Peripheral blood biomarkers associated with combination of immune checkpoint blockade plus chemotherapy in NSCLC.

BACKGROUND: Biomarkers predicting clinical outcomes of treating non-small cell lung cancer (NSCLC) with combination of immune checkpoint inhibitors (ICIs) and chemotherapy would be valuable. OBJECTIVE: This study aims to seek predictors of combination of ICI/chemotherapy response in NSCLC patients using peripheral blood samples. METHODS: Patients diagnosed with advanced NSCLC between July 2019 and May 2021 receiving combination of ICI/chemotherapy were included and assessed for partial responses (PR), stable disease (SD) or progressive disease (PD). We measured circulating immune cells, plasma cytokines and chemokines. RESULTS: Nineteen patients were enrolled. The proportions of circulating natural killer (NK) cells within CD45 + cells, programmed death 1 (PD-1) + Tim-3 + T cells within CD4 + cells, and the amount of chemokine C-X-C ligand (CXCL10) in the plasma were significantly elevated in PR relative to SD/PD patients (median 8.1%-vs-2.1%, P= 0.0032; median 1.2%-vs-0.3%, P= 0.0050; and median 122.6 pg/ml-vs-76.0 pg/ml, P= 0.0125, respectively). Patients with 2 or 3 elevated factors had longer progression-free survival than patients with 0 or only one (not reached-vs-5.6 months, P= 0.0002). CONCLUSIONS: We conclude that NK cells, CD4 + PD-1 + Tim-3 + T cells, and CXCL10 levels in pre-treatment peripheral blood may predict the efficacy of combination of ICI/chemotherapy in NSCLC.

Associations between birth weight and lung function in a Japanese adult population: The tohoku medical megabank community-based cohort study.

BACKGROUND: Birth weight, as a measure of intrauterine growth, is commonly used in epidemiological studies and is reported to be associated with adult lung function. However, findings regarding this association in previous studies have been inconsistent. Furthermore, no studies have reported associations stratified by age or smoking status, or adjusted for eosinophil count or other parameters related to type 2 airway inflammation. METHODS: This cross-sectional study included 2632 men and 7237 women aged ≥20 years living in Miyagi Prefecture, Japan. Lung function was assessed based on spirometry. Birth weight data were obtained through a questionnaire survey. Analysis of covariance was used to evaluate the associations between birth weight and lung function, adjusting for potential confounders. Stratified analyses by age and smoking status were also conducted, together with a sub-analysis for low birth-weight participants. RESULTS: Birth weight was positively associated with forced expiratory volume in 1 s (FEV1) for both sexes and with vital capacity in women, after adjusting for height, age, smoking status, and parameters related to type 2 airway inflammation. The stratified analysis for smoking status revealed associations in never-smokers and ex-smokers. When stratified by age, the associations were confirmed in middle-aged participants. The effect of smoking status on the FEV1 of low birth-weight participants was not significant. CONCLUSIONS: Our analysis of a large, Japanese adult population showed that birth weight was independently and positively associated with adult lung function, even after adjustment for age, height, smoking status, and parameters related to type 2 airway inflammation.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Decreased expression of airway epithelial Axl is associated with eosinophilic inflammation in severe asthma.

BACKGROUND: Airway epithelium-derived cytokines are critical to provoke and perpetuate type 2 inflammation in asthma. Yet it is poorly understood how this epithelial cell-driven inflammatory response is negatively regulated. We previously reported that Axl receptor tyrosine kinase was expressed by basal cells in the airway epithelium and had a role in defining their stem cell identity. However, whether and how Axl regulates airway type 2 inflammation remains unknown. METHODS: We performed immunofluorescence staining to compare Axl expression in airway epithelium between non-asthmatic subjects, mild-moderate asthma and severe asthma. We confirmed this result by interrogating public databases of global gene expression in endobronchial biopsies. We then quantified eosinophil numbers infiltrating into the trachea of wild-type or Axl-knockout mice that were intranasally treated with house dust mite extracts (HDM). Cell-based assays using siRNA targeting Axl were further performed to identify molecules involved in Axl-mediated regulation of inflammation. RESULTS: Histological assessments and transcriptome analyses revealed decreases in protein and mRNA of Axl in airway basal cells of severe asthmatics. This reduction of Axl expression was correlated with infiltration of eosinophils and mast cells in severe asthmatics. Eosinophil infiltration was more evident in the trachea of Axl-knockout mice in response to repetitive HDM administration. siRNA-mediated knockdown of Axl increased mRNA and protein expression of granulocyte macrophage-colony stimulating factor (GM-CSF) in human bronchial epithelial cells. CONCLUSIONS: Axl kinase expressed by basal cells may suppress excessive eosinophilic inflammation via inhibition of GM-CSF in the airway. Axl reduction has clinical implications for the pathogenesis of severe asthma.

Increased LHX9 expression in alveolar epithelial type 2 cells of patients with chronic obstructive pulmonary disease.

BACKGROUND: Alveolar epithelial type 2 (AT2) cells serve as stem cells in alveolar epithelium and are assumed to lose their stem cell function in the lungs of chronic obstructive pulmonary disease (COPD). Although we previously reported that LHX9 mRNA expression was up-regulated in AT2 cells of COPD lung tissues, it is yet to be elucidated how LHX9 is associated with the vulnerability of AT2 cells in COPD. METHODS: AT2 cells were isolated from lung tissues of 10 non-COPD subjects and 11 COPD patients. LHX9 mRNA expression was determined by quantitative RT-PCR. To identify up-stream molecules, an alveolar epithelial cell line A549 was exposed to pro-inflammatory cytokines in vitro. siRNA-mediated Lhx9 knockdown was performed to determine how Lhx9 affected the cellular viability and the cell-division cycle. RESULTS: LHX9 mRNA expression was increased in AT2 cells from COPD lung tissues, compared to those from non-COPD tissues. The airflow obstruction was independently correlated with the increase in LHX9 expression. Among several pro-inflammatory cytokines, interferon-γ was a strong inducer of LHX9 expression in A549 cells. Lhx9 was involved in the increased susceptibility to serum starvation-induced death of A549 cells. CONCLUSIONS: Our data suggest that IFN-γ predominantly increases the LHX9 expression which enhances the susceptibility to cell death. Considering the independent association of the increased LHX9 expression in AT2 cells with airflow obstruction, the IFN-γ-Lhx9 axis might contribute to the vulnerability of AT2 cells in the lungs of COPD patients.

Upregulation of leukocyte immunoglobulin-like receptor B4 on interstitial macrophages in COPD; their possible protective role against emphysema formation.

BACKGROUND: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is one of the inhibitory receptors in various types of immune cells including macrophages. Previous reports suggested that LILRB4 could be involved in a negative feedback system to prevent excessive inflammatory responses. However, its role has been unclear in chronic obstructive pulmonary disease (COPD), in which macrophages play a crucial role in the pathogenesis. In this study, we aimed to examine the changes of LILRB4 on macrophages both in the lung specimens of COPD patients and the lungs of a mouse emphysema model. We then tried to compare the differences in both inflammation and emphysematous changes of the model between wild-type and LILRB4-deficient mice in order to elucidate the role of LILRB4 in the pathogenesis of COPD. METHODS: We prepared single-cell suspensions of resected lung specimens of never-smokers (n = 21), non-COPD smokers (n = 16), and COPD patients (n = 14). The identification of LILRB4-expressing cells and the level of LILRB4 expression were evaluated by flow cytometry. We analyzed the relationships between the LILRB4 expression and clinical characteristics including respiratory function. In the experiments using an elastase-induced mouse model of emphysema, we also analyzed the LILRB4 expression on lung macrophages. We compared inflammatory cell accumulation and emphysematous changes induced by elastase instillation between wild-type and LILRB4-deficient mice. RESULTS: The levels of surface expression of LILRB4 are relatively high on monocyte linage cells including macrophages in the human lungs. The percentage of LILRB4+ cells in lung interstitial macrophages was increased in COPD patients compared to non-COPD smokers (p = 0.018) and correlated with the severity of emphysematous lesions detected by CT scan (rs = 0.559, p < 0.001), whereas the amount of smoking showed no correlation with LILRB4 expression. Increased LILRB4 on interstitial macrophages was also observed in elastase-treated mice (p = 0.008). LILRB4-deficient mice showed severer emphysematous lesions with increased MMP-12 expression in the model. CONCLUSIONS: LILRB4 on interstitial macrophages was upregulated both in human COPD lungs and in a mouse model of emphysema. This upregulated LILRB4 may have a protective effect against emphysema formation, possibly through decreasing MMP-12 expression in the lungs.

ACO (Asthma-COPD Overlap) Is Independent from COPD, a Case in Favor: A Systematic Review.

Asthma and chronic obstructive pulmonary disease (COPD) are now recognized to be able to co-exist as asthma-COPD overlap (ACO). It is clinically relevant to evaluate whether patients with COPD concurrently have components of asthma in primary care. This is because: (i) ACO is a relatively common condition among asthma (over 40 years of age) or COPD irrespective of its diagnosis criteria; (ii) patients with ACO can have higher frequency of exacerbation and more rapid decline in lung function than those with asthma or COPD; and (iii) asthmatic features such as eosinophilic airway inflammation are promising indicators for prediction of inhaled corticosteroid-responsiveness in COPD. The aim of this review to evaluate diagnostic markers for ACO. We searched PubMed for articles related to ACO published until 2020. Articles associated with diagnostic biomarkers were included. We identified a total of 25 studies, some of which have revealed that a combination of biomarkers such as fractional exhaled nitric oxide and serum immunoglobulin E is useful to discern type 2 inflammation in the airways of COPD. Here, we review the current understanding of the clinical characteristics, biomarkers and molecular pathophysiology of ACO in the context of how ACO can be differentiated from COPD.

PGC-1α regulates airway epithelial barrier dysfunction induced by house dust mite.

BACKGROUND: The airway epithelial barrier function is disrupted in the airways of asthmatic patients. Abnormal mitochondrial biogenesis is reportedly involved in the pathogenesis of asthma. However, the role of mitochondrial biogenesis in the airway barrier dysfunction has not been elucidated yet. This study aimed to clarify whether the peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), a central regulator of mitochondrial biogenesis, is involved in the disruption of the airway barrier function induced by aeroallergens. METHODS: BEAS-2B cells were exposed to house dust mite (HDM) and the expressions of PGC-1α and E-cadherin, a junctional protein, were examined by immunoblotting. The effect of SRT1720, a PGC-1α activator, was investigated by immunoblotting, immunocytochemistry, and measuring the transepithelial electrical resistance (TEER) on the HDM-induced reduction in mitochondrial biogenesis markers and junctional proteins in airway bronchial epithelial cells. Furthermore,the effects of protease activated receptor 2 (PAR2) inhibitor, GB83, Toll-like receptor 4 (TLR4) inhibitor, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), protease inhibitors including E64 and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) on the HDM-induced barrier dysfunction were investigated. RESULTS: The amounts of PGC-1α and E-cadherin in the HDM-treated cells were significantly decreased compared to the vehicle-treated cells. SRT1720 restored the expressions of PGC-1α and E-cadherin reduced by HDM in BEAS-2B cells. Treatment with SRT1720 also significantly ameliorated the HDM-induced reduction in TEER. In addition, GB83, LPS-RS, E64 and AEBSF prevented the HDM-induced reduction in the expression of PGC1α and E-cadherin. CONCLUSIONS: The current study demonstrated that HDM disrupted the airway barrier function through the PAR2/TLR4/PGC-1α-dependent pathway. The modulation of this pathway could be a new approach for the treatment of asthma.

Improving the viability of tissue-resident stem cells using an organ-preservation solution.

Human clinical specimens are a valuable source of tissue-resident stem cells, but such cells need to be collected immediately after tissue collection. To extend the timescale for collection from fresh human samples, we developed a new extracellular fluid (ECF)-type preservation solution based on a high-sodium and low-potassium solution containing low-molecular-weight dextran and glucose, which is used for preservation of organs for transplantation. In this study, we compared the preservation of tissue-resident stem cells using our ECF solution with that using three other solutions: PBS, Dulbecco's modified Eagle's medium and Euro-Collins solution. These solutions represent a common buffer, a common culture medium and a benchmark organ-preservation solution, respectively. Lung tissues were removed from mice and preserved for 72 h under low-temperature conditions. Of the solutions tested, only preservation in the ECF-type solution could maintain the proliferation and differentiation capacity of mouse lung tissue-resident stem cells. In addition, the ECF solution could preserve the viability and proliferation of human alveolar epithelial progenitor cells when stored for more than 7 days at 4 °C. The mean viability of human alveolar type II cells at 2, 5, 8 and 14 days of low-temperature preservation was 90.9%, 84.8%, 85.7% and 66.3%, respectively, with no significant differences up to 8 days. Overall, our findings show that use of our ECF-type preservation solution may maintain the viability and function of tissue-resident stem cells. Use of this preservation solution may facilitate the investigation of currently unobtainable human tissue specimens for human stem cell biology.

Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases.

Epithelial cell proliferation, division, and differentiation are critical for barrier repair following inflammation, but the initial trigger for this process is unknown. Here we define that sensing of apoptotic cells by the TAM receptor tyrosine kinase Axl is a critical indicator for tracheal basal cell expansion, cell cycle reentry, and symmetrical cell division. Furthermore, once the pool of tracheal basal cells has expanded, silencing of Axl is required for their differentiation. Genetic depletion of Axl triggers asymmetrical cell division, leading to epithelial differentiation and ciliated cell regeneration. This discovery has implications for conditions associated with epithelial barrier dysfunction, basal cell hyperplasia, and continued turnover of dying cells in patients with chronic inflammatory pulmonary diseases.

Nitrosative stress in patients with asthma-chronic obstructive pulmonary disease overlap.

BACKGROUND: Asthma-chronic obstructive pulmonary disease overlap (ACO) has frequent exacerbations and a poor quality of life and prognosis compared with those of chronic obstructive pulmonary disease alone. However, the pathogenesis of ACO has not been fully elucidated yet. OBJECTIVES: The aim of this study was to investigate nitrosative stress, which causes a redox imbalance and tissue inflammation in the airways of patients with ACO, and to evaluate the relationship between nitrosative stress and the clinical course in study subjects. METHODS: Thirty healthy subjects and 56 asthmatic patients participated in this study. The asthmatic patients were divided into 33 asthmatic patients and 23 patients with ACO. The study subjects had been followed prospectively for 2 years to evaluate the clinical course. Nitrosative stress was evaluated based on the production of 3-nitrotyrosine (3-NT) in sputum cells. RESULTS: Production of 3-NT was significantly enhanced in patients with ACO compared with that in asthmatic patients. Amounts of reactive persulfides and polysulfides, newly identified powerful antioxidants, were significantly decreased in the ACO group. Baseline levels of 3-NT were significantly correlated with the frequency of exacerbations and decrease in FEV1 adjusted by age, smoking history, and blood eosinophil count. The 3-NT-positive cells were also significantly correlated with amounts of proinflammatory chemokines and cytokines. CONCLUSIONS: These findings suggested that greater nitrosative stress occurred in the airways of patients with ACO, and the degree of nitrosative stress was correlated with an impairment in the clinical course. Nitrosative stress might be related to the pathogenesis of ACO.

Axl kinase drives immune checkpoint and chemokine signalling pathways in lung adenocarcinomas.

Axl receptor tyrosine kinase is involved in the growth and metastasis and is an indicator of poor prognosis in several cancers including lung cancers. Although a mitogen-activated protein kinase (MAPK) pathway and an epithelial-to-mesenchymal transition (EMT) program are critical, molecular mechanisms underlying the Axl-driven cancer progression have not been fully elucidated. We aimed to identify molecules up-regulated by Axl kinase in lung adenocarcinomas. Through the global gene expression analysis and the functional annotation clustering, we found that AXL expression positively correlated with mRNA expressions of immune checkpoint molecules and chemokine receptors in non-small-cell lung cancers. Validation cohorts including our biobank confirmed that the AXL expression significantly correlated with expression of genes encoding programmed death-ligand1 (PD-L1) and CXC chemokine receptor 6 (CXCR6) in lung adenocarcinoma, especially in epidermal growth factor receptor (EGFR) mutation-positive adenocarcinoma. Pharmacological inhibition of Axl kinase activity decreased mRNA expressions of PD-L1 and CXCR6 in EGFR mutation-positive cell lines. Our data indicates the novel role of Axl kinase as a driver of immune checkpoint molecules and chemokine signalling pathways in the progression of lung adenocarcinomas. This study also highlights the necessity of clinical trials in order to test the efficacy of Axl kinase inhibition in the Axl-highly expressing subset of lung adenocarcinomas. .

Expression of cytochrome P450 mRNAs in Type II alveolar cells from subjects with chronic obstructive pulmonary disease.

Inhaled drugs are critical for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). To develop better therapeutics for pulmonary disease it is of potential importance to understand molecular mechanisms of local biotransformation in the lung. Alveolar epithelial type II (ATII) cells have a key role in homeostasis in the lung, but little is known about expression patterns of genes encoding cytochrome P450 (CYP) enzymes in ATII cells. In addition, alteration of CYP gene expression has not been fully defined in COPD. We previously established a method to purify ATII cells from the adult human lung using fluorescence-activated cell sorting. By employing this technique we determined gene expression patterns of 14 CYP enzymes in ATII cells from nonsmokers (n = 4) and smokers (n = 4), both having normal pulmonary function. Although most CYP genes are highly expressed in primary hepatocytes, we found that CYP1B1 mRNA expression was 7.2-fold higher in ATII compared to hepatocytes (P = .0275). Additionally we noted a 3.0-fold upregulation of CYP2C19 and 50% reduction in CYP2J2 mRNA expressions in ATII cells isolated from patients with COPD (n = 3) compared to smokers without COPD (n = 4). These data, for the first time, detail a comprehensive set of genes encoding CYP enzymes in human ATII cells and highlights differentially expressed CYP mRNAs of patients with COPD. Such understanding may have important implications for the development of novel inhaled drugs.

Oxidative stress enhances the expression of IL-33 in human airway epithelial cells.

BACKGROUND: Interleukin-33 (IL-33) is a cytokine belonging to the IL-1 family, and its possible involvement in the pathophysiology of COPD and viral-induced exacerbations has been demonstrated. IL-33 has been shown to be increased in the airway epithelial cells from COPD patients, but the regulating mechanism of IL-33 expression in airway epithelial cells remains largely unknown. In the current study, we examined whether oxidative stress, which participates in the pathogenesis of COPD, affects the expression of IL-33 in airway epithelial cells and also evaluated the effect during viral infection. METHODS: The involvement of oxidative stress in the expression of IL-33, and its signal pathway was examined after stimulation with hydrogen peroxide (H2O2), with or without stimulation by polyinosinic-polycytidylic acid [poly (I:C)], a synthetic analogue of dsRNA that mimics viral infection, or rhinovirus infection in NCI-H292 cells and primary human bronchial epithelial cells (HBECs). In addition, the effect of antioxidant, N-acetylcysteine (NAC) in the expression of IL-33 was compared between HBECs from healthy subjects and those from COPD patients. RESULTS: Treatment with H2O2 significantly potentiated IL-33 expression in NCI-H292 cells, and the potentiation was reversed by NAC treatment. Mitogen-activated protein kinase (MAPK) inhibitors, but not nuclear factor-kappa B inhibitors, also significantly decreased the H2O2-potentiated IL-33 expression. In addition, H2O2 significantly potentiated the poly (I:C)- or rhinovirus-stimulated IL-33 expression. In HBECs from healthy subjects, H2O2-potentiated IL-33 expression and its reversal by NAC was also confirmed. Under the condition without H2O2-stimulation, treatment with NAC significantly decreased the expression of IL-33 in HBECs from COPD patients, but not in those from healthy subjects. CONCLUSIONS: These results demonstrate that oxidative stress involves in the expression of IL-33 in airway epithelial cells via MAPK signal pathway and it augments IL-33 expression during viral infection. This mechanism may participate in the regulation of IL-33 expression in airway epithelial cells in COPD and the viral-induced exacerbations. Modulation of this pathway could become a therapeutic target for viral-induced exacerbations of COPD.

Production of reactive persulfide species in chronic obstructive pulmonary disease.

BACKGROUND: Oxidative stress is a major aetiological factor driving chronic obstructive pulmonary disease (COPD). Recently recognised as potent antioxidants, reactive persulfide and polysulfide species are biosynthesised by cystathionine ß-synthase and cystathionine γ-lyase. The production of reactive persulfide and polysulfide species in the lungs of patients with COPD remain unknown. OBJECTIVES: The aim of this study was to examine the production of reactive persulfides and polysulfides, such as glutathione persulfide (GSSH), cysteine persulfide (CysSSH) and glutathione trisulfide (GSSSH), in lung-resident cells and epithelial lining fluid (ELF) obtained from patients with mild to moderate COPD. METHODS: Lung tissues, primary lung cells, ELF and sputum were obtained. The amounts of reactive persulfides and polysulfides in the cells and ELF were measured by liquid chromatography-tandem mass spectrometry with ß-(4-hydroxyphenyl) ethyl iodoacetamide as a trapping agent for hydroper/polysulfides. The amounts of synthases in the lung tissues, sputum and primary cells were quantified. RESULTS: The amounts of GSSH, CysSSH and GSSSH were decreased in the lung cells and ELF from patients with COPD. The amounts of reactive persulfides and polysulfides in the lung cells had a positive correlation with the degree of airflow limitation. By contrast, the amounts of the synthases were increased in the lung tissues and sputum cells of patients with COPD. CONCLUSIONS: We have identified a decrease in reactive persulfide and polysulfide species in the lungs of patients with COPD. These data suggest that the newly detected antioxidants reactive persulfides and polysulfides could be associated with the redox balance in the lungs of patients with COPD.

Phenotypic screening identifies Axl kinase as a negative regulator of an alveolar epithelial cell phenotype.

Loss of epithelial barrier integrity is implicated in a number of human lung diseases. However, the molecular pathways underlying this process are poorly understood. In a phenotypic screen, we identified Axl kinase as a negative regulator of epithelial phenotype and function. Furthermore, suppression of Axl activity by a small molecule kinase inhibitor or downregulation of Axl expression by small interfering RNA led to: (1) the increase in epithelial surfactant protein expression; (2) a cell morphology transition from front-rear polarity to cuboidal shape; (3) the cytoskeletal re-organization resulting in decreased cell mobility; and (4) the acquisition of epithelial junctions. Loss of Axl activity reduced activation of the Axl canonical pathway members, Akt and extracellular signal-regulated kinase-1/2 and resulted in the loss of gene expression of a unique profile of epithelial-to-mesenchymal transition transcription factors including SNAI2, HOXA5, TBX2 or TBX3. Finally, we observed that Axl was activated in hyperplasia of epithelial cells in idiopathic pulmonary fibrosis where epithelial barrier integrity was lost. These results suggest that the Axl kinase signaling pathway is associated with the loss integrity of alveolar epithelium in pathological remodeling of human lung diseases.

Decrease in an anti-ageing factor, growth differentiation factor 11, in chronic obstructive pulmonary disease.

RATIONALE: Cellular senescence is observed in the lungs of patients with COPD and may contribute to the disease pathogenesis. Growth differentiation factor 11 (GDF11) belongs to the transforming growth factor ß superfamily and was recently reported to be a circulating protein that may have rejuvenating effects in mice. We aimed to investigate the amounts of GDF11 in the plasma and the lungs of patients with COPD and elucidate the possible roles of GDF11 in cellular senescence. METHODS: The plasma levels of GDF11 were investigated in two separate cohorts by western blotting. The localisation and expression of GDF11 in the lungs were investigated by immunohistochemistry and quantitative reverse transcription PCR, respectively. The effects of GDF11 on both cigarette smoke extract (CSE)-induced cellular senescence in vitro and on elastase-induced cellular senescence in vivo were investigated. RESULTS: The levels of plasma GDF11 in the COPD group were decreased compared with the control groups in the two independent cohorts. The levels of plasma GDF11 were significantly positively correlated with pulmonary function data. The mRNA expression of GDF11 in mesenchymal cells from the COPD group was decreased. Chronic exposure to CSE decreased the production of GDF11. Treatment with GDF11 significantly inhibited CSE-induced cellular senescence and upregulation of inflammatory mediators, partly through Smad2/3 signalling in vitro. Daily GDF11 treatment attenuated cellular senescence and airspace enlargement in an elastase-induced mouse model of emphysema. CONCLUSIONS: The decrease in GDF11 may be involved in the cellular senescence observed in COPD.

Serum extracellular vesicular miR-21-5p is a predictor of the prognosis in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis. Although the median survival is 3 years, the clinical course varies to a large extent among IPF patients. To date, there has been no definitive prognostic marker. Extracellular vesicles (EVs) are known to hold nucleic acid, including microRNAs, and to regulate gene expression in the recipient cells. Moreover, EVs have been shown to express distinct surface proteins or enveloped microRNAs depending on the parent cell or pathological condition. We aimed to identify serum EV microRNAs that would be prognostic for IPF. METHODS: To determine target microRNAs in IPF, we measured serum EV microRNA expression profiles using microRNA PCR arrays in a bleomycin mouse model and validated the microRNAs in additional mice using RT-PCR. Secondly, we enrolled 41 IPF patients and conducted a 30-month prospective cohort study. Expression of serum EV miR-21-5p was normalized by dividing by the EV amount. The relative amount of EVs was measured using the ExoScreen method. We calculated the correlations between baseline serum EV miR-21-5p expression and other clinical variables. Furthermore, we determined if serum EV miR-21-5p can predict mortality during 30 months using the Cox hazard model. According to the median level, we divided the IPF patients into two groups. Then we compared the survival rate during 30 months between the two groups using the Kaplan-Meier method. RESULTS: Serum EV miR-21-5p was elevated in both the acute inflammatory phase (day 7) and the chronic fibrotic phase (day 28) in the mouse model. In the clinical setting, serum EV miR-21-5p was significantly higher in IPF patients than in healthy control subjects. The baseline serum EV miR-21-5p was correlated with the rate of decline in vital capacity over 6 months. Furthermore, serum EV miR-21-5p was independently associated with mortality during the following 30 months, even after adjustment for other variables. In the survival analysis, IPF patients whose baseline serum EV miR-21-5p was high had a significantly poorer prognosis over 30 months. CONCLUSIONS: Our results suggest that serum EV miR-21-5p has potential as a prognostic biomarker for IPF.