-induced lung injury remains unknown. The aim of this study was to investigate whether HMGB1/RAGE and its activated downstream pathways are involved in the process of arsenic exposure-induced lung injury in rats. In this study, an animal model of oral exposure to arsenic was induced using 2.5, 5 and 10 mg/kg NaAsO2. The results showed that capillary permeability (LDH, TP, ACP, and AKP) was increased in the arsenic exposure groups, resulting in cell damage; this was accompanied by acute inflammation marked by significant neutrophil infiltration. Meanwhile, obvious histopathological damage, including thickening of the lung epithelium, increased infiltration of inflammatory cells, rupture of the alveolar wall, swelling of the mitochondria, and chromatin agglutination was observed by H&E staining and transmission electron microscopy. Furthermore, the results confirmed that the expressions of HMGB1 and RAGE in lung tissue were enhanced, and protein expression of PI3K, p-AKT, IL-1β, IL-18, and MMP-9 was increased in lung homogenates from the arsenic-exposed groups compared to the control group. Finally, Masson's staining results revealed arsenic-induced fibrosis and collagen deposition. Moreover, a significant increase in key fibrosis factors, including TGF-β1, p-SMAD2, p-SMAD3, and SMAD4 was observed in the lung homogenates in arsenic-exposed groups. In conclusion, the current study demonstrates that sub-chronic arsenic exposure triggers the inflammatory response and collagen fiber deposition in rat lung tissue. The potential mechanism may be closely related to activation of the pro-inflammatory-related HMGB1/RAGE pathway and initiation of the PI3K/AKT and TGF-β1/SMAD pathways.

Antifibrotic and anti-inflammatory effects of the selective nonsteroidal MR antagonist finerenone in preclinical pulmonary fibrosis

Abstract Background Patients with idiopathic pulmonary fibrosis (IPF) are at increased risk of developing coronary artery disease (CAD) with a median survival of 3.5 years after diagnosis. Cardiac and pulmonary fibrosis share several common signaling pathways including TGF-β-related collagen synthesis. Pirfenidone and nintedanib were the first approved medical antifibrotic IPF therapies that inhibit TGF-β–related collagen synthesis or multiple receptor tyrosine kinases, respectively. However, the role of mineralocorticoid receptor blockade in IPF is less well understood. Purpose To test the hypothesis that the selective nonsteroidal MR antagonist finerenone has antifibrotic and anti-inflammatory activity in preclinical IPF. Methods Anti-fibrotic/anti-inflammatory efficacy was investigated in two different mouse models of pulmonary fibrosis. Lung fibrosis was induced by either intranasal application of bleomycin (1 mg/kg) or intratracheal application of silica particles (2.5 mg/mouse). Bleomycin-treated C57BL6 mice (8 weeks old male, n=10–15/group) were administered once daily orally from day 7 to 21 with vehicle (ethanol/solutol/water), finerenone (10 mg/kg) or nintedanib (60 mg/kg) in vehicle. Silica-treated C57BL6 mice (8 weeks old male, n=10–12/group) were administered once daily orally from day 7 to 30 with vehicle (ethanol/solutol/water), finerenone (1 or 10 mg/kg) or nintedanib (100 mg/kg) in vehicle, or with pirfenidone (800mg/kg/d) via chow. At day 21 and 30 respectively, histological examination of pulmonary fibrosis using immunohistochemistry (COL1A1 positive tissue area in %) and Ashcroft score as well as profibrotic and pro-inflammatory cytokine and COL1A1 protein level (ELISA) analysis in lung homogenate were performed. Results Bleomycin caused a significant increase in lung COL1A1 positive tissue area and Ashcroft Score and silica-induced lung pathology additionally showed elevated IL-1beta, IL-6, IL-10, IL-12p70, TNFalpha and COL1A1 protein levels in lung tissue. Therapeutic treatment with finerenone as well as nintedanib from day 7–21 significantly reduced the percentage of lung COL1A1 positive tissue and Ashcroft score in the bleomycin mice model without a significant difference between treatment groups. Furthermore, finerenone significantly reduced lung COL1A1 as well as IL-1beta, IL-6, IL-10, IL-12p70 and TNFalpha protein levels in lung homogenate after a therapeutic treatment from day 7–30 in the silica mice model, whereas nintedanib showed only a reduction in lung COL1A1 and IL-10 levels and treatment with pirfenidone was without significant effects. There was a significant difference in the reduction of lung IL-12p70 and TNFalpha protein levels between finerenone 10 mg/kg and nintedanib as well as pirfenidone treatment. Conclusion Nonsteroidal MR antagonism by finerenone showed anti-fibrotic/anti-inflammatory efficacy in two animal models of pulmonary fibrosis and may impact the progressive course of fibrotic diseases. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): BAYER AG

A formulated poly (I:C)/CCL21 as an effective mucosal adjuvant for gamma-irradiated influenza vaccine

BackgroundSeveral studies on gamma-irradiated influenza A virus (γ-Flu) have revealed its superior efficacy for inducing homologous and heterologous virus-specific immunity. However, many inactivated vaccines, notably in nasal delivery, require adjuvants to increase the quality and magnitude of vaccine responses.MethodsTo illustrate the impacts of co-administration of the gamma-irradiated H1N1 vaccine with poly (I:C) and recombinant murine CCL21, either alone or in combination with each other, as adjuvants on the vaccine potency, mice were inoculated intranasally 3 times at one-week interval with γ-Flu alone or with any of the three adjuvant combinations and then challenged with a high lethal dose (10 LD50) of A/PR/8/34 (H1N1) influenza virus. Virus-specific humoral, mucosal, and cell-mediated immunity, as well as cytokine profiles in the spleen (IFN-γ, IL-12, and IL-4), and in the lung homogenates (IL-6 and IL-10) were measured by ELISA. The proliferative response of restimulated splenocytes was also determined by MTT assay.ResultsThe findings showed that the co-delivery of the γ-Flu vaccine and CCL21 or Poly (I:C) significantly increased the vaccine immunogenicity compared to the non-adjuvanted vaccine, associated with more potent protection following challenge infection. However, the mice given a combination of CCL21 with poly (I:C) had strong antibody- and cell-mediated immunity, which were considerably higher than responses of mice receiving the γ-Flu vaccine with each adjuvant separately. This combination also reduced inflammatory mediator levels (notably IL-10) in lung homogenate samples.ConclusionsThe results indicate that adjuvantation with the CCL21 and poly (I:C) can successfully induce vigorous vaccine-mediated protection, suggesting a robust propensity for CCL21 plus poly (I:C) as a potent mucosal adjuvant.

Circulating exosomes induced by respiratory viral infections in lung transplant recipients activate cellular stress, innate immune pathways and epithelial to mesenchymal transition

BackgroundChronic lung transplant rejection occurs in over 50% of lung transplant recipients and mechanism of chronic rejection is unknown. Evaluation of potential mechanism of exosomes from lung transplant recipients diagnosed with respiratory viral infection (RVI) in inducing chronic lung allograft dysfunction (CLAD). MethodExosomes were isolated from lung transplant recipients followed by DNA and RNA isolation from exosomes. Cell signaling mechanisms were studied by co-culturing exosomes with human epithelial cells. Mice were immunized with exosomes and lung homogenates were studied for immune signaling proteins. ResultsExosomes from lung transplant recipients with RVI carry nucleic acids which are capable of inducing innate immune signaling, endoplasmic reticulum stress, and epithelial mesenchymal transition. ConclusionTherefore, we propose that RVI can lead to induction of exosomes that initiate the process leading to CLAD in mice models. These novel findings identified the molecular mechanisms by which RVI increases the risk of CLAD.

AEROBIC EXERCISE REDUCES SEVERE ASTHMA PHENOTYPE

TOPIC: Allergy and Airway TYPE: Original Investigations PURPOSE: Severe asthma is characterized by higher rates of airway hyperresponsiveness and concomitantly increased eosinophilic and neutrophilic airway and systemic inflammation in which bradykinin plays a central role. Aerobic exercise (AE) is the main component in a pulmonary rehabilitation inhibiting airway inflammation and hyperresponsiveness in mild and moderate asthmatics. In addition, AE in mouse models of allergic asthma is proven to reduce eosinophilic airway inflammation, airway remodeling, and hyperresponsiveness, by controlling exacerbated Th2 immune response. However, whether AE could inhibit a severe asthma phenotype is unknown METHODS: House dust mite (HDM; 200ug/mouse; 3x/week; 6 weeks) Dermatophagoides pteronisynuss was used. Eight C57Bl/6 male mice (Control; n=2x10), (Exercise; n=2x10), (Asthma; n=2x10) and (Asthma+Exercise; n=2x10) were assessed after 6 weeks protocol (2 initial weeks of intra-tracheal HDM without AE) followed by 4 weeks of treadmill AE (1h/session, 5x/week, moderate intensity) + (4 weeks of intra-tracheal HDM) RESULTS: The results showed that AE reduced HDM-increased total leukocytes, eosinophils, neutrophils, lymphocytes, and macrophages (p<0.01) in bronchoalveolar lavage (BAL) and total leukocytes, eosinophils, neutrophils, lymphocytes, and monocytes (p<0.01) in whole blood. The cytokines IL-1beta, IL-4, IL-5, IL-13, IL-17, IL-33, TNF-alpha (p<0.01) were reduced both in BAL and in plasma in HDM+Ex group compared with HDM group, while IL-10 was increased. The density of peribronchial eosinophils, neutrophils, lymphocytes and macrophages (p<0.01) and the percentage of airway collagen fibers, epithelial and smooth muscle thickness (p<0.01) were reduced in HDM+Ex group compared with HDM group. The levels of bradykinin in BAL and in lung homogenate (p<0.01) and the expression at mRNA levels by qRT-PCR of B1 receptor (p<0.001) were reduced in HDM+Ex group compared with HDM group. The lung mechanics revealed that exercise improved airway resistance (RAW; p<0.001), tissue resistance (HTIS; p<0.001), and tissue elastance (GTIS; p<0.001). In addition, once AE-induced IL-10 release, we tested the hypothesis that AE-induced IL-10 could inhibit bradykinin-induced airway epithelial cells (BEAS-2B), lung fibroblast cells (MRC5), and freshly isolated human eosinophils and neutrophils activation, measured by IL-1beta release. The results demonstrated that IL-10 inhibited bradykinin-induced IL-1beta release by BEAS-2B, MRC5, human eosinophils and neutrophils. In addition, IL-10 inhibited bradykinin-induced increased expression of B1 receptor in BEAS-2B, MRC5, human eosinophils, and neutrophils CONCLUSIONS: In conclusion, AE can inhibit severe asthma phenotype in vivo and in vitro, and further clinical trials to confirm these findings in severe asthmatic patients are guaranteed. CLINICAL IMPLICATIONS: The possible involvement of Bradikinin may be one of the keys to understanding the mechanisms of severe asthma and aerobic physical exercise may inhibit the action of the severe asthma phenotype. DISCLOSURES: no disclosure on file for Francine Almeida; No relevant relationships by Nilsa Regina Damaceno-Rodrigues, source=Web Response no disclosure on file for Fernanda Degobbi Tenorio Quirino Santos Lopes; No relevant relationships by RENILSON FERREIRA, source=Web Response No relevant relationships by Sara Hamaguchi, source=Web Response no disclosure on file for João Bosco Pesquero; No relevant relationships by 119 Maysa Alves Rodrigues Rangel, source=Web Response No relevant relationships by Anamei Silva-Reis, source=Web Response no disclosure on file for Rodolfo de Paula Vieira

The ethanol extract of flower buds of Tussilago farfara L. attenuates cigarette smoke-induced lung inflammation through regulating NLRP3 inflammasome, Nrf2, and NF-κB

Ethnopharmacological relevanceThe flower buds of Tussilago farfara L. (Abbreviated as FTF) were widely used in traditional Chinese medicine (TCM) to treat respiratory diseases, including asthma, dry throat, great thirst, turbid saliva, stinky pus, and coughs caused by various causes. Aim of studyThe aim of study is to explore the efficiency of FTF in vitro and in vivo for the treatment of lung inflammation, and to illustrate the possible mechanisms of FTF in treating inflammation-related respiratory diseases targeting NOD-like receptor 3 (NLRP3) inflammasome, nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear transcription factor-κB (NF-κB). MethodsLung inflammation model in vivo was induced by exposure of mice to cigarette smoke (CS) for two weeks. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), inflammatory factors, and histology in lung tissues were investigated in presence or absence of ethanol extract of the flower buds of T. farfara L. (FTF-EtOH). In the cell-based models, nitric oxide (NO) assay, flow cytometry assay, enzyme-linked immunosorbent assay (Elisa), and glutathione (GSH) assay were used to explore the anti-inflammatory and anti-oxidant effects of FTF-EtOH. Possible anti-inflammatory mechanisms of FTF targeting NLRP3 inflammasome, Nrf2, and NF-κB have been determined using western blot, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), immunofluorescence assay, nuclear and cytoplasmic extraction, and ubiqutination assay. ResultsFTF-EtOH suppressed CS-induced overproduction of inflammatory factors [e.g., tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)], and upregulation of the content of intracellular MDA in the lung homogenate of mice. In cell-based models, FTF-EtOH reduced the lipopolysaccharide (LPS)-induced overproduction of inflammatory factors, and attenuated the CS extract-induced overgeneration of reactive oxygen species (ROS). Furthermore, FTF-EtOH up-regulated Nrf2 and its downstream genes through enhancing the stability of Nrf2 protein, and inhibited the activation of NF-κB and NLRP3 inflammasome, which have been confirmed by detecting the protein levels in the mouse model. ConclusionsFTF-EtOH effectively attenuated lung inflammation in vitro and in vivo. The protection of FTF-EtOH against inflammation was produced by activation of Nrf2 and inhibitions of NF-κB and NLRP3 inflammasome. These datas definitely support the ethnopharmacological use of FTF as an anti-inflammatory drug for treating respiratory diseases in TCM.

Anti-inflammatory activity of lefamulin versus azithromycin and dexamethasone in vivo and in vitro in a lipopolysaccharide-induced lung neutrophilia mouse model.

Several antibiotics demonstrate both antibacterial and anti-inflammatory/immunomodulatory activities and are used to treat inflammatory pulmonary disorders. Lefamulin is a pleuromutilin antibiotic approved to treat community-acquired bacterial pneumonia (CABP). This study evaluated lefamulin anti-inflammatory effects in vivo and in vitro in a lipopolysaccharide-induced lung neutrophilia model in which mouse airways were challenged with intranasal lipopolysaccharide. Lefamulin and comparators azithromycin and dexamethasone were administered 30min before lipopolysaccharide challenge; neutrophil infiltration into BALF and inflammatory mediator induction in lung homogenates were measured 4h postchallenge. Single subcutaneous lefamulin doses (10‒140mg/kg) resulted in dose-dependent reductions of BALF neutrophil cell counts, comparable to or more potent than subcutaneous azithromycin (10‒100mg/kg) and oral/intraperitoneal dexamethasone (0.5/1mg/kg). Lipopolysaccharide-induced pro-inflammatory cytokine (TNF-α, IL-6, IL-1β, and GM-CSF), chemokine (CXCL-1, CXCL-2, and CCL-2), and MMP-9 levels were significantly and dose-dependently reduced in mouse lung tissue with lefamulin; effects were comparable to or more potent than with dexamethasone or azithromycin. Pharmacokinetic analyses confirmed exposure-equivalence of 30mg/kg subcutaneous lefamulin in mice to a single clinical lefamulin dose to treat CABP in humans (150mg intravenous/600mg oral). In vitro, neither lefamulin nor azithromycin had any relevant influence on lipopolysaccharide-induced cytokine/chemokine levels in J774.2 mouse macrophage or human peripheral blood mononuclear cell supernatants, nor were any effects observed on IL-8‒induced human neutrophil chemotaxis. These in vitro results suggest that impediment of neutrophil infiltration by lefamulin in vivo may not occur through direct interaction with macrophages or neutrophilic chemotaxis. This is the first study to demonstrate inhibition of neutrophilic lung infiltration and reduction of pro-inflammatory cytokine/chemokine concentrations by clinically relevant lefamulin doses. This anti-inflammatory activity may be beneficial in patients with acute respiratory distress syndrome, cystic fibrosis, or severe inflammation-mediated lung injury, similar to glucocorticoid (eg, dexamethasone) activity. Future lefamulin anti-inflammatory/immunomodulatory activity studies are warranted to further elucidate mechanism of action and evaluate clinical implications.

Interleukin 16 Enhances the Host Susceptibility to Influenza A Virus Infection.

Influenza A virus (IAV) is a major respiratory pathogen that causes seasonal and pandemic flu, being a threat to global health. Various viral and cellular factors have been characterized to support or limit IAV infection. Interleukin 16 (IL16) has been known as one of the blood signature biomarkers discriminating systemic inflammation due to viral infection vs. other etiologies. Here, we report that the level of IL16 was elevated in the serum samples, lung homogenates, and bronchoalveolar lavage fluid of IAV-infected mice. IL16 overexpression facilitated IAV replication. Conversely, loss of IL16 reduced the host susceptibility to IAV infection in vitro and in vivo. Furthermore, IL16 deficiency blocked IAV-induced body weight loss and attenuated lung injury in the infected mice. Molecular mechanism analyses further revealed that IL16 could directly inhibit IFN-β transcription and suppress the expression of IFN-β and IFN-stimulated gene. In conclusion, these findings demonstrate that IL16 is a supporting factor for IAV infection.

Leonurine protects against influenza A virus infection-induced pneumonia in mice.

Influenza A virus (H1N1), a swine-origin influenza A virus, causes seasonal epidemics that result in severe illnesses and deaths. Leonurine has been reported to function as an anti-inflammatory agent with protective effects on nervous, urinary and cardiovascular systems. However, the therapeutic effects of leonurine on the pneumonia caused by H1N1 infection remain unclear. Hematoxylin and eosin staining was performed to evaluate the lung injuries of mice infected by H1N1. The amount of immune cells was analyzed by flow cytometry. Enzyme-linked immunosorbent assay was used to evaluate the alteration of multiple cytokines in lung tissues. Real-time quantitative polymerase chain reaction assay was performed to investigate the ribonucleic acid (RNA) levels of certain genes. The protein levels in toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR4/NF-κB) signaling were estimated by western blot assay. Leonurine treatment significantly inhibited the mortality caused by H1N1 infection. Leonurine treatment (60 mg/kg) alleviated the lung injuries caused by virus infection. The inflammatory cell accumulation and cytokine expression were inhibited by the leonurine administration. Leonurine inhibited the mRNA expression of pro-inflammatory cytokines in the lung homogenates at day 5 postinfection. Leonurine regulated the TLR4/NF-κB signaling in the lung homogenates of H1N1-infected mice at day 5 postinfection. Leonurine protects against H1N1 infection-induced pneumonia in mice.

The impact of airborne endotoxin exposure on rheumatoid arthritis-related joint damage, autoantigen expression, autoimmunity, and lung disease

Airborne biohazards are risk factors in the development and severity of rheumatoid arthritis (RA) and RA-associated lung disease, yet the mechanisms explaining this relationship remain unclear. Lipopolysaccharide (LPS, endotoxin) is a ubiquitous inflammatory agent in numerous environmental and occupational air pollutant settings recognized to induce airway inflammation. Combining repetitive LPS inhalation exposures with the collagen induced arthritis (CIA) model, DBA1/J mice were assigned to either: sham (saline injection/saline inhalation), CIA (CIA/saline), LPS (saline/LPS 100 ng inhalation), or CIA + LPS for 5 weeks. Serum anti-citrullinated (CIT) protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) antibodies were strikingly potentiated with co-exposure (CIA + LPS). CIT- and MAA-modified lung proteins were increased with co-exposure and co-localized across treatment groups. Inhaled LPS exacerbated arthritis with CIA + LPS > LPS > CIA versus sham. Periarticular bone loss was demonstrated in CIA and CIA + LPS but not in LPS alone. LPS induced airway inflammation and neutrophil infiltrates were reduced with co-exposure (CIA + LPS). Potentially signaling transition to pro-fibrotic processes, there were increased infiltrates of activated CD11c+CD11b+ macrophages and transitioning CD11c+CD11bint monocyte-macrophage populations with CIA + LPS. Moreover, several lung remodeling proteins including fibronectin and matrix metalloproteinases as well as complement C5a were potentiated with CIA + LPS compared to other treatment groups. IL-33 concentrations in lung homogenates were enhanced with CIA + LPS with IL-33 lung staining driven by LPS. IL-33 expression was also significantly increased in lung tissues from patients with RA-associated lung disease (N = 8) versus controls (N = 7). These findings suggest that patients with RA may be more susceptible to developing interstitial lung disease following airborne biohazard exposures enriched in LPS.

Increased airway epithelial cell-derived exosomes activate macrophage-mediated allergic inflammation via CD100 shedding.

Airway epithelial cells (AECs) participate in allergic airway inflammation by producing mediators in response to allergen stimulation. Whether ovalbumin (OVA) challenge promotes exosome release from AECs (OVA‐challenged AEC‐derived exosomes (OAEs)), thereby affecting airway inflammation, as well as the underlying mechanisms, is unknown. Our study showed that AECs released an increased number of exosomes after OVA challenge, and the expression of Plexin B2 (PLXNB2; a natural CD100 ligand) was increased by a massive 85.7‐fold in OAEs than in PBS‐treated AEC‐derived exosomes (PAEs). CD100+F4/80+ macrophages engulfed OAEs to trigger the transcription of pro‐inflammatory chemokines and cytokines. Plxnb2 transcripts increased in asthmatic lungs, and similarly, PLXNB2 protein was highly enriched in exosomes purified from asthmatic bronchoalveolar lavage (BAL) fluid. Furthermore, aspiration of PLXNB2 or OAEs increased the recruitment of lung neutrophils, monocytes, eosinophils and dendritic cells in OVA‐challenged mice. Mechanistically, OAE aspiration enhanced the cleavage of CD100 by MMP14, which manifested as an increase in the soluble CD100 (sCD100) level in BAL fluid and lung homogenates. Knockdown of Mmp14 in macrophages prevented the cleavage of CD100 and reduced Ccl2, Ccl5 and Cxcl2 transcription. These data indicate that PLXNB2‐containing OAEs aggravate airway asthmatic inflammation via cleavage of CD100 by MMP14, suggesting potential therapeutic targets of OAE‐mediated asthma exacerbations.

Postnatal Ozone Exposure Disrupts Alveolar Development, Exaggerates Mucoinflammatory Responses, and Suppresses Bacterial Clearance in Developing Scnn1b-Tg+ Mice Lungs

Increased levels of ambient ozone, one of the six criteria air pollutants, result in respiratory tract injury and worsening of ongoing lung diseases. However, the effect of ozone exposure on the respiratory tract undergoing active lung development and simultaneously experiencing mucoinflammatory lung diseases, such as cystic fibrosis, remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice, a mouse model of cystic fibrosis-like lung disease, and littermate wild-type (WT) mice to ozone from postnatal days (PND) 3-20 and examined the lung phenotypes at PND21. As compared with filtered air (FA)-exposed WT mice, the ozone-exposed WT mice exhibited marked alveolar space enlargement, in addition to significant eosinophilic infiltration, type 2 inflammation, and mucous cell metaplasia. Ozone-exposed Scnn1b-Tg+ mice also exhibited significantly increased alveolar space enlargement, which was also accompanied by exaggerated granulocytic infiltration, type 2 inflammation, and a greater degree of mucus obstruction. The alveolar space enlargement in ozone-exposed WT, FA-exposed Scnn1b-Tg+, and ozone-exposed Scnn1b-Tg+ mice was accompanied by elevated levels of MMP12 protein in macrophages and Mmp12 mRNA in the lung homogenates. Finally, although bacterial burden was largely resolved by PND21 in FA-exposed Scnn1b-Tg+ mice, ozone-exposed Scnn1b-Tg+ mice exhibited compromised bacterial clearance, which was also associated with increased levels of IL-10, an immunosuppressive cytokine, and marked mucus obstruction. Taken together, our data show that ozone exposure results in alveolar space remodeling during active phases of lung development and markedly exaggerates the mucoinflammatory outcomes of pediatric-onset lung disease, including bacterial infections, granulocytic inflammation, mucus obstruction, and alveolar space enlargement.

Therapeutic Effects of Hyaluronic Acid Against Cytotoxic Extracellular Vesicles Released During Pseudomonas Aeruginosa Pneumonia.

Extracellular vesicles (EVs) have now been recognized as important mediators of cellular communication during injury and repair. We previously found that plasma EVs isolated from ex vivo perfused human lungs injured with Escherichia coli bacterial pneumonia were inflammatory, and exogenous administration of high molecular weight (HMW) hyaluronic acid (HA) as therapy bound to these EVs, decreasing inflammation and injury. In the current study, we studied the role of EVs released during severe Pseudomonas aeruginosa (PA) pneumonia in mice and determined whether intravenous administration of exogenous HMW HA would have therapeutic effects against the bacterial pneumonia. EVs were collected from the bronchoalveolar lavage fluid (BALF) of mice infected with PA103 by ultracentrifugation and analyzed by NanoSight and flow cytometry. In a cytotoxicity assay, administration of EVs released from infected mice (I-EVs) decreased the viability of A549 cells compared to EV isolated from sham control mice (C-EVs). Either exogenous HMW HA or an anti-CD44 antibody, when co-incubated with I-EVs, significantly improved the viability of the A549 cells. In mice with PA103 pneumonia, administration of HMW HA improved pulmonary edema and bacterial count in the lungs and decreased TNF-α and caspase-3 levels in the supernatant of lung homogenates. In conclusion, EVs isolated from BALF of mice with P. aeruginosa pneumonia were cytotoxic and inflammatory, and intravenous HMW HA administration was protective against P. aeruginosa pneumonia.

Chlamydia trachomatis Cross-Serovar Protection during Experimental Lung Reinfection in Mice.

Chlamydia trachomatis causes most bacterial sexually transmitted diseases worldwide. Different major outer membrane proteins (MOMPs) define various serovars of this intracellular pathogen: In women, D to L3 can cause urethritis, cervicitis, salpingitis, and oophoritis, and, thus, infertility. Protective immunity might be serovar-specific since chlamydial infection does not appear to induce an effective acquired immunity and reinfections occur. A better understanding of induced cross-serovar protection is essential for the selection of suitable antigens in vaccine development. In our mouse lung infection screening model, we evaluated the urogenital serovars D, E, and L2 in this regard. Seven weeks after primary infection or mock-infection, respectively, mice were infected a second time with the identical or one of the other serovars. Body weight and clinical score were monitored for 7 days. Near the peak of the second lung infection, bacterial load, myeloperoxidase, IFN-γ, and TNF-α in lung homogenate, as well as chlamydia-specific IgG levels in blood were determined. Surprisingly, compared with mice that were infected then for the first time, almost independent of the serovar combination used, all acquired parameters of disease were similarly diminished. Our reinfection study suggests that efficient cross-serovar protection could be achieved by a vaccine combining chlamydial antigens that do not include nonconserved MOMP regions.

Isoforskolin Alleviates AECOPD by Improving Pulmonary Function and Attenuating Inflammation Which Involves Downregulation of Th17/IL-17A and NF-κB/NLRP3.

Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide, is widely considered to be related to cigarette smoke (CS), and viral infections trigger acute exacerbation of COPD (AECOPD). Isoforskolin (ISOF) is a bioactive component from the plant Coleus forskohlii, native to Yunnan in China. It has been demonstrated that ISOF has anti-inflammatory effect on acute lung injury animal models. In the present study, we investigated the efficacy and mechanism of ISOF for the prevention and treatment of AECOPD. Mice were exposed to CS for 18 weeks and then infected with influenza virus A/Puerto Rico/8/34 (H1N1). ISOF (0.5, 2 mg/kg) was intragastrically administered once a day after 8 weeks of exposure to cigarette smoke when the body weight and lung function of model mice declined significantly. The viral load, pulmonary function, lung morphology, Th17 cells, and inflammatory cytokines in lung tissues were evaluated. The expression of nuclear factor κB (NF-κB) and NOD-like receptor pyrin domain–containing protein 3 (NLRP3) inflammasome pathways were detected. The results showed that ISOF treatment reduced the viral load in the lung homogenate, decreased the lung index of model mice, and lung pathological injuries were alleviated. ISOF also improved the pulmonary function with increased FEV0.1/FVC and decreased Rn and Rrs. The levels of inflammatory mediators (TNF-α, IL-1β, IL-6, IL-17A, MCP-1, MIG, IP-10, and CRP) in the lung homogenate were reduced after ISOF treatment. ISOF decreased the proportion of Th17 cells in the lung tissues by the flow cytometry test, and the protein expression levels of RORγt and p-STAT3 were also decreased. Furthermore, ISOF significantly inhibited the activation of NF-κB signaling and NLRP3 inflammasome in the lung tissues of model mice. In conclusion, ISOF alleviates AECOPD by improving pulmonary function and attenuating inflammation via the downregulation of proinflammatory cytokines, Th17/IL-17 A, and NF-κB/NLRP3 pathways.

The effects of edaravone, a free-radical scavenger in lung injury induced by valproic acid demonstrated via different biochemical parameters.

In this study, we aimed to evaluate whether edaravone (EDA) has a protective role against valproic acid (VPA)-induced lung damage via its antioxidative activity. Male Sprague-Dawley rats were split into four groups. Control (n = 8) rats; rats given EDA (30 mg kg-1 day-1 ; n = 10); rats given only (VPA, 500 mg kg-1 day-1 ; n = 10); rats given VPA + EDA (in the same dose and time) for 7 days. EDA and VPA were applied intraperitoneally. After 8 days, lung tissues were immediately taken from the rats. In lung homogenates, reduced glutathione, total antioxidant status levels, and superoxide dismutase, glutathione peroxidase, sodium/potassium ATPase, paraoxonase1, and carbonic anhydrase activities significantly abated, whereas catalase, glutathione reductase, glutathione-S-transferase activities insignificantly decreased in the VPA-treated group. In contrast, lipid peroxidation, reactive oxygen species, and total oxidant status levels, glycoprotein and protein carbonyl contents, nitric oxide, hydroxyproline levels, and xanthine oxidase, lactate dehydrogenase, arginase, and prolidase activities significantly increased in the VPA-given group. Administration of EDA caused the reverse effects. As a consequence, EDA prevented oxidative stress-mediated lung injury via its robust antioxidant effects.

The Efficacy of Phage Therapy in a Murine Model of Pseudomonas aeruginosa Pneumonia and Sepsis.

The emergence of multi-drug resistant Pseudomonas aeruginosa necessitates the search for treatment options other than antibiotic use. The use of bacteriophages is currently being considered as an alternative to antibiotics for the treatment of bacterial infections. A number of bacteriophages were introduced to treat pneumonia in past reports. However, there are still lack of knowledge regarding the dosages, application time, mechanism and safety of phage therapy against P. aeruginosa pneumonia. We used the bacteriophage KPP10 against P. aeruginosa strain D4-induced pneumonia mouse models and observed their outcomes in comparison to control models. We found that the nasal inhalation of highly concentrated KPP10 (MOI = 80) significantly improved survival rate in pneumonia models (P < 0.01). The number of viable bacteria in both lungs and in serum were significantly decreased (P < 0.01) in phage-treated mice in comparison to the control mice. Pathological examination showed that phage-treated group had significantly reduced bleeding, inflammatory cell infiltration, and mucus secretion in lung interstitium. We also measured inflammatory cytokine levels in the serum and lung homogenates of mice. In phage-treated models, serum TNFα, IL-1β, and IFN-γ levels were significantly lower (P < 0.05, P < 0.01, and P < 0.05, respectively) than those in the control models. In the lung homogenate, the mean IL-1β level in phage-treated models was significantly lower (P < 0.05) than that of the control group. We confirmed the presence of phage in blood and lungs, and evaluated the safety of bacteriophage use in living models since bacteriophage mediated bacterial lysis arise concern of endotoxic shock. The study results suggest that phage therapy can potentially be used in treating lung infections caused by Pseudomonas aeruginosa.

Progranulin attenuated asthma inflammation by inhibiting the expression of IL-6

Objective: To investigate the role and mechanism of progranulin (PGRN) in asthma. Methods: Control group and model group were set up in wild and IL-6 knockout (IL-6 ko) mice, respectively. For asthma model, mice were intraperitoneally sensitized with 100 μg OVA on days 0 and 7, followed by aerosol challenges with 5% OVA for 30 min per day from day 14 to 21, and mice were sacrificed 24 h after the last challenge. The mice in control group were treated in the same way with PBS. Bronchoalveolar lavage fluid (BALF) was collected for leukocytes count and differential count. The pathological changes of lung tissues were observed by H&E staining. The cytokines in lung homogenate, serum and BALF were detected by Q-PCR and ELISA. The in vitro model of asthma was induced by stimulating A549 or BEAS-2B cells with IL-13. Each group was replicated in three wells and four groups were designed: PBS group, IL-13 treatment group, IL-13 + rhPGRN treatment group, inhibitors of p38 phosphorylation (SB203508) treatment group. The cells or supernatant were collected after 0~48 h. PGRN and IL-6 levels were determined by Q-PCR and ELISA, the level of p38 phosphorylation was tested by Western blot (WB). Results: Compared with control group, PGRN levels were decreased in lung homogenate and BALF (P＜0.05), and PGRN presented a downtrend in serum, however, the level of IL-6 in BALF was increased in asthma mice (P＜0.01). In IL-6 ko asthma mice, compared with the wild asthma mice, leukocytes, especially neutrophils in BALF were decreased (P＜0.05), but PGRN was increased (P＜0.05), lung pathological damage was significantly alleviated. In vitro experiments, compared with PBS group, PGRN level was decreased (P＜0.05), IL-6 level was increased (P＜0.01), phosphorylation of p38 was activated in IL-13 treatment group. Compared with IL-13 treatment group, in IL-13 + PGRN treatment group, IL-6 level was decreased (P＜0.05); phosphorylation of p38 was inhibited (P＜0.05); and the production of IL-6 (P＜0.05) was decreased after treatment with inhibitor of p38 phosphorylation. Conclusion: PGRN inhibited the production of IL-6 by suppressing the p38 phosphorylation to alleviate asthmatic airway inflammation.

Toxic effect of cooking oil fume (COF) on lungs: Evidence of endoplasmic reticulum stress in rat

BackgroundCooking oil fumes (COF) is one of the primary sources of indoor air pollution in China, which is associated with respiratory diseases such as acute lung injury and lung cancer. However, evidence of COF toxic effect was few. ObjectivesThe research was aimed to investigate the toxic effect and the underlying mechanisms induced by COF. MethodsThe female Wistar rats were randomly divided into several groups, including control group, COF exposure group and VE protection group, and instilled intratracheally with different COF suspensions (0.2, 2, 20 mg/kg) or saline once every 3 days for 30 days. After 24 h of final exposure, all rat were anesthetic euthanasia to draw materials. The alveolar lavage fluid (BALF) was for inflammatory cell count. The lung homogenate was to determine the biochemical indexes such as oxidative stress, apoptosis factors, carcinogenic toxicity and endoplasmic reticulum (ER) stress. The left lung was made for immunohistochemical and histopathological analysis. ResultsThe results showed that the levels of oxidative stress (ROS), apoptosis factors (NF-κB), carcinogenic toxicity (P53 and 8-OhdG), ER stress (IRE-1α and Caspase-12) in 2 mg/kg and 20 mg/kg COF exposure groups were significantly increased compared with the saline groups. The above pathological changes were improved after vitamin E (VE) supplementation. In addition, the immunohistochemical and histopathological analysis found the same trend. ConclusionThe COF had health risk of heredity and potential carcinogenicity. Besides, COFs can not only induce oxidative stress, but also induce ER stress in lung and airway epithelial cells of female rats through the unfolded protein reaction (UPR) pathway. It revealed that the oxidative stress and ER stress interacted in aggravating lung injury. VE could effectively alleviate the lung injury causing by COF exposure.