Interleukin-2/anti-interleukin-2 complex attenuates inflammation in a mouse COPD model by expanding CD4+ CD25+ Foxp3+ regulatory T cells.

BACKGROUND AND PURPOSE: Chronic, nonspecific inflammation of the alveoli and airways is an important pathological feature of chronic obstructive pulmonary disease (COPD), while sustained inflammatory reactions can cause alveolar damage. Regulatory T cells (Tregs) inhibit inflammation, whereas the interleukin-2/anti-interleukin-2 complex (IL-2C) increases the number of Tregs; however, whether the IL-2C has a therapeutic role in COPD remains unknown. Therefore, this study investigated whether IL-2C alleviates lung inflammation in COPD by increasing the number of Tregs. EXPERIMENTAL APPROACH: A mouse COPD model was created by exposing mice to lipopolysaccharides (LPS) and cigarette smoke (CS), and the effects of IL-2C treatment on COPD were evaluated. The number of Tregs in the spleen and lung, pulmonary pathological changes, and inflammatory damage were examined through flow cytometry, histopathology, and immunofluorescence, respectively. KEY RESULTS: IL-2C increased the number of Treg cells in the spleen and lungs after exposure to CS and LPS, reduced the number of T helper 17 (Th17) cells in lung tissue, and improved the Th17/Treg balance. IL-2C decreased the number of inflammatory cells and reduced the levels of pro-inflammatory cytokines IL-6, TNF-α, IL-1ß, CCL5, KC, and MCP-1 in bronchoalveolar lavage fluid and serum. IL-2C significantly reduced the pathological scores for lung inflammation, as well as decreased airway mucus secretion and infiltration of neutrophils and macrophages in the lungs. The depletion of Tregs using anti-CD25 antibodies eliminated the beneficial effects of IL-2C. CONCLUSIONS AND IMPLICATIONS: IL-2C is a potential therapeutic agent for alleviating excessive inflammation in the lungs of patients with COPD.

MiR-23a-5p alleviates chronic obstructive pulmonary disease through targeted regulation of RAGE-ROS pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease and represents the third leading cause of death worldwide. This study aimed to investigate miRNA regulation of Receptor for Advanced Glycation End-products (RAGE), a causal receptor in the pathogenesis of cigarette smoke (CS)-related COPD, to guide development of therapeutic strategies. METHODS: RAGE expression was quantified in lung tissue of COPD patients and healthy controls, and in mice with CS-induced COPD. RNA-sequencing of peripheral blood from COPD patients with binding site prediction was used to screen differentially expressed miRNAs that may interact with RAGE. Investigation of miR-23a-5p as a potential regulator of COPD progression was conducted with miR-23a-5p agomir in COPD mice in vivo using histology and SCIREQ functional assays, while miR-23a-5p mimics or RAGE inhibitor were applied in 16-HBE human bronchial epithelial cells in vitro. RNA-sequencing, ELISA, and standard molecular techniques were used to characterize downstream signaling pathways in COPD mice and 16-HBE cells treated with cigarette smoke extract (CSE). RESULTS: RAGE expression is significantly increased in lung tissue of COPD patients, COPD model mice, and CSE-treated 16-HBE cells, while inhibiting RAGE expression significantly reduces COPD severity in mice. RNA-seq analysis of peripheral blood from COPD patients identified miR-23a-5p as the most significant candidate miRNA interaction partner of RAGE, and miR-23a-5p is significantly downregulated in mice and cells treated with CS or CSE, respectively. Injection of miR-23a-5p agomir leads to significantly reduced airway inflammation and alleviation of symptoms in COPD mice, while overexpressing miR-23a-5p leads to improved lung function. RNA-seq with validation confirmed that reactive oxygen species (ROS) signaling is increased under CSE-induced aberrant upregulation of RAGE, and suppressed in CSE-stimulated cells treated with miR-23a-5p mimics or overexpression. ERK phosphorylation and subsequent cytokine production was also increased under RAGE activation, but inhibited by increasing miR-23a-5p levels, implying that the miR-23a-5p/RAGE/ROS axis mediates COPD pathogenesis via ERK activation. CONCLUSIONS: This study identifies a miR-23a-5p/RAGE/ROS signaling axis required for pathogenesis of COPD. MiR-23a-5p functions as a negative regulator of RAGE and downstream activation of ROS signaling, and can inhibit COPD progression in vitro and in vivo, suggesting therapeutic targets to improve COPD treatment.

Serum Proteomic Profiling in Patients with Chronic Obstructive Pulmonary Disease.

Purpose: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with high morbidity and mortality rates. This study used proteomic profiling of serum to identify the differentially expressed proteins in COPD patients compared with healthy controls, to expand the knowledge of COPD pathogenesis and to ascertain potential new targets for diagnosis and treatment of COPD. Methods: Serum samples were collected from 56 participants (COPD group n = 28; Healthy Control group n = 28). A data-independent acquisition quantitative proteomics approach was used to identify differentially expressed proteins (DEPs) between the two groups. Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment, and protein-protein interaction analyses of DEPs were conducted to identify their relevant biological processes, cellular components, and related pathways. We used a parallel reaction monitoring (PRM)-based targeted quantitative proteomics approach to validate those findings. Results: Of 8484 peptides identified by searching the UniProtKB/Swiss-Prot knowledgebase, 867 proteins were quantifiable, of which 20 were upregulated and 35 were downregulated in the COPD group. GO functional annotation indicated that the subcellular localization of most DEPs was extracellular. The top three molecular functions of the DEPs were signaling receptor binding, antigen binding, and immunoglobulin receptor binding. The most relevant biological process was immune response. The transforming growth factor-ß signaling pathway, Staphylococcus aureus infection, and hematopoietic cell lineage were the top three pathways identified in the KEGG pathway functional enrichment. Our PRM analyses confirmed the identification of 11 DEPs identified in our data-independent acquisition analyses, 8 DEPs were upregulated and 3 DEPs were downregulated. Conclusion: This study using data-independent acquisition analyses with PRM confirmation of findings identified 11 DEPs in the serum of patients with COPD. These DEPs are potential diagnostic or prognostic biomarkers or may be future targets for the treatment of COPD.

Inhibition of Nur77 expression and translocation by compound B6 reduces ER stress and alleviates cigarette smoke-induced inflammation and injury in bronchial epithelial cells.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide with inflammation and injury in airway epithelial cells. However, few treatment options effectively reduce severity. We previously found that Nur77 is involved in lipopolysaccharide-induced inflammation and injury of lung tissue. Here, we established an in vitro model of COPD-related inflammation and injury in 16-HBE cells induced by cigarette smoke extract (CSE). In these cells, Nur77 expression and localization to the endoplasmic reticulum (ER) increased following CSE treatment, as did ER stress marker (BIP, ATF4, CHOP) expression, inflammatory cytokine expression, and apoptosis. The flavonoid derivative, named B6, which was shown to be a modulator of Nur77 in previous screen, molecular dynamics simulation revealed that B6 binds strongly to Nur77 through hydrogen bonding and hydrophobic interactions. Treating CSE-stimulated 16-HBE cells with B6 resulted in a reduction of both inflammatory cytokine expression and secretion, as well as attenuated apoptosis. Furthermore, B6 treatment resulted in a decrease in Nur77 expression and translocation to the ER, which was accompanied by a concentration-dependent reduction in the expression of ER stress markers. Meanwhile, B6 played a similar role in CSE-treated BEAS-2B cells. These combined effects suggest that B6 could inhibit inflammation and apoptosis in airway epithelial cells after cigarette smoke stimulation, and support its further development as a candidate intervention for treating COPD-related airway inflammation.

Influenza and pneumococcal vaccination coverage and associated factors in patients hospitalized with acute exacerbations of COPD in China: Findings from the real-world data.

BACKGROUND: Influenza and pneumococcal vaccination are a priority in patients with chronic obstructive pulmonary disease (COPD). However, limited information is available on vaccination coverage among patients with acute exacerbations of COPD (AECOPD) in China. This study aimed to determine the rates and associated factors of influenza and pneumococcal vaccination in patients hospitalized with AECOPD. METHODS: Baseline data from a national, multicenter, hospital-based study that included adult inpatients with AECOPD between 2017 and 2021 were analyzed. The outcomes of interest were the influenza vaccination in the past year and the pneumococcal vaccination in the past 5 years. To ensure national representativeness, rates were weighted according to the distribution of hospital levels and types enrolled in this study. Multivariable Poisson regression based on mixed-effects models were used to determine the associated factors. The independent variables included the region and hospital features where the participants were located, sociodemographic characteristics (age, sex, rural/urban residence, education, etc.), and clinical indicators (COPD disease history, lung function parameters, comorbidities, etc.). The treatment profiles of the vaccinated and unvaccinated participants were compared. RESULTS: Of 6949 eligible participants, the weighted rates of influenza/pneumococcal, influenza, and pneumococcal vaccination were 2.72% (95% confidence interval [CI]: 2.34-3.10%), 2.09% (95% CI: 1.76-2.43%), and 1.25% (95% CI: 0.99-1.51%), respectively. In multivariable models, age ≥60 years (60-69 years, odds ratio [OR]: 1.90, 95% CI: 1.11-3.25; ≥80 years, OR: 2.00, 95% CI: 1.06-3.78), geographical regions (Northern China relative to Eastern China, OR: 5.09, 95% CI: 1.96-13.21), urban residence (OR: 1.69, 95% CI: 1.07-2.66), a higher education level (junior high school, OR: 1.77, 95% CI: 1.21-2.58; senior high school or above, OR: 2.61, 95% CI: 1.69-4.03), former smoking (OR: 1.79, 95% CI: 1.15-2.79), and regular inhaled medication treatment (OR: 3.28, 95% CI: 2.29-4.70) were positively associated with vaccination. Patients who had experienced severe exacerbations in the past year were less likely to be vaccinated (OR: 0.65, 95% CI: 0.45-0.96). Compared with unvaccinated participants, vaccinated participants adhered better to pharmacological and non-pharmacological treatment. CONCLUSIONS: Influenza and pneumococcal vaccination coverage are extremely low. Urgent measures are necessary to increase vaccination coverage among inpatients with AECOPD in China.

Design, synthesis, and evaluation of novel benzoylhydrazone derivatives as Nur77 modulators with potent antitumor activity against hepatocellular carcinoma.

Nur77 modulators have emerged as a promising therapeutic approach for hepatocellular carcinoma. In this study, a structure-based rational drug design approach was used to design and synthesise a series of 4-((8-hydroxy-2-methylquinolin-4-yl)amino)benzoylhydrazone derivatives based on the binding characteristics of our previously reported 10g and the native ligand 3NB at the binding Site C of Nur77. Cell-based cytotoxicity assays revealed that compound TMHA37 demonstrated the highest cytotoxicity against all tested cancer cells. The induced fit docking and binding pose metadynamics simulation suggested that TMHA37 was the most promising Nur77 binder at Site C. Molecular dynamics simulation validated the stable binding of TMHA37 to Nur77's Site C but not to Sites A or B. Specifically, TMHA37 bound strongly to Nur77-LBD (KD = 445.3 nM) and could activate Nur77's transcriptional activity. Furthermore, TMHA37 exhibited antitumor effects by blocking the cell cycle at G2/M phase and inducing cell apoptosis in a Nur77-dependent manner.

The national COPD screening programme in China: rationale and design.

Background: COPD is the most prevalent chronic respiratory disease in China. It is estimated that there is a large, as-yet undetected, high-risk population who will develop in COPD in future. Methods and design: In this context, a nationwide COPD screening programme was launched on 9 October 2021. This multistage sequential screening programme incorporates a previously validated questionnaire (i.e. COPD Screening Questionnaire) and pre- and post-bronchodilator spirometry to target the COPD high-risk population. The programme plans to recruit 800 000 participants (eligible age 35-75 years) from 160 districts or counties of 31 provinces, autonomous regions or municipalities across China. The filtered COPD high-risk population and early-detected COPD patients will receive integrated management and be followed-up for ≥1 year. Discussion: This is the first large-scale prospective study to determine the net benefit of mass screening for COPD in China. Whether the smoking cessation rate, morbidity, mortality and health status of individuals at high risk of COPD could be improved along with this systematic screening programme will be observed and validated. Moreover, the diagnostic accuracy, cost-effectiveness and superiority of the screening programme will also be assessed and discussed. The programme marks a remarkable achievement in the management of chronic respiratory disease in China.

Dynamic analysis of gene signatures in the progression of COPD.

Aims: Oxidative stress is an important amplifying mechanism in COPD; however, it is unclear how oxidative stress changes and what its exact amplification mechanism is in the pathological process. We aimed to dynamically analyse the progression of COPD and further elucidate the characteristics of each developmental stage and unveil the underlying mechanisms. Methods: We performed a holistic analysis by integrating Gene Expression Omnibus microarray datasets related to smoking, emphysema and Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification based on the concept of gene, environment and time (GET). Gene ontology (GO), protein-protein interaction (PPI) networks and gene set enrichment analysis (GSEA) were used to explore the changing characteristics and potential mechanisms. Lentivirus was used to promote HIF3A overexpression. Results: In smokers versus nonsmokers, the GO term mainly enriched in "negative regulation of apoptotic process". In later transitions between stages, the main enriched terms were continuous progression of "oxidation-reduction process" and "cellular response to hydrogen peroxide". Logistic regression showed that these core differentially expressed genes (DEGs) had diagnostic accuracy in test (area under the curve (AUC)=0.828) and validation (AUC=0.750) sets. GSEA and PPI networks showed that one of the core DEGs, HIF3A, strongly interacted with the ubiquitin-mediated proteolysis pathway. Overexpression of HIF3A restored superoxide dismutase levels and alleviated the reactive oxygen species accumulation caused by cigarette smoke extract treatment. Conclusion: Oxidative stress was continuously intensified from mild emphysema to GOLD 4; thus, special attention should be paid to the identification of emphysema. Furthermore, the downregulated HIF3A may play an important role in the intensified oxidative stress in COPD.

Identification of potential ATP-competitive cyclin-dependent kinase 1 inhibitors: De novo drug generation, molecular docking, and molecular dynamics simulation.

Cyclin-dependent kinases 1 (CDK1) has been identified as a potential target for the search for new antitumor drugs. However, no clinically effective CDK1 inhibitors are now available for cancer treatment. Therefore, this study aimed to offer potential CDK1 inhibitors using de novo drug generation, molecular docking, and molecular dynamics (MD) simulation studies. We first utilized the BREED algorithm (a de novo drug generation approach) to produce a novel library of small molecules targeting CDK1. To initially obtain novel potential CDK1 inhibitors with favorable physicochemical properties and excellent druggability, we performed a virtual rule-based rational drug screening on our generated library and found ten initial hits. Then, the molecular interactions and dynamic stability of these ten initial hits and CDK1 complexes during their all-atom MD simulations (total 18 µs) and binding pose metadynamics simulations were investigated, resulting in five final hits. Furthermore, another MD simulation (total 2.1 µs) with different force fields demonstrated the binding ability of the five hits to CDK1. It was found that these five hits, CBMA001 (ΔG = -29.88 kcal/mol), CBMA002 (ΔG = -34.89 kcal/mol), CBMA004 (ΔG = -32.47 kcal/mol), CBMA007 (ΔG = -31.16 kcal/mol), and CBMA008 (ΔG = -34.78 kcal/mol) possessed much greater binding affinity to CDK1 than positive compound Flavopiridol (FLP, ΔG = -25.38 kcal/mol). Finally, CBMA002 and CBMA004 were identified as excellent selective CDK1 inhibitors in silico. Together, this study provides a workflow for rational drug design and two promising selective CDK1 inhibitors that deserve further investigation.

Adenovirus-mediated Overexpression of FcγRIIB Attenuates Pulmonary Inflammation and Fibrosis.

Progressive fibrosing interstitial lung diseases (PF-ILDs) result in high mortality and lack effective therapies. The pathogenesis of PF-ILDs involves macrophages driving inflammation and irreversible fibrosis. Fc-γ receptors (FcγRs) regulate macrophages and inflammation, but their roles in PF-ILDs remain unclear. We characterized the expression of FcγRs and found upregulated FcγRIIB in human and mouse lungs after exposure to silica. FcγRIIB deficiency aggravated lung dysfunction, inflammation, and fibrosis in silica-exposed mice. Using single-cell transcriptomics and in vitro experiments, FcγRIIB was found in alveolar macrophages, where it regulated the expression of fibrosis-related genes Spp1 and Ctss. In mice with macrophage-specific overexpression of FcγRIIB and in mice treated with adenovirus by intratracheal instillation to upregulate FcγRIIB, silica-induced functional and histological changes were ameliorated. Our data from three genetic models and a therapeutic model suggest that FcγRIIB plays a protective role that can be enhanced by adenoviral overexpression, representing a potential therapeutic strategy for PF-ILDs.

Discovery of a Nur77-mediated cytoplasmic vacuolation and paraptosis inducer (4-PQBH) for the treatment of hepatocellular carcinoma.

Nur77, an orphan nuclear receptor, has antitumor activity in hepatocellular carcinoma (HCC). However, its antitumor mechanisms of action in HCC are complicated and rarely reported. Our recent work demonstrated that certain quinoline-Schiff-base derivatives were good Nur77 mediators that exerted excellent anti-HCC activities in vitro and in vivo. Interestingly, these compounds shared similar chemical structures, but they displayed different Nur77-targeted anticancer mechanisms of action. As a continuous work, we synthesized a series of 4-(quinoline-4-amino) benzoylhydrazide derivatives and evaluated their anti-HCC activity and binding affinity to Nur77 in vitro. Compound 4-PQBH emerged as the best Nur77 binder (KD = 1.17 µM) and has potentially selective cytotoxicity to HCC cells. Mechanistically, 4-PQBH extensively induced caspase-independent cytoplasmic vacuolization and paraptosis through Nur77-mediated ER stress and autophagy. Moreover, 4-PQBH exhibited an effective xenograft tumor inhibition by modulating Nur77-dependent cytoplasmic vacuolation and paraptosis. This paper is the first to disclose that chemotherapeutic agents targeting Nur77-mediated cytoplasmic vacuolization and paraptosis may provide a promising strategy to combat HCC that frequently evade the apoptosis program.

Tetrandrine alleviates silicosis by inhibiting canonical and non-canonical NLRP3 inflammasome activation in lung macrophages.

Silicosis caused by inhalation of silica particles leads to more than ten thousand new occupational exposure-related deaths yearly. Exacerbating this issue, there are currently few drugs reported to effectively treat silicosis. Tetrandrine is the only drug approved for silicosis treatment in China, and despite more than decades of use, its efficacy and mechanisms of action remain largely unknown. Here, in this study, we established silicosis mouse models to investigate the effectiveness of tetrandrine of early and late therapeutic administration. To this end, we used multiple cardiopulmonary function test, as well as markers for inflammation and fibrosis. Moreover, using single cell RNA sequencing and transcriptomics of lung tissue and quantitative microarray analysis of serum from silicosis and control mice, our results provide a novel description of the target pathways for tetrandrine. Specifically, we found that tetrandrine attenuated silicosis by inhibiting both the canonical and non-canonical NLRP3 inflammasome pathways in lung macrophages. Taken together, our work showed that tetrandrine yielded promising results against silicosis-associated inflammation and fibrosis and further lied the groundwork for understanding its molecular targets. Our results also facilitated the wider adoption and development of tetrandirne, potentially accelerating a globally accepted therapeutic strategy for silicosis.

Synthesis, SAR study, and bioactivity evaluation of a series of Quinoline-Indole-Schiff base derivatives: Compound 10E as a new Nur77 exporter and autophagic death inducer.

We previously reported 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole- 2-carbohydrazide derivatives as new Nur77 modulators. In this study, we explored whether the 8-methoxy-2-methylquinoline moiety and bicyclic aromatic rings at the N'-methylene position were critical for their antitumor activity against hepatocellular carcinoma (HCC). For this purpose, a small library of 5-substituted 1H-indole-2-carbohydrazide derivatives was designed and synthesized. We found that the 8-methoxy-2-methylquinoline moiety was a fundamental structure for its biological function, while the introduction of the bicyclic aromatic ring into the N'-methylene greatly improved its anti-tumor effect. We found that the representative compound 10E had a high affinity to Nur77. The KD values were in the low micromolar (2.25-4.10 µM), which were coincident with its IC50 values against the tumor cell lines (IC50 < 3.78 µM). Compound 10E could induce autophagic cell death of liver cancer cells by targeting Nur77 to mitochondria while knocking down Nur77 greatly impaired anti-tumor effect. These findings provide an insight into the structure-activity relation of Quinoline-Indole-Schiff base derivatives and further demonstrate that antitumor agents targeting Nur77 may be considered as a promising strategy for HCC therapy.

Design, synthesis, and biological evaluation of 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide derivatives as novel Nur77 modulators.

Nur77 is a potential target for the treatment of cancer such as HCC. Herein, we detailed the discovery of a novel series of 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide derivatives as potential Nur77 modulators. The studies of antiproliferative activity and Nur77-binding affinity of target compounds resulted in the discovery of a lead candidate (10g), which was a good Nur77 binder (KD = 3.58 ± 0.16 µM) with a broad-spectrum antiproliferative activity against all tested hepatoma cells (IC50 < 2.0 µM) and was low toxic to normal LO2 cells. 10g could up-regulate Nur77 expression and mediate sub-cellular localization of Nur77 to induce apoptosis in hepatocellular carcinoma cell lines, which relied on 10g inducing Nur77-dependent autophagy and endoplasmic reticulum stress as the upstream of apoptosis. Moreover, the in vivo assays verified that 10g significantly inhibited xenograft tumor growth. These results indicate that 10g has the potential to be developed as a novel Nur77-targeting anti-hepatoma drug.

Synthesis and biological evaluations of N'-substituted methylene-4-(quinoline-4-amino) benzoylhydrazides as potential anti-hepatoma agents.

In the effort to develop novel quinoline derivatives for the treatment of liver cancer, we synthesized a series of N'-Substituted methylene-4-(quinoline-4-amino) benzoylhydrazides and evaluated their biological activities as anticancer agents. Compounds 5h and 5j were found to be the potent antiproliferative agents against HepG2 cell line with an IC50 value of 12.6 ± 0.1 µM and 27.3 ± 1.7 µM, respectively. The most effective compound 5h also exhibited potent cytotoxicity against SMMC-7721 and Huh7 cells with IC50 values of 9.6 ± 0.7 µM and 6.3 ± 0.2 µM, respectively. Inspiringly, both 5h and 5j exhibited lower cytotoxic property in normal cells than hepatic carcinoma cells. Compounds 5h and 5j could down-regulate mRNA level of c-Myc and expression level of c-Myc. Meanwhile, they decreased expression level of anti-apoptotic protein Bcl-2 and increased expression levels of pro-apoptotic protein Bax and cleaved PARP with reference to tubulin. So various assays including cell colony formation, cell cycle distribution, as well as cell apoptosis and migration were performed to understand their antitumor role. It was confirmed that 5h and 5j inhibited the growth of HepG2 cells due to their anti-survival effect, induction of cell cycle arrest and cell apoptosis, and inhibition of cell migration. These results demonstrated that 5h might be as potential lead compounds to develop anticancer agents for the treatment of hepatocellular carcinoma.