Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis.

BACKGROUND: Influenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood. METHODS: COPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole. RESULTS: The present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV20,50/FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1ß attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1ß/STAT1 signaling via MTs. CONCLUSIONS: These findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1ß/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD.

Dexamethasone protects against asthma via regulating Hif-1α-glycolysis-lactate axis and protein lactylation.

PURPOSE: Asthma can not be eradicated till now and its control primarily relies on the application of corticosteroids. Recently, glycolytic reprogramming has been reportedly contributed to asthma, this study aimed to reveal whether the effect of corticosteroids on asthma control is related to their regulation of glycolysis and glycolysis-dependent protein lactylation. METHODS: Ovalbumin (OVA) aeroallergen was used to challenge mice and stimulate human macrophage cell line THP-1 following dexamethasone (DEX) treatment. Airway hyperresponsiveness, airway inflammation, the expressions of key glycolytic enzymes and pyroptosis markers, the level of lactic acid, real-time glycolysis and oxidative phosphorylation (OXPHOS), and protein lactylation were analyzed. RESULTS: DEX significantly attenuated OVA-induced eosinophilic airway inflammation, including airway hyperresponsiveness, leukocyte infiltration, goblet cell hyperplasia, Th2 cytokines production and pyroptosis markers expression. Meanwhile, OVA-induced Hif-1α-glycolysis axis was substantially downregulated by DEX, which resulted in low level of lactic acid. Besides, key glycolytic enzymes in the lungs of asthmatic mice were notably co-localized with F4/80-positive macrophages, indicating metabolic shift to glycolysis in lung macrophages during asthma. This was confirmed in OVA-stimulated THP-1 cells that DEX treatment resulted in reductions in pyroptosis, glycolysis and lactic acid level. Finally, protein lactylation was found significantly increased in the lungs of asthmatic mice and OVA-stimulated THP-1 cells, which were both inhibited by DEX. CONCLUSION: Our present study revealed that the effect of DEX on asthma control was associated with its suppressing of Hif-1α-glycolysis-lactateaxis and subsequent protein lactylation, which may open new avenues for the therapy of eosinophilic asthma.

Melatonin regulates circadian clock proteins expression in allergic airway inflammation.

Asthma demonstrates a strong circadian rhythm with disrupted molecular clock. Melatonin which can directly regulate circadian rhythm has been reported to alleviate asthma, but whether this effect is related to its regulation on circadian clock has not yet been known. Here, female C57BL/6 mice were challenged with ovalbumin (OVA) to establish allergic airway inflammation, and were treated with melatonin or Luzindole to investigate whether the expressions of circadian clock proteins were changed in response to OVA and were affected by exogenous/endogenous melatonin. Airway inflammation, mucus secretion, protein expressions of circadian proteins (Bmal1, Per1, Clock, Timeless, Cry1 and Cry2), melatonin biosynthetase (ASMT, AANAT) and melatonin receptor (Mel-1A/B-R) were analyzed accordingly. The results showed that in the successfully established allergic airway inflammation model, inflammatory cells infiltration, expressions of circadian clock proteins in the lung tissues of OVA-challenged mice were all notably up-regulated as compared to that of the vehicle mice. Meanwhile, the protein expression of ASMT and the level of melatonin in the lung tissues were reduced in allergic mice, while the expression of melatonin receptor Mel-1A/B-R was markedly increased. After addition of exogenous melatonin, the OVA-induced airway inflammation was pronouncedly ameliorated, while simultaneously the OVA-induced expressions of Per1 and Clock were further increased. However, a melatonin receptor antagonist Luzindole further augmented the OVA-induced airway inflammation, accompanied with remarkably decreased expressions of Per1, Bmal1, Cry1 and Cry2 but notably increased expression of Timeless. Collectively, our results demonstrated that the expression of circadian clock proteins was increased in the lungs during allergic airway inflammation, and Per1 was a clock protein that can be regulated by both exogenous and endogenous melatonin, suggesting Per1 may be an important potential circadian clock target for melatonin as a negative regulatory factor against Th2-type airway inflammation.

Exosome-derived circKIF20B suppresses gefitinib resistance and cell proliferation in non-small cell lung cancer.

BACKGROUND: The gefitinib resistance mechanism in non-small cell lung cancer (NSCLC) remains unclear, albeit exosomal circular RNA (circRNA) is known to possibly play a vital role in it. METHODS: We employed high-throughput sequencing techniques to detect the expressions of exosomal circRNA both in gefitinib-resistant and gefitinib-sensitive cells in this study. The circKIF20B expression was determined in serum exosomes and tissues of patients by qRT-PCR. The structure, stability, and intracellular localization of circKIF20B were verified by Sanger sequencing, Ribonuclease R (RNase R)/actinomycin D (ACTD) treatments, and Fluorescence in situ hybridization (FISH). The functions of circKIF20B were investigated by 5-Ethynyl-20-deoxyuridine (EdU), flow cytometry, Cell Counting Kit-8 (CCK-8), oxygen consumption rate (OCR), and xenograft model. Co-culture experiments were performed to explore the potential ability of exosomal circKIF20B in treating gefitinib resistance. The downstream targets of circKIF20B were determined by luciferase assay, RNA pulldown, and RNA immunoprecipitation (RIP). RESULTS: We found that circKIF20B was poorly expressed in the serum exosomes of gefitinib-resistant patients (n = 24) and the tumor tissues of patients with NSCLC (n = 85). CircKIF20B was negatively correlated with tumor size and tumor stage. Decreasing circKIF20B was found to promote gefitinib resistance by accelerating the cell cycle, inhibiting apoptosis, and enhancing mitochondrial oxidative phosphorylation (OXPHOS), whereas increasing circKIF20B was found to restore gefitinib sensitivity. Mechanistically, circKIF20B is bound to miR-615-3p for regulating the MEF2A and then altering the cell cycle, apoptosis, and mitochondrial OXPHOS. Overexpressing circKIF20B parental cells can restore sensitivity to gefitinib in the recipient cells by upregulating the exosomal circKIF20B expression. CONCLUSIONS: This study revealed a novel mechanism of circKIF20B/miR-615-3p/MEF2A signaling axis involving progression of gefitinib resistance in NSCLC. Exosomal circKIF20B is expected to be an easily accessible and alternative liquid biopsy candidate and potential therapeutic target in gefitinib-resistant NSCLC. The schematic diagram of mechanism in this study. Exosomal circKIF20B inhibits gefitinib resistance and cell proliferation by arresting the cell cycle, promoting apoptosis, and reducing OXPHOS via circKIF20B/miR-615-3p/MEF2A axis in NSCLC.

Differential Characteristics of Patients for Hospitalized Severe COVID-19 Infected by the Omicron Variants and Wild Type of SARS-CoV-2 in China.

Background: As multiple mutations of SARS-Cov-2 exist, there are now many viral variants with regional differences in distribution. The clinical characteristics of patients hospitalized with the virus also vary significantly, with those of the Omicron variants being strikingly different from those of the earliest wild-type variant. However, comprehensive data on this subject is lacking. It is therefore crucial to explore these differences to develop better clinical strategies for the management of COVID-19. Methods: A total of 554 confirmed COVID-19 cases in China were clinically classified as mild, moderate, severe, and critical according to their diagnoses and treatment plans. We compared the demographics and clinical characteristics of patients infected with the Omicron vs wild-type strains, between severe and non-severe cases. Bacterial co-infections with SARS-CoV-2 and correlation between inflammatory factors and T cells were analyzed. Results: Compared to the wild-type cases, the severe Omicron cases were older (median age 48.36 vs 73.24), and had more upper-respiratory symptoms and comorbidities. Decreased leukocyte counts were less pronounced, although more instances of significantly decreased CD4+ and CD8+ T-cell counts, elevated infection-related biomarkers (eg procalcitonin and C-reactive protein), and abnormal coagulation factors (including increased D-dimer and fibrinogen levels) were detected in the severe Omicron cases. The mean length of hospital stay was significantly shorter in the severe Omicron cases. CD4+ and CD8+ T cell numbers were negatively correlated with neutrophil-to-lymphocyte ratios, as well as serum interleukin-6, procalcitonin, and C-reactive protein levels. Conclusion: There were significant clinical differences between patients hospitalized with severe cases of Omicron- variant COVID-19 vs wild-type. The Omicron cases tended to be older and had more upper respiratory tract symptoms, comorbidities and bacterial co-infections. Elevated levels of inflammatory cytokines with T-cell depletion correlated with poor disease progression and prognosis. We hope these data provide a theoretical basis for future integrated prevention and control plans for COVID-19.

TLR2-hif1α-mediated glycolysis contributes to pyroptosis and oxidative stress in allergic airway inflammation.

As a pattern recognition receptor which activates innate immune system, toll-like receptor 2 (TLR2) has been reportedly mediates allergic airway inflammation (AAI), yet the underlying mechanism remains elusive. Here, in a murine AAI model, TLR2-/- mice showed decreased airway inflammation, pyroptosis and oxidative stress. RNA-sequencing revealed that allergen-induced hif1 signaling pathway and glycolysis were significantly downregulated when TLR2 was deficient, which were confirmed by lung protein immunoblots. Glycolysis inhibitor 2-Deoxy-d-glucose (2-DG) inhibited allergen-induced airway inflammation, pyroptosis, oxidative stress and glycolysis in wild type (WT) mice, while hif1α stabilizer ethyl 3,4-dihydroxybenzoate (EDHB) restored theses allergen-induced changes in TLR2-/- mice, indicating TLR2-hif1α-mediated glycolysis contributes to pyroptosis and oxidative stress in AAI. Moreover, upon allergen challenge, lung macrophages were highly activated in WT mice but were less activated in TLR2-/- mice, 2-DG replicated while EDHB reversed such effect of TLR2 deficiency on lung macrophages. Likewise, both in vivo and ex vivo WT alveolar macrophages (AMs) exhibited higher TLR2/hif1α expression, glycolysis and polarization activation in response to ovalbumin (OVA), which were all inhibited in TLR2-/- AMs, suggesting AMs activation and metabolic switch are dependent on TLR2. Finally, depletion of resident AMs in TLR2-/- mice abolished while transfer of TLR2-/- resident AMs to WT mice replicated the protective effect of TLR2 deficiency on AAI when administered before allergen challenge. Collectively, we suggested that loss of TLR2-hif1α-mediated glycolysis in resident AMs ameliorates allergic airway inflammation that inhibits pyroptosis and oxidative stress, therefore the TLR2-hif1α-glycolysis axis in resident AMs may be a novel therapeutic target for AAI.

Itaconate Suppresses the Activation of Mitochondrial NLRP3 Inflammasome and Oxidative Stress in Allergic Airway Inflammation.

Itaconate has emerged as a novel anti-inflammatory and antioxidative endogenous metabolite, yet its role in allergic airway inflammation (AAI) and the underlying mechanism remains elusive. Here, the itaconate level in the lung was assessed by High Performance Liquid Chromatography (HPLC), and the effects of the Irg1/itaconate pathway on AAI and alveolar macrophage (AM) immune responses were evaluated using an ovalbumin (OVA)-induced AAI model established by wild type (WT) and Irg1-/- mice, while the mechanism of this process was investigated by metabolomics analysis, mitochondrial/cytosolic protein fractionation and transmission electron microscopy in the lung tissues. The results demonstrated that the Irg1 mRNA/protein expression and itaconate production in the lung were significantly induced by OVA. Itaconate ameliorated while Irg1 deficiency augmented AAI, and this may be attributed to the fact that itaconate suppressed mitochondrial events such as NLRP3 inflammasome activation, oxidative stress and metabolic dysfunction. Furthermore, we identified that the Irg1/itaconate pathway impacted the NLRP3 inflammasome activation and oxidative stress in AMs. Collectively, our findings provide evidence for the first time, supporting the conclusion that in the allergic lung, the itaconate level is markedly increased, which directly regulates AMs' immune responses. We therefore propose that the Irg1/itaconate pathway in AMs is a potential anti-inflammatory and anti-oxidative therapeutic target for AAI.

RKIP suppresses the influenza A virus­induced airway inflammatory response via the ERK/MAPK pathway.

Raf kinase inhibitor protein (RKIP) is an inflammation­inhibiting mediator that is involved in several diseases; however, the potential mechanism of action of RKIP on the inflammatory response induced by influenza A virus (IAV) remains unclear. The present study aimed to investigate whether RKIP regulated the inflammatory response via the ERK/MAPK pathway. The present study detected the expression levels of RKIP and alterations in the inflammatory response in human normal bronchial epithelial BEAS­2B cells, diseased human bronchial epithelial cells and primary human bronchial epithelial cells infected with IAV. Cells were treated with locostatin to inhibit the expression of RKIP. RKIP was overexpressed by lentivirus transduction and the small molecule inhibitor SCH772984 was applied to specifically inhibit activation of the ERK/MAPK pathway. In addition, C57BL/6 mice were infected with IAV to further confirm the role of RKIP in regulation of the inflammatory response via ERK/MAPK in vivo. Western blotting, reverse transcription­quantitative PCR, ELISA, 5­ethynyl­-2'­deoxyuridine assay, immunofluorescence staining, Cell Counting Kit­8, cell cycle assay, hematoxylin and eosin staining, and immunohistochemistry were used to detect all of the changes. Notably, RKIP attenuated the inflammatory response that was triggered by IAV infection in airway epithelial cells, which was characterized by augmented inflammatory cytokines and cell cycle arrest. Furthermore, the ERK/MAPK pathway was revealed to be activated by IAV infection and downregulation of RKIP aggravated the airway inflammatory response. By contrast, overexpression of RKIP effectively ameliorated the airway inflammatory response induced by IAV. These findings demonstrated that RKIP may serve a protective role in airway epithelial cells by combating inflammation via the ERK/MAPK pathway. Collectively, the present findings suggested that RKIP may negatively regulate airway inflammation and thus may constitute a promising therapeutic strategy for airway inflammatory­related diseases that are induced by IAV.

Melatonin Suppresses Macrophage M1 Polarization and ROS-Mediated Pyroptosis via Activating ApoE/LDLR Pathway in Influenza A-Induced Acute Lung Injury.

Influenza virus infection is one of the strongest pathogenic factors for the development of acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS). However, the underlying cellular and molecular mechanisms have not been clarified. In this study, we aim to investigate whether melatonin modulates macrophage polarization, oxidative stress, and pyroptosis via activating Apolipoprotein E/low-density lipoprotein receptor (ApoE/LDLR) pathway in influenza A-induced ALI. Here, wild-type (WT) and ApoE-/- mice were instilled intratracheally with influenza A (H3N2) and injected intraperitoneally with melatonin for 7 consecutive days. In vitro, WT and ApoE-/- murine bone marrow-derived macrophages (BMDMs) were pretreated with melatonin before H3N2 stimulation. The results showed that melatonin administration significantly attenuated H3N2-induced pulmonary damage, leukocyte infiltration, and edema; decreased the expression of proinflammatory M1 markers; enhanced anti-inflammatory M2 markers; and switched the polarization of alveolar macrophages (AMs) from M1 to M2 phenotype. Additionally, melatonin inhibited reactive oxygen species- (ROS-) mediated pyroptosis shown by downregulation of malonaldehyde (MDA) and ROS levels as well as inhibition of the NLRP3/GSDMD pathway and lactate dehydrogenase (LDH) release. Strikingly, the ApoE/LDLR pathway was activated when melatonin was applied in H3N2-infected macrophages and mice. ApoE knockout mostly abrogated the protective impacts of melatonin on H3N2-induced ALI and its regulatory ability on macrophage polarization, oxidative stress, and pyroptosis. Furthermore, recombinant ApoE3 (re-ApoE3) inhibited H3N2-induced M1 polarization of BMDMs with upregulation of MT1 and MT2 expression, but re-ApoE2 and re-ApoE4 failed to do this. Melatonin combined with re-ApoE3 played more beneficial protective effects on modulating macrophage polarization, oxidative stress, and pyroptosis in H3N2-infected ApoE-/- BMDMs. Our study indicated that melatonin attenuated influenza A- (H3N2-) induced ALI by inhibiting the M1 polarization of pulmonary macrophages and ROS-mediated pyroptosis via activating the ApoE/LDLR pathway. This study suggested that melatonin-ApoE/LDLR axis may serve as a novel therapeutic strategy for influenza virus-induced ALI.

Interleukin-6 neutralizing antibody attenuates the hypersecretion of airway mucus via inducing the nuclear translocation of Nrf2 in chronic obstructive pulmonary disease.

Airway mucus hypersecretion is a vital pathophysiologic feature in chronic obstructive pulmonary disease (COPD) patients in which airflow limitation result, and it is key to strategizing in the management of COPD. To investigate the mechanisms underlying the action of interleukin-6 neutralizing antibody (IL-6 Ab) in attenuating airway mucus hypersecretion in COPD, human and mouse primary bronchial epithelial cells from COPD patients and mice were isolated, human organoid model of trachea was established and all treated with IL-6 and/or IL-6 Ab. The differential expression of Muc5ac and Nrf2 were determined in pDHBE compared to pNHBE cells via high-throughput sequencing of transcriptome. The serum concentration of Muc5ac was significantly elevated and positively correlated with IL-6 in COPD patients using ELISA, and the excessive mucus secretion was observed in the trachea of COPD patients using HE, AB-PAS and IHC staining. The levels of Muc5ac were significantly elevated in the IL-6-treated group, and diminished with IL-6 Ab treatment, both in vitro and in the organoid model using qRT-PCR, WB and IF. The expression levels of protein Muc5ac were significantly reduced in cells transfected with the IL-6 small interfering RNA (siRNA-IL-6), which was in contrast to the levels of protein Nrf2, and the protective effects of IL-6 Ab were inhibited in cells transfected with Nrf2 short hairpin RNA (shRNA-Nrf2). IL-6 Ab significantly attenuated hypersecretion of airway mucus by inducing nuclear translocation of Nrf2 in COPD. These findings indicated that IL-6 Ab may constitute a novel therapeutic agent for IL-6-induced airway mucus hypersecretion by improving airflow limitation in COPD patients.

Influenza a virus triggers acute exacerbation of chronic obstructive pulmonary disease by increasing proinflammatory cytokines secretion via NLRP3 inflammasome activation.

BACKGROUND: Influenza A virus (IAV) triggers acute exacerbation of chronic obstructive pulmonary disease (AECOPD), but the molecular mechanisms remain unclear. In this study, we investigated the role of IAV induced NLRP3 inflammasome activation to increase airway inflammation response in the progression of AECOPD. METHODS: Human bronchial epithelial cells were isolated and cultured from normal and COPD bronchial tissues and co-cultured with IAV. The NLRP3 inflammasome associated genes were identified using RNA sequencing, and the expressions of NLRP3 inflammasome components were measured using qRT-PCR and western blot after cells were transfected with siRNA and treated with MCC950. Moreover, IAV-induced COPD rat models were established to confirm the results; 37 AECOPD patients were included to measure the serum and bronchoalveolar lavage fluid (BALF) of interleukin (IL)-18 and IL-1ß. RESULTS: Increased levels of NLRP3 inflammasome components were not seen until 6 h post-inoculation in normal cells. However, both cell groups reached peak NLRP3 level at 12 h post-inoculation and maintained it for up to 24 h. ASC, Caspase-1, IL-1ß and IL-18 were also elevated in a similar time-dependent pattern in both cell groups. The mRNA and protein expression of the NLRP3 inflammasome components were decreased when COPD cells treated with siRNA and MCC950. In COPD rats, the NLRP3 inflammasome components were elevated by IAV. MCC950 alleviated lung damage, improved survival time, and reduced NLRP3 inflammasome components expression in COPD rats. Additionally, the serum and BALF levels of IL-1ß and IL-18 were increased in AECOPD patients. CONCLUSIONS: NLRP3 inflammasome is activated in COPD patients as a pre-existing condition that is further exacerbated by IAV infection.

Melatonin attenuates LPS-induced pyroptosis in acute lung injury by inhibiting NLRP3-GSDMD pathway via activating Nrf2/HO-1 signaling axis.

Acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) is featured by intensive inflammatory responses and oxidative stress, which lead to cytokine storms and pyroptosis. Here, we aimed to investigate whether melatonin was capable of alleviating LPS-induced ALI via activating the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling axis and inhibiting pyroptosis. Mice were injected with melatonin (30 mg/kg) intraperitoneally for consecutive five days before LPS instillation intratracheally, and human alveolar epithelial cell (AECⅡ) A549 cell lines and murine macrophages Raw264.7 cell lines were pretreated with melatonin (400 µM) before LPS (10 µg/ml) stimulation. The result demonstrated that LPS induced obvious lung injury characterized by alveolar damage, neutrophil infiltration and lung edema as well as the reduction of the survival rate of mice, which were totally reversed by melatonin pretreatment. Mechanistically, melatonin pretreatment activated nuclear factor erythroid2-related factor (Nrf) 2 signaling, subsequently, drove antioxidant pathways including significant increases in the expression of Nrf2, HO-1, NQO1, Mn-SOD and Catalase in vivo and in vitro. Simultaneously, melatonin inhibited ROS and MDA overproduction, iNOS expression as well as TNF-α and IL-1ß expression and release. Furthermore, melatonin inhibited LPS-induced pyroptosis by reversing the overexpression of NLRP3, Caspase-1, IL-1ß, IL-18 and GSDMD-N, as well as LDH release and TUNEL-positive cells in A549 cells and Raw264.7 cells. Overall, the current study suggests that melatonin exerts protective roles on LPS-induced ALI and pyroptosis by inhibiting NLRP3-GSDMD pathway via activating Nrf2/HO-1 signaling axis.

CD19 and POU2AF1 are Potential Immune-Related Biomarkers Involved in the Emphysema of COPD: On Multiple Microarray Analysis.

Purpose: Emphysema is the main cause of the progression of chronic obstructive pulmonary disease (COPD). This study aimed to identify the key genes involved in COPD-related emphysema. Patients and Methods: GSE76925 was downloaded from Gene Expression Omnibus database. Protein-protein interaction networks of differentially expressed genes (DEGs) between control and COPD groups were constructed to identify hub genes using Cytoscape. Diagnostic performance of hub genes was evaluated using receiver operating characteristic analysis. Correlation analysis was performed to identify the key genes by analyzing the relationship between the hub genes and lung function and computed tomography (CT) indexes of emphysema. COPD patients were then divided into two groups based on the median expression of key genes and DEGs between these two groups were identified. Enrichment analysis of DEGs and correlation analysis between key genes and the infiltration of the immune cells were also analyzed. Finally, the role of key genes was evaluated in a lung tissues dataset (GSE47460) and a blood dataset (GSE76705). Additionally, the expression of key genes was validated by quantitative real-time polymerase chain reaction and immunohistochemistry. Results: CD19 and POU2AF1 had diagnostic efficacy for COPD and were significantly correlated with lung function and CT indexes of emphysema. Enrichment and immune analyses revealed that CD19 and POU2AF1 were correlated with the B cells in COPD. These results were consistent in GSE47460. The expression of CD19 and POU2AF1 in blood was the opposite of that in lung tissues, and CD19 and POU2AF1 were both significantly upregulated in COPD lung tissues at both the mRNA and protein levels. Conclusion: CD19 and POU2AF1 may serve as key regulators of emphysema and contribute to the progression of COPD by regulating the B-cell immunology. Targeting B cells may be a promising strategy for treating COPD.

The Influenza A Virus H3N2 Triggers the Hypersusceptibility of Airway Inflammatory Response via Activating the lncRNA TUG1/miR-145-5p/NF-κB Pathway in COPD.

Background: Patients with chronic obstructive pulmonary disease (COPD) are more susceptible to influenza A virus (IAV) with more severe symptoms, yet the underlying molecular mechanisms of the hypersusceptibility of airway inflammatory response remain unclear. Methods: The primary human bronchial epithelial cells (pHBECs) were isolated from normal and COPD bronchial tissues (NHBE and DHBE) and cultured with/without IAV infection in vitro. DHBE cells were exposed to IAV for 24 h after knockdown of lncRNA TUG1 with short hairpin RNA (shRNA). Gain-of-function assays were performed with the miR-145-5p inhibitor and NF-κBp65 transfection. The expressions of lncRNA TUG1, miR-145-5p, phospho-NF-κBp65, NF-κBp65, TNF-α, and (Interleukin) IL-1ß were examined with qRT-PCR, Western blotting, and ELISA. The interactions of lncRNA TUG1, miR-145-5p, and NF-κB were verified with luciferase reporter assay. Results: The expressions of lncRNA TUG1, phospho-NF-κBp65, TNF-α, and IL-1ß were increased significantly in pHBECs after being infected with IAV for 24 h (all p0.05). The detailed time analysis revealed that the NF-κBp65 in DHBE was activated earlier than that in NHBE by Western blotting and immunofluorescence. Knockdown of lncRNA TUG1 and miR-145-5p mimic attenuated the expressions of NF-κBp65, TNF-α, and IL-1ß significantly. The miR-145-5p inhibitor and NF-κBp65 transfection reversed the attenuated expressions of NF-κBp65, TNF-α, and IL-1ß. Conclusion: The IAV causes the hypersusceptibility of airway inflammatory response, which may be closely associated with more severe symptoms in AECOPD patients. The lncRNA TUG1 inhibitor may be a promising therapeutic strategy for AECOPD caused by IAV.

Abscisic acid suppresses the activation of NLRP3 inflammasome and oxidative stress in murine allergic airway inflammation.

Abscisic acid (ABA), a well-known natural phytohormone reportedly exerts anti-inflammatory and anti-oxidative properties in diabetes and colitis. However, the efficacy of ABA against allergic airway inflammation and the underlying mechanism remain unknown. Herein, an OVA-induced murine allergic airway inflammation model was established and treated with ABA in the presence or absence of PPAR-γ antagonist GW9662. The results showed that ABA effectively stunted the development of airway inflammation, and concordantly downregulated OVA-induced activation of NLRP3 inflammasome, suppressed oxidative stress and decreased the expression of mitochondrial fusion/fission markers including Optic Atrophy 1 (OPA1), Mitofusion 2 (Mfn2), dynamin-related protein 1 (DRP1) and Fission 1 (Fis1). Moreover, ABA treatment further increased OVA-induced expression of PPAR-γ, while GW9662 abrogated the inhibitory effect of ABA on allergic airway inflammation as well as on the activation of NLRP3 inflammasome and oxidative stress. Consistently, ABA inhibited the activation of NLRP3 inflammasome, suppressed oxidative stress and mitochondrial fusion/fission in LPS-stimulated Raw264.7 cells via PPAR-γ. Collectively, ABA ameliorates OVA-induced allergic airway inflammation in a PPAR-γ dependent manner, and such effect of ABA may be associated with its inhibitory effect on NLRP3 inflammasome and oxidative stress. Our results suggest the potential of ABA or ABA-rich food in protecting against asthma.

Correlation between sestrin2 expression and airway remodeling in COPD.

BACKGROUND: Airway remodeling is a major pathological characteristic of chronic obstructive pulmonary disease (COPD), and has been shown to be associated with oxidative stress. Sestrin2 has recently drawn attention as an important antioxidant protein. However, the underlying correlation between sestrin2 and airway remodeling in COPD has yet to be clarified. METHODS: A total of 124 subjects were enrolled in this study, including 62 control subjects and 62 COPD patients. The pathological changes in airway tissues were assessed by different staining methods. The expression of sestrin2 and matrix metalloproteinase 9 (MMP9) in airway tissues was monitored by immunohistochemistry. Enzyme-linked immunosorbent assays (ELISAs) were used to detect the serum concentrations of sestrin2 and MMP9. The airway parameters on computed tomography (CT) from all participants were measured for evaluating airway remodeling. The relationship between serum sestrin2 and MMP9 concentration and airway parameters in chest CT was also analyzed. RESULTS: In patients with COPD, staining of airway structures showed distinct pathological changes of remodeling, including cilia cluttered, subepithelial fibrosis, and reticular basement membrane (Rbm) fragmentation. Compared with control subjects, the expression of sestrin2 and MMP9 was significantly increased in both human airway tissues and serum. Typical imaging characteristics of airway remodeling and increased airway parameters were also found by chest CT. Additionally, serum sestrin2 concentration was positively correlated with serum MMP9 concentration and airway parameters in chest CT. CONCLUSION: Increased expression of sestrin2 is related to airway remodeling in COPD. We demonstrated for the first time that sestrin2 may be a novel biomarker for airway remodeling in patients with COPD.

Shufeng Jiedu capsules for treating acute exacerbations of chronic obstructive pulmonary disease: a systematic review and meta-analysis.

BACKGROUND: Chinese herbal medicine is widely used in combination with usual care for acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in China. Chinese patent medicine Shufeng Jiedu (SFJD) capsules is widely used for respiratory infectious diseases. This review aims to evaluate effectiveness and safety of SFJD for AECOPD. METHODS: A systematic review of randomised controlled trials (RCTs) in patients with AECOPD, who received SFJD as a single intervention or as add-on treatment to usual care. PubMed, the Cochrane Library, EMBASE, Scopus, Web of Science and four Chinese databases were searched from inception to April 2019. Two authors screened trials, extracted data, and assessed risk of bias, independently. Meta-analysis was performed using RevMan 5.3 software. We performed subgroup analyses and sensitivity analyses according to the predefined protocol. Quality of evidence was assessed using GRADE. RESULTS: Thirteen RCTs (1036 patients, with 936 inpatients) were included, all compared SFJD in combination with usual care (including antibiotics) to usual care alone. The mean age of participants ranged from 52 to 67 years, with approximately 60% male. Due to lack of blinding and other factors, all trials were of high risk of bias. SFJD was associated with a significant reduction in treatment failure, from 20.1 to 8.3% (11 trials; 815 patients; relative risk 0.43, 95% confidence interval [CI] 0.30 to 0.62), and duration of hospital stay (2 trials; 79 patients; mean difference - 4.32 days, 95% CI - 5.89 to - 2.75 days). No significant difference in adverse events was found between SFJD and control groups. CONCLUSION: Low certainty evidence suggests SFJD may bring additional benefit in reducing treatment failure, shorten hospital stay, and improving symptoms. Further large, high quality RCTs are needed to confirm its benefit and safety. TRIAL REGISTRATION: PROSPERO CRD42019133682.

TLR2-Melatonin Feedback Loop Regulates the Activation of NLRP3 Inflammasome in Murine Allergic Airway Inflammation.

Toll-like receptor 2 (TLR2) is suggested to initiate the activation of NLRP3 inflammasome, and considered to be involved in asthma. The findings that melatonin modulates TLRs-mediated immune responses, together with the suppressing effect of TLRs on endogenous melatonin synthesis, support the possibility that a feedback loop exists between TLRs system and endogenous melatonin synthesis. To determine whether TLR2-melatonin feedback loop exists in allergic airway disease and regulates NLRP3 inflammasome activity, wild-type (WT) and TLR2-/- mice were challenged with OVA to establish allergic airway disease model. Following OVA challenge, WT mice exhibited increased-expression of TLR2, activation of NLRP3 inflammasome and marked airway inflammation, which were all effectively inhibited in the TLR2-/- mice, indicating that TLR2-NLRP3 mediated airway inflammation. Meanwhile, melatonin biosynthesis was reduced in OVA-challenged WT mice, while such reduction was notably rescued by TLR2 deficiency, suggesting that TLR2-NLRP3-mediated allergic airway inflammation was associated with decreased endogenous melatonin biosynthesis. Furthermore, addition of melatonin to OVA-challenged WT mice pronouncedly ameliorated airway inflammation, decreased TLR2 expression and NLRP3 inflammasome activation, further implying that melatonin in turn inhibited airway inflammation via suppressing TLR2-NLRP3 signal. Most interestingly, although melatonin receptor antagonist luzindole significantly reduced the protein expressions of ASMT, AANAT and subsequent level of melatonin in OVA-challenged TLR2-/- mice, it exhibited null effect on leukocytes infiltration, Th2-cytokines production and NLRP3 activity. These results indicate that a TLR2-melatonin feedback loop regulates NLRP3 inflammasome activity in allergic airway inflammation, and melatonin may be a promising therapeutic medicine for airway inflammatory diseases such as asthma.