Bioinformatics analysis

We used the Venn tool (https://bioinfogp.cnb.csic.es/tools/venny/) from the DisGeNET database (http://www.disgenet.org/) to identify the common genes in the two patient groups. Information related to functional enrichment, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, was obtained from David online tool (https://david.ncifcrf.gov/) [17]. We developed a hub-key genes and protein–protein interaction (PPI) network through the software Cytoscape 3.8.0 and STRING (https://string-db.org/).

Participant’s enrollment and human lung tissue samples collection

In the current study, a total of 68 patients aged 50–75 years were screened and eventually included in the analysis from January 1, 2021, to November 31, 2021, at Fudan University, affiliated with Huadong Hospital. COPD was diagnosed according to guidelines from the European Respiratory Society and the American Thoracic Society. The diagnostic criteria of COPD was FEV1 (forced expiratory volume in one second), less than 70% of the predicted value after bronchodilators. The diagnostic criteria of HUA were defined as SUA > 420 µmol/L. Patients with the following conditions should be excluded from the study: (1) different degrees of acute exacerbation of COPD in the past 3 months. (2) suffered from infectious airway disease in the past 3 months. (3) severe underlying disorders of kidney, liver, immune, cerebrovascular or hematopoietic system, cancer, or mental disorders. (4) participated in other clinical trials in the past 3 months. (5) serum creatinine level was > 1.5 mg/dL. (6) ALT level is more than twice the normal upper limit. (7) severe deformity or stiffness because of gouty arthropathy. (8) severe arrhythmia. (9) patients who consumed medications that contained aspirin (> 325 mg/d) or salicylate, azathioprine, 6-mercaptopurine, or hypouricemic medications. The study was approved by the Ethics Committee of Huadong Hospital, Affiliated with Fudan University (No. 20190037). All written informed consents were available, and this trial is registered with the Chinese Clinical Trial Registry (ChiCTR2000038257). The written informed consent was available according to the Declaration of Helsinki. The lung tissue samples were collected from patients undergoing pulmonary benign tumor approval from the Institutional Ethics Committee of Shanghai Huadong Hospital (2018K024).

HBE Cells culture and CSE preparation

We acquired HBE cells from American Type Culture Collection (ATCC) and cultivated them in RPMI1640 medium with fetal bovine serum (all containing 5% FBS, Gibco, Invitrogen) at 37 °C in the presence of 5% CO2. CSE was obtained using the smoke of two cigarettes (Hong Shuang xi, a cigarette brand produced by Shanghai Tobacco Group, China). Tar (12 mg) and nicotine (1.0 mg) were collected in 15 mL of culture media using a 50 mL syringe. The resultant CSE solution was defined as 100% cigarette smoke extraction. It was frozen before preserving at -80 °C and filtered through a 0.22um filter to remove large particles before use. Subsequently, the CSE solution was diluted to the required concentration using culture media.

Western blot

WB was performed on extracellular vesicle (EV) lysates obtained from plasma, BALF, and sputum of patients. Radioimmunoprecipitation assay (RIPA) buffer was used to prepare lysates of EVs. Thereafter, each sample was run in the NOVEX 10%–20% Tris–glycine gel (Invitrogen). In addition, we employed iBlot2 (Invitrogen) in transferring proteins onto polyvinylidene fluoride (PVDF) membranes (Millipore Sigma), followed by blocking with 5% bovine serum albumin (BSA, Sangon Biotech, Shanghai, China) within 0.1% Tween 20 (TBST) as blocking buffer for one hour.Then, we incubated membranes with diluted primary antibodies. The Image Lab software (Bio-Rad V5.2.1) was used for WB quantification, with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being the reference. In the present work, the following primary antibodies (all were diluted at 1:1000) were adopted, including rabbit pAb Antitrypsin, rabbit pAb MUC1, rabbit pAb ELANE, rabbit pAb IL-6, rabbit pAb IL-8, rabbit pAb CD63, rabbit pAb TSG101, rabbit pAb Calnexin, rabbit pAb CDKN1A, rabbit pAb CDKN2A, rabbit pAb IL-1A, and rabbit pAb 53BP1. All antibodies were obtained from the brand ABclonal (Wuhan, China) (https://abclonal.com.cn/).

The isolation of peripheral blood mononuclear cells (PBMCs)

PBMCs were obtained from the patients and separated with Ficoll density-gradient centrifugation. Later, we cultivated cells in RPMI-1640 medium supplemented with 10% FBS (Gibco).

Reverse transcription-polymerase chain reaction (RT-PCR)

We isolated total cellular RNA by using a guanidinium thiocyanate reagent. Subsequently, cDNA was prepared from the isolated total RNA following specific instructions. We conducted RT-PCR with the green Polymerase Chain Reaction kit (std. Synergy Brands, Inc.) and amplified PCR products using ABI 7300. Then 2-ΔΔCt approach was utilized to determine gene level, with GAPDH being the reference. Primers used for RT-PCRs were: IL6, Fwd:5'-CCTTCTCCACAATACCCCCAGG-3'; Rev:5'-TGTGCCCAGTGGACAGGTTT-3'; IL8, Fwd: 5'-GTGCTGTGTTGAATTACGGA; Rev:5'-TTGACTGTGGAGTTTTGGC-3'; Antitrypsin, Fwd: 5'-ATGATGAAGCGTTTAGGCA-3'; Rev:5'-CAGGCAGGAAGAAGATGG-3'; ELANE, Fwd: 5'-CGACCCCGTAAACTTGCT-3'; Rev:5'-ACGTTGGCGTTGATGGT-3'; GAPDH, Fwd:5'-TGGGGTGATGCAGGTGCTAC-3'; Rev:5'-GGACACGGAAGGCCATACCA-3'.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was used to determine interleukin-6 (IL-6), IL-8, Antitrypsin and Elane levels in the plasma (X–Y Biotechnology, Shanghai, China). Plasma collected from patients was subjected to 3-min centrifugation at 500xg to remove debris. The kit contents were brought to the ambient temperature before starting the test. Sample diluent and standard were added to each blank well, followed by the addition of standards or samples of diverse doses (100 µL/well) in the rest wells. We used sealing tape to seal each reaction well, followed by a 90-min incubation at 36 °C in an incubator. We made the biotinylated antibody working solution 20 min before adding, which was diluted and added into each blank well, while the non-diluted solution was added into all rest wells (100 µL/well). The reaction well was sealed, followed by a 60-min incubation at 36 °C. Enzyme conjugate working solution was prepared 20 min before use at 22–25 °C in the dark, which was diluted and added into each blank well, whereas the non-diluted solution was added to all rest wells (100 µL/well) and incubated for 30-min at 36 °C in the dark. After heating, we used the microplate reader to measure absorbance. Then we added chromogenic substrate (TMB, 100 µL/well) into each well and incubated it for 15-min at 36 °C in the dark, followed by a stop solution (100 µL/well) into each well. After mixing, the OD450 value was recorded immediately (in 3 min), and target substance concentrations were measured in the samples.

Cell viability assay on cells treated with different doses of CSE and EVs

After the isolation of CSE, the quality control of 100% CSE was performed by measuring the optical density at 320λ wavelength and a value of 1.158, and the intervention time and concentration was determined according to the previous research [18]. CBA assay was performed to evaluate the level of EVs from different subjects' plasma, and PBS was used to dilute. A Cell Counting Kit-8 (CCK-8, Beijing Solarbio Science and Technology Co., Ltd.) assay was conducted to assess cell viability after CES and EVs treatments at diverse doses. Cells (1 × 104 cells/ well) were cultivated within the 96-well plate for 24–96 h (Supplement 1). The microplate reader was used to measure the absorbance (OD) at 450 nm. All experiments were conducted three times. Therefore, we treated the HBE cells with CSE for 24 h and then EVs from subjects' plasma for another 24 h. The results of CCK8 have been show in the supplement.

Immunohistochemistry (IHC), Masson staining, and Multiplex IF of lung tissues

We analyzed eight samples of lung tissue from COPD patients, including four samples of patients with normal SUA and four samples of patients with HUA. Lung tissue samples were collected from patients with benign lung tumors approved by the institutional ethics committee of Shanghai Huadong Hospital and voluntarily signed the informed consent. The specific inclusion criteria were consistent with those of the clinical cohort patients in this study. To analyze histology, we embedded lung tissues fixed with 4% paraformaldehyde (PFA) in paraffin and prepared the samples into 5-µm sections for HE and Masson staining. After thoroughly rinsing with normal saline, we fixed left lung tissue using 10% formalin solution neutral buffered. After xylene deparaffinization thrice (for 15, 5, and 10 min separately), we removed benzene with the 100%, 90%, 80%, and 70% ethanol gradient (10 min each). Each section was then rinsed for 5 min twice with distilled water, followed by 15-min hematoxylin (Recordbio, Shanghai, China) staining and washing with distilled water. Later, decolorization was done with 0.5% hydrochloric acid alcohol, rinsed for a 15-min period by distilled water, immersed in 70% and 80% ethanol in succession for a 10-min period, followed by 1-min eosin re-staining and 10-s 90% ethanol differentiation. Thereafter, we dehydrated sections for 10-min twice with 95% ethanol and then for 15-min twice with 100% ethanol. After clearing with xylene thrice (10, 15, and 15 min), we observed each section after neutral resin mounting. Before staining, we soaked each section for 30–60-s in distilled water, followed by the addition of hematoxylin nuclear staining solution to stain for 60-s, solution discarding and rinsing for 30-s. Sections were stained for a 30–60-s period by fuchsin acid staining solution, followed by solution discarding and rinsing for 30-s by the cleaning solution. Sections were then treated with the phosphomolybdic acid solution, followed by solution discarding following separation for 6–8 m. Sections were re-stained for a 5-min period with aniline blue re-staining solution; after that, the solution was discarded, and the sample was rinsed with anhydrous ethanol. We then sealed the dried sections for microscopic observation. The fibrosis area (%) in every group was measured through ImageJ. We took images under the inverted microscope (LSM 780) (Carl Zeiss, Jena, Germany) to identify the degree of chronic pulmonary inflammation, fibrosis, and emphysema. The three-color Fluorescence kit (Shanghai Recordbio Biological Technology, Shanghai, China) was utilized to co-stain elastase, antitrypsin, and 4’,6-diamidino-2-phenylindole (DAPI) through tyramide signal amplification (TSA) in line with the manufacturer’s instruction.