Protective Effects of Resveratrol Against Airway Hyperreactivity, Oxidative Stress, and Lung Inflammation in a Rat Pup Model of Bronchopulmonary Dysplasia.

Oxygen therapy provides an important treatment for preterm and low-birth-weight neonates, however, it has been shown that prolonged exposure to high levels of oxygen (hyperoxia) is one of the factors contributing to the development of bronchopulmonary dysplasia (BPD) by inducing lung injury and airway hyperreactivity. There is no effective therapy against the adverse effects of hyperoxia. Therefore, this study was undertaken to test the hypothesis that natural phytoalexin resveratrol will overcome hyperoxia-induced airway hyperreactivity, oxidative stress, and lung inflammation. Newborn rats were exposed to hyperoxia (fraction of inspired oxygen - FiO2>95 % O2) or ambient air (AA) for seven days. Resveratrol was supplemented either in vivo (30 mg·kg-1·day-1) by intraperitoneal administration or in vitro to the tracheal preparations in an organ bath (100 mikroM). Contractile and relaxant responses were studied in tracheal smooth muscle (TSM) using the in vitro organ bath system. To explain the involvement of nitric oxide in the mechanisms of the protective effect of resveratrol against hyperoxia, a nitric oxide synthase inhibitor - Nomega-nitro-L-arginine methyl ester (L-NAME), was administered in some sets of experiments. The superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels in the lungs were determined. Resveratrol significantly reduced contraction and restored the impaired relaxation of hyperoxia-exposed TSM (p<0.001). L-NAME reduced the inhibitory effect of resveratrol on TSM contractility, as well as its promotion relaxant effect (p<0.01). Resveratrol preserved the SOD and GPx activities and decreased the expression of TNF-alpha and IL-1beta in hyperoxic animals. The findings of this study demonstrate the protective effect of resveratrol against hyperoxia-induced airway hyperreactivity and lung damage and suggest that resveratrol might serve as a therapy to prevent the adverse effects of neonatal hyperoxia. Keywords: Bronchopulmonary dysplasia, Hyperoxia, Airway hyperreactivity, Resveratrol, Pro-inflammatory cytokines.

Simvastatin attenuates silica-induced pulmonary inflammation and fibrosis in rats via the AMPK-NOX pathway.

BACKGROUND: Simvastatin (Sim), a hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been widely used in prevention and treatment of cardiovascular diseases. Studies have suggested that Sim exerts anti-fibrotic effects by interfering fibroblast proliferation and collagen synthesis. This study was to determine whether Sim could alleviate silica-induced pulmonary fibrosis and explore the underlying mechanisms. METHODS: The rat model of silicosis was established by the tracheal perfusion method and treated with Sim (5 or 10 mg/kg), AICAR (an AMPK agonist), and apocynin (a NOX inhibitor) for 28 days. Lung tissues were collected for further analyses including pathological histology, inflammatory response, oxidative stress, epithelial mesenchymal transformation (EMT), and the AMPK-NOX pathway. RESULTS: Sim significantly reduced silica-induced pulmonary inflammation and fibrosis at 28 days after administration. Sim could reduce the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α and transforming growth factor-ß1 in lung tissues. The expressions of hydroxyproline, α-SMA and vimentin were down-regulated, while E-cad was increased in Sim-treated rats. In addition, NOX4, p22pox, p40phox, p-p47phox/p47phox expressions and ROS levels were all increased, whereas p-AMPK/AMPK was decreased in silica-induced rats. Sim or AICAR treatment could notably reverse the decrease of AMPK activity and increase of NOX activity induced by silica. Apocynin treatment exhibited similar protective effects to Sim, including down-regulating of oxidative stress and inhibition of the EMT process and inflammatory reactions. CONCLUSIONS: Sim attenuates silica-induced pulmonary inflammation and fibrosis by downregulating EMT and oxidative stress through the AMPK-NOX pathway.

Inhibition of GBP5 activates autophagy to alleviate inflammatory response in LPS-induced lung injury in mice.

BACKGROUND: Pulmonary emphysema is a condition that causes damage to the lung tissue over time. GBP5, as part of the guanylate-binding protein family, is dysregulated in mouse pulmonary emphysema. However, the role of GBP5 in lung inflammation in ARDS remains unveiled. METHODS: To investigate whether GBP5 regulates lung inflammation and autophagy regulation, the study employed a mouse ARDS model and MLE-12 cell culture. Vector transfection was performed for the genetic manipulation of GBP5. Then, RT-qPCR, WB and IHC staining were conducted to assess its transcriptional and expression levels. Histological features of the lung tissue were observed through HE staining. Moreover, ELISA was conducted to evaluate the secretion of inflammatory cytokines, autophagy was assessed by immunofluorescent staining, and MPO activity was determined using a commercial kit. RESULTS: Our study revealed that GBP5 expression was altered in mouse ARDS and LPS-induced MLE-12 cell models. Moreover, the suppression of GBP5 reduced lung inflammation induced by LPS in mice. Conversely, overexpression of GBP5 diminished the inhibitory impact of LPS on ARDS during autophagy, leading to increased inflammation. In the cell line of MLE-12, GBP5 exacerbates LPS-induced inflammation by blocking autophagy. CONCLUSION: The study suggests that GBP5 facilitates lung inflammation and autophagy regulation. Thus, GBP5 could be a potential therapeutic approach for improving ARDS treatment outcomes, but further research is required to validate these findings.

Modulation of alveolar macrophage and mitochondrial fitness by medicinal plant-derived nanovesicles to mitigate acute lung injury and viral pneumonia.

Acute lung injury (ALI) is generally caused by severe respiratory infection and characterized by overexuberant inflammatory responses and inefficient pathogens-containing, the two major processes wherein alveolar macrophages (AMs) play a central role. Dysfunctional mitochondria have been linked with distorted macrophages and hence lung disorders, but few treatments are currently available to correct these defects. Plant-derive nanovesicles have gained significant attention because of their therapeutic potential, but the targeting cells and the underlying mechanism remain elusive. We herein prepared the nanovesicles from Artemisia annua, a well-known medicinal plant with multiple attributes involving anti-inflammatory, anti-infection, and metabolism-regulating properties. By applying three mice models of acute lung injury caused by bacterial endotoxin, influenza A virus (IAV) and SARS-CoV-2 pseudovirus respectively, we showed that Artemisia-derived nanovesicles (ADNVs) substantially alleviated lung immunopathology and raised the survival rate of challenged mice. Macrophage depletion and adoptive transfer studies confirmed the requirement of AMs for ADNVs effects. We identified that gamma-aminobutyric acid (GABA) enclosed in the vesicles is a major molecular effector mediating the regulatory roles of ADNVs. Specifically, GABA acts on macrophages through GABA receptors, promoting mitochondrial gene programming and bioenergy generation, reducing oxidative stress and inflammatory signals, thereby enhancing the adaptability of AMs to inflammation resolution. Collectively, this study identifies a promising nanotherapeutics for alleviating lung pathology, and elucidates a mechanism whereby the canonical neurotransmitter modifies AMs and mitochondria to resume tissue homeostasis, which may have broader implications for treating critical pulmonary diseases such as COVID-19.

Mechanistic insights into targeting caspase-3 activation and alveolar macrophage pyroptosis by Ephedra and bitter almond compounds for treating pediatric pneumonia via network pharmacology and bioinformatics.

This study investigates the molecular mechanism of Ma Huang-Ku Xing Ren, a traditional Chinese medicine formula, in treating pediatric pneumonia. The focus is on the regulation of caspase-3 activation and reduction of alveolar macrophage necrosis through network pharmacology and bioinformatics analyses of Ephedra and bitter almond components. Active compounds and targets from ephedrine and bitter almond were obtained using TCMSP, TCMID, and GeneCards databases, identifying pediatric pneumonia-related genes. A protein-protein interaction (PPI) network was constructed, and core targets were screened. GO and KEGG pathway enrichment analyses identified relevant genes and pathways. An acute pneumonia mouse model was created using the lipopolysaccharide (LPS) inhalation method, with caspase-3 overexpression induced by a lentivirus. The mice were treated with Ephedra and bitter almond through gastric lavage. Lung tissue damage, inflammatory markers (IL-18 and IL-1ß), and cell death-related gene activation were assessed through H&E staining, ELISA, western blot, flow cytometry, and immunofluorescence. The study identified 128 active compounds and 121 gene targets from Ephedra and bitter almond. The PPI network revealed 13 core proteins, and pathway analysis indicated involvement in inflammation, apoptosis, and cell necrosis, particularly the caspase-3 pathway. In vivo results showed that Ephedra and bitter almond treatment significantly mitigated LPS-induced lung injury in mice, reducing lung injury scores and inflammatory marker levels. It also decreased caspase-3 activity and cell death in alveolar macrophages. In conclusion, the active ingredients of Ma Huang-Ku Xing Ren, particularly targeting caspase-3, may effectively treat pediatric pneumonia by reducing apoptosis in alveolar macrophages, as demonstrated by both network pharmacology, bioinformatics analyses, and experimental data.

CLCA1 exacerbates lung inflammation via p38 MAPK pathway in acute respiratory distress syndrome.

Recent research has revealed that airway epithelial calcium-activated chloride channel-1 (CLCA1) is implicated in the inflammation of multiple human respiratory diseases, but the specific role in acute respiratory distress syndrome (ARDS) remains unknown. To investigate the role of CLCA1 in ARDS, 80 participants, including 26 ARDS patients, 26 patients with community-acquired pneumonia (CAP) and 28 control subjects, were enrolled in this study. As the result shows, the level of CLCA1 was significantly increased in ARDS patients and positively correlated with neutrophil infiltration and the poor prognosis of ARDS. Then, the level of CLCA1 also elevated in the LPS-induced ARDS mouse model, and the administration of CLCA1 significantly regulated the phenotypes of ARDS in mice, such as lung injury score, BALF protein concentration, neutrophils infiltration and the secretions of inflammatory factors. Furthermore, administration of CLCA1 substantially altered the phosphorylation of p38 in the ARDS mouse model, whereas repressing the expression of CLCA1 or inhibiting the activation of p38 both alleviated the inflammatory response of ARDS. In summary, CLCA1 was notably correlated with ARDS and exacerbated the ARDS phenotypes through the p38 MAPK pathway.

TRIM27-Induced Protective Autophagy: A Novel Therapeutic Approach for Pneumonia.

BACKGROUND: Pneumonia is a prevalent respiratory ailment involving complex physiological and pathological mechanisms. The tripartite motif containing 27 (TRIM27) plays a crucial role in regulating inflammation mechanisms. Therefore, the purpose of this study is to further explore the therapeutic potential of TRIM27 in pneumonia, based on its regulatory mechanisms in inflammation and autophagy. METHODS: This study established a mouse pneumonia animal model through lipopolysaccharide (LPS) administration, designating it as the LPS model group. Subsequently, adenovirus-mediated TRIM27 overexpression was implemented in the animals of the LPS model group, creating the TRIM27 treatment group. After a 7-day treatment period, lung tissues from the mice were collected. Various techniques, including immunohistochemistry, quantitative reverse transcription PCR (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA), and electron microscopy were utilized to analyze the impact of TRIM27 overexpression on inflammatory factors, oxidative stress, autophagy, and inflammatory processes in pulmonary tissues. Finally, an in vitro LPS cell model was established, and the effects of TRIM27 overexpression and autophagy inhibition on inflammatory cytokines and autophagosomes in LPS-induced inflammatory cells were examined through RT-qPCR and immunofluorescence techniques. RESULTS: The research findings demonstrate a significant reduction in the elevated levels of interleukin-6 (IL-6), IL-1ß, and Tumor necrosis factor-alpha (TNF-α) induced by LPS with TRIM27 overexpression (p < 0.01). Conversely, the autophagy inhibitor 3-Methyladenine (3-MA) diminished the effects induced by TRIM27 overexpression. Moreover, TRIM27 overexpression enhanced the expression of Microtubule-associated protein 1A/1B light chain 3 (LC3) II/I and Beclin-1 proteins in mice subjected to LPS stimulation (p < 0.01), while reducing the expression of the p62 protein (p < 0.01). The addition of 3-MA, however, decreased Beclin-1 expression and inhibited autophagy (p < 0.01). Additionally, TRIM27 overexpression decreased the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved caspase-1, IL-1ß, and Gasdermin D N-terminal fragment (GSDMD-N) proteins in LPS-stimulated mice (p < 0.05). TRIM27 overexpression also decreased the levels of malondialdehyde (MDA), Activating Transcription Factor 6 (ATF6), and C/EBP-homologous protein (CHOP), while increasing the levels of superoxide dismutase (SOD) and glutathione (GSH) in mice exposed to LPS (p < 0.01). CONCLUSION: The induction of TRIM27 overexpression emerges as a potential and effective pneumonia treatment. The underlying mechanism may involve inducing protective autophagy, thereby reducing oxidative stress and cell pyroptosis.

Feruloylated Oligosaccharides Prevented Influenza-Induced Lung Inflammation via the RIG-I/MAVS/TRAF3 Pathway.

Uncontrolled inflammation contributes significantly to the mortality in acute respiratory infections. Our previous research has demonstrated that maize bran feruloylated oligosaccharides (FOs) possess notable anti-inflammatory properties linked to the NF-kB pathway regulation. In this study, we clarified that the oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines (IFN-α, IFN-ß, IL-6, IL-10, and IL-23) in the lungs. The mechanism involves FOs suppressing the transduction of the RIG-I/MAVS/TRAF3 signaling pathway, subsequently lowering the expression of NF-κB. In silico analysis suggests that FOs have a greater binding affinity for the RIG-I/MAVS signaling complex. This indicates that FOs have potential as promising targets for immune modulation. Moreover, in MAVS knockout mice, we confirmed that the anti-inflammatory function of FOs against influenza depends on MAVS. Comprehensive analysis using 16S rRNA gene sequencing and metabolite profiling techniques showed that FOs have the potential to restore immunity by modulating the gut microbiota. In conclusion, our study demonstrates that FOs are effective anti-inflammatory phytochemicals in inhibiting lung inflammation caused by influenza. This suggests that FOs could serve as a potential nutritional strategy for preventing the H1N1 virus infection and associated lung inflammation.

Urban airborne PM2.5 induces pulmonary fibrosis through triggering glycolysis and subsequent modification of histone lactylation in macrophages.

Airborne fine particulate matter (PM2.5) can cause pulmonary inflammation and even fibrosis, however, the underlying molecular mechanisms of the pathogenesis of PM2.5 exposure have not been fully appreciated. In the present study, we explored the dynamics of glycolysis and modification of histone lactylation in macrophages induced by PM2.5-exposure in both in vivo and in vitro models. Male C57BL/6 J mice were anesthetized and administrated with PM2.5 by intratracheal instillation once every other day for 4 weeks. Mouse RAW264.7 macrophages and alveolar epithelial MLE-12 cells were treated with PM2.5 for 24 h. We found that PM2.5 significantly increased lactate dehydrogenase (LDH) activities and lactate contents, and up-regulated the mRNA expression of key glycolytic enzymes in the lungs and bronchoalveolar lavage fluids of mice. Moreover, PM2.5 increased the levels of histone lactylation in both PM2.5-exposed lungs and RAW264.7 cells. The pro-fibrotic cytokines secreted from PM2.5-treated RAW264.7 cells triggered epithelial-mesenchymal transition (EMT) in MLE-12 cells through activating transforming growth factor-ß (TGF-ß)/Smad2/3 and VEGFA/ERK pathways. In contrast, LDHA inhibitor (GNE-140) pretreatment effectively alleviated PM2.5-induced pulmonary inflammation and fibrosis via inhibiting glycolysis and subsequent modification of histone lactylation in mice. Thus, our findings suggest that PM2.5-induced glycolysis and subsequent modification of histone lactylation play critical role in the PM2.5-associated pulmonary fibrosis.

Neutrophil-derived Activin-A moderates their pro-NETotic activity and attenuates collateral tissue damage caused by Influenza A virus infection.

Background: Pre-neutrophils, while developing in the bone marrow, transcribe the Inhba gene and synthesize Activin-A protein, which they store and release at the earliest stage of their activation in the periphery. However, the role of neutrophil-derived Activin-A is not completely understood. Methods: To address this issue, we developed a neutrophil-specific Activin-A-deficient animal model (S100a8-Cre/Inhba fl/fl mice) and analyzed the immune response to Influenza A virus (IAV) infection. More specifically, evaluation of body weight and lung mechanics, molecular and cellular analyses of bronchoalveolar lavage fluids, flow cytometry and cell sorting of lung cells, as well as histopathological analysis of lung tissues, were performed in PBS-treated and IAV-infected transgenic animals. Results: We found that neutrophil-specific Activin-A deficiency led to exacerbated pulmonary inflammation and widespread hemorrhagic histopathology in the lungs of IAV-infected animals that was associated with an exuberant production of neutrophil extracellular traps (NETs). Moreover, deletion of the Activin-A receptor ALK4/ACVR1B in neutrophils exacerbated IAV-induced pathology as well, suggesting that neutrophils themselves are potential targets of Activin-A-mediated signaling. The pro-NETotic tendency of Activin-A-deficient neutrophils was further verified in the context of thioglycollate-induced peritonitis, a model characterized by robust peritoneal neutrophilia. Of importance, transcriptome analysis of Activin-A-deficient neutrophils revealed alterations consistent with a predisposition for NET release. Conclusion: Collectively, our data demonstrate that Activin-A, secreted by neutrophils upon their activation in the periphery, acts as a feedback mechanism to moderate their pro-NETotic tendency and limit the collateral tissue damage caused by neutrophil excess activation during the inflammatory response.

Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Macrophage-derived exosomal HMGB3 regulates silica-induced pulmonary inflammation by promoting M1 macrophage polarization and recruitment.

BACKGROUND: Chronic inflammation and fibrosis are characteristics of silicosis, and the inflammatory mediators involved in silicosis have not been fully elucidated. Recently, macrophage-derived exosomes have been reported to be inflammatory modulators, but their role in silicosis has not been explored. The purpose of the present study was to investigate the role of macrophage-derived exosomal high mobility group box 3 (HMGB3) in silica-induced pulmonary inflammation. METHODS: The induction of the inflammatory response and the recruitment of monocytes/macrophages were evaluated by immunofluorescence, flow cytometry and transwell assays. The expression of inflammatory cytokines was examined by RT-PCR and ELISA, and the signalling pathways involved were examined by western blot analysis. RESULTS: HMGB3 expression was increased in exosomes derived from silica-exposed macrophages. Exosomal HMGB3 significantly upregulated the expression of inflammatory cytokines, activated the STAT3/MAPK (ERK1/2 and p38)/NF-κB pathways in monocytes/macrophages, and promoted the migration of these cells by CCR2. CONCLUSIONS: Exosomal HMGB3 is a proinflammatory modulator of silica-induced inflammation that promotes the inflammatory response and recruitment of monocytes/macrophages by regulating the activation of the STAT3/MAPK/NF-κB/CCR2 pathways.

Favipiravir ameliorates bleomycin-induced pulmonary fibrosis by reprogramming M1/M2 macrophage polarization.

Corona Virus Disease 2019 (COVID-19) is an infectious disease that seriously endangers human life and health. The pathological anatomy results of patients who died of the COVID-19 showed that there was an excessive inflammatory response in the lungs. It is also known that most of the COVID-19 infected patients will cause different degrees of lung damage after infection, and may have pulmonary fibrosis remaining after cure. Macrophages are a type of immune cell population with pluripotency and plasticity. In the early and late stages of infection, the dynamic changes of the balance and function of M1/M2 alveolar macrophages have a significant impact on the inflammatory response of the lungs. In the early stage of pulmonary fibrosis inflammation, the increase in the proportion of M1 type is beneficial to clear pathogenic microorganisms and promote the progress of inflammation; in the later stage of fibrosis, the increase in the number of M2 type macrophages can inhibit the inflammatory response and promote the degradation of fibrosis. As a potential treatment drug for new coronavirus pneumonia, favipiravir is in the process of continuously carried out relevant clinical trials. This study aims to discuss whether the antiviral drug favipiravir can suppress inflammation and immune response by regulating the M1/M2 type of macrophages, thereby alleviating fibrosis. We established a bleomycin-induced pulmonary fibrosis model, using IL-4/13 and LPS/IFN-γ cell stimulating factor to induce macrophage M1 and M2 polarization models, respectively. Our study shows that favipiravir exerts anti-fibrotic effects mainly by reprogramming M1/M2 macrophages polarization, that is, enhancing the expression of anti-fibrotic M1 type, reducing the expression of M2 type pro-fibrotic factors and reprogramming it to anti-fibrotic phenotype. Aspects of pharmacological mechanisms, favipiravir inhibits the activation of JAK2-STAT6 and JAK2-PI3K-AKT signaling by targeting JAK2 protein, thereby inhibiting pro-fibrotic M2 macrophages polarization and M2-induced myofibroblast activation. In summary, favipiravir can reduce the progression of pulmonary fibrosis, we hope to provide a certain reference for the treatment of pulmonary fibrosis.

Ischemia and reperfusion-injured liver-derived exosomes elicit acute lung injury through miR-122-5p regulated alveolar macrophage polarization.

Acute lung injury (ALI) is a common postoperative complication, particularly in pediatric patients after liver transplantation. Hepatic ischemia-reperfusion (HIR) increases the release of exosomes (IR-Exos) in peripheral circulation. However, the role of IR-Exos in the pathogenesis of ALI induced by HIR remains unclear. Here, we explored the role of exosomes derived from the HIR-injured liver in ALI development. Intravenous injection of IR-Exos caused lung inflammation in naive rats, whereas pretreatment with an inhibitor of exosomal secretion (GW4869) attenuated HIR-related lung injury. In vivo and in vitro results show that IR-Exos promoted proinflammatory responses and M1 macrophage polarization. Furthermore, miRNA profiling of serum identified miR-122-5p as the exosomal miRNA with the highest increase in young rats with HIR compared with controls. Additionally, IR-Exos transferred miR-122-5p to macrophages and promoted proinflammatory responses and M1 phenotype polarization by targeting suppressor of cytokine signaling protein 1(SOCS-1)/nuclear factor (NF)-κB. Importantly, the pathological role of exosomal miR-122-5p in initiating lung inflammation was reversed by inhibition of miR-122-5p. Clinically, high levels of miR-122-5p were found in serum and correlated to the severity of lung injury in pediatric living-donor liver transplant recipients with ALI. Taken together, our findings reveal that IR-Exos transfer liver-specific miR-122-5p to alveolar macrophages and elicit ALI by inducing M1 macrophage polarization via the SOCS-1/NF-κB signaling pathway.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

RATIONALE: Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. OBJECTIVES: To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. METHODS: In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. RESULTS: GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. CONCLUSIONS: GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

NLRP3 knockout in mice provided protection against Serratia marcescens-induced acute pneumonia by decreasing PD-L1 and PD-1 expression in macrophages.

Antibiotic-resistant Serratia marcescens (Sm) is known to cause bloodstream infections, pneumonia, etc. The nod-like receptor family, pyrin domain-containing 3 (NLRP3), has been implicated in various lung infections. Yet, its role in Sm-induced pneumonia was not well understood. In our study, we discovered that deletion of Nlrp3 in mice significantly improved Sm-induced survival rates, reduced bacterial loads in the lungs, bronchoalveolar lavage fluid (BALF), and bloodstream, and mitigated the severity of acute lung injury (ALI) compared to wild-type (WT) mice. Mechanistically, we observed that 24 h post-Sm infection, NLRP3 inflammasome activation occurred, leading to gasdermin D NH2-terminal (GSDMD-NT)-induced pyroptosis in macrophages and IL-1ß secretion. The NLRP3 or NLRP3 inflammasome influenced the expression PD-L1 and PD-1, as well as the count of PD-L1 or PD-1-expressing macrophages, alveolar macrophages, interstitial macrophages, PD-L1-expressing neutrophils, and the count of macrophage receptors with collagenous structure (MARCO)-expressing macrophages, particularly MARCO+ alveolar macrophages. The frequency of MARCO+ alveolar macrophages, PD-1 expression, particularly PD-1+ interstitial macrophages were negatively or positively correlated with the Sm load, respectively. Additionally, IL-1ß levels in BALF correlated with three features of acute lung injury: histologic score, protein concentration and neutrophil count in BALF. Consequently, our findings suggest that Nlrp3 deletion offers protection agaisnt acute Sm pneumonia in mice by inhibiting inflammasome activation and reducing Sm infection-induced PD-L1/PD-1 or MARCO expression, particularly in macrophages. This highlights potential therapeutic targets for Sm and other gram-negative bacteria-induced acute pneumonia.

IL-4 activates ULK1/Atg9a/Rab9 in asthma, NLRP3 inflammasomes, and Golgi fragmentation by increasing autophagy flux and mitochondrial oxidative stress.

During asthma, there is an intensification of pulmonary epithelial inflammation, mitochondrial oxidative stress, and Golgi apparatus fragmentation. However, the underlying mechanism remains largely unknown. Therefore, this study investigated the roles of ULK1, Atg9a, and Rab9 in epithelial inflammation, mitochondrial oxidative stress, and Golgi apparatus fragmentation. We found that ULK1 gene knockout reduced the infiltration of inflammatory cells, restored the imbalance of the Th1/Th2 ratio, and inhibited the formation of inflammatory bodies in the lung tissue of house dust mite-induced asthma mice. Moreover, we demonstrated that Atg9a interacted with ULK1 at S467. ULK1 phosphorylated Atg9a at S14. Treatment with ULK1 activator (LYN-1604) and ULK1 inhibitor (ULK-101) respectively promoted and inhibited inflammasome activation, indicating that the activation of inflammasome induced by house dust mite in asthma mice is dependent on ULK1. For validation of the in vivo results, we then used a lentivirus containing ULK1 wild type and ULK1-S467A genes to infect Beas-2b-ULK1-knockout cells and establish a stable cell line. The results suggest that the ULK1 S467 site is crucial for IL-4-induced inflammation and oxidative stress. Experimental verification confirmed that Atg9a was the superior signaling pathway of Rab9. Interestingly, we found for the first time that Rab9 played a very important role in inflammation-induced fragmentation of the Golgi apparatus. Inhibiting the activation of the ULK1/Atg9a/Rab9 signaling pathways can inhibit Golgi apparatus fragmentation and mitochondrial oxidative stress in asthma while reducing the production of NLRP3-mediated pulmonary epithelial inflammation.

Fine particulate matter 2.5 induces susceptibility to Pseudomonas aeruginosa infection via expansion of PD-L1high neutrophils in mice.

BACKGROUND: Exposure to PM2.5 has been implicated in a range of detrimental health effects, particularly affecting the respiratory system. However, the precise underlying mechanisms remain elusive. METHODS: To address this objective, we collected ambient PM2.5 and administered intranasal challenges to mice, followed by single-cell RNA sequencing (scRNA-seq) to unravel the heterogeneity of neutrophils and unveil their gene expression profiles. Flow cytometry and immunofluorescence staining were subsequently conducted to validate the obtained results. Furthermore, we assessed the phagocytic potential of neutrophils upon PM2.5 exposure using gene analysis of phagocytosis signatures and bacterial uptake assays. Additionally, we utilized a mouse pneumonia model to evaluate the susceptibility of PM2.5-exposed mice to Pseudomonas aeruginosa infection. RESULTS: Our study revealed a significant increase in neutrophil recruitment within the lungs of PM2.5-exposed mice, with subclustering of neutrophils uncovering subsets with distinct gene expression profiles. Notably, exposure to PM2.5 was associated with an expansion of PD-L1high neutrophils, which exhibited impaired phagocytic function dependent upon PD-L1 expression. Furthermore, PM2.5 exposure was found to increase the susceptibility of mice to Pseudomonas aeruginosa, due in part to increased PD-L1 expression on neutrophils. Importantly, monoclonal antibody targeting of PD-L1 significantly reduced bacterial burden, dissemination, and lung inflammation in PM2.5-exposed mice upon Pseudomonas aeruginosa infection. CONCLUSIONS: Our study suggests that PM2.5 exposure promotes expansion of PD-L1high neutrophils with impaired phagocytic function in mouse lungs, contributing to increased vulnerability to bacterial infection, and therefore targeting PD-L1 may be a therapeutic strategy for reducing the harmful effects of PM2.5 exposure on the immune system.

The combined effect of oxidative stress and TRPV1 on temperature-induced asthma: Evidence in a mouse model.

Temperature is one of the possible activators for asthma. As global warming continues, the health hazard of high temperatures is increasing. It is unclear, nevertheless, how high temperatures affect asthma. The research aims to examine how asthma is affected by high temperatures and underlying molecular mechanisms. The BALB/c mice were adopted in a model of asthma. The mice were exposed at 24 °C, 38 °C and 40 °C for 4h on weekdays from day 1 to day 30. After the experiment, the lung function was measured in vivo, and then serum protein, pulmonary inflammation and immunohistochemistry assay was assessed in vitro. As the temperature increased from 24 °C to 40 °C, there was a significant increase in serum protein, while there is no discernible difference in serum protein of OVA-sIgE and OVA-sIgG between the OVA (38 °C) group and OVA (24 °C) group. The immunohistochemistry assay showed a change in the pro-inflammatory cytokines. The histopathological analysis exhibited the change of airway structure after high-temperature exposure, especially for exposure at 40 °C. The results of signals protein showed a remarkable rise of TRPV1 for OVA+40 °C. Our results revealed that high temperatures may make asthmatic airway dysfunction severe, and the higher the temperature, the more serious asthma. The oxidative stress and TRPV1 receptor can be a potential drug target for asthma. It will provide a new tool for precision medicine in asthma.