Normal lung specimens were collected from patients who underwent surgical resection for pulmonary lump in Tongji Hospital, Wuhan, China, between 2017 and 2021. BALF samples were intentionally obtained from patients who underwent bronchoscopy in Tongji Hospital between 2015 and 2017. COPD was diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (Vestbo et al., 2013). Patients with a post-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity ratio of less than 70% were enrolled. Age- and gender-matched non-smokers and smokers without COPD were also recruited as control subjects. Participants were excluded if they suffered from asthma, severe lung infections, or other obstructive lung diseases. This study was approved by the Ethics Committees of the Tongji Hospital (TJ-IRB 20140415 and TJ-IRB20210346) and written informed consent was obtained from all subjects.

Alveolar macrophages in BALF were obtained by performing bronchoscopy following the international guidelines (Meyer et al., 2012). A total of 120 ml sterile isotonic saline solution at 37°C were separated into four aliquots and flushed into the right middle lobe. After each flush, the fluid was aspirated immediately and gently, collected in sterile centrifuge tubes, and kept on ice. Approximately 50% BALF was recovered. Samples were then filtered through a 40-μm cell strainer and centrifuged at 1,000 × g for 10 min at 4°C. Cell pellets were resuspended in RPMI-1640 medium containing 20% fetal bovine serum, 200 U/ml penicillin, and 200 ug/ml streptomycin. Slides were prepared by cytocentrifugation at 1,200 rpm for 5 min and then fixed in 4% formaldehyde for immunofluorescence staining. Cells were cultured in 12-well culture plates in a 5% CO₂ humidified incubator at 37°C for 2 h. Then, non-adherent cells were removed by washing the plates with RPMI-1640 medium, yielding monolayers that contained at least 95% macrophages. Cell morphology was analyzed and proteins were extracted.

Cigarette smoke extract was prepared by bubbling the smoke from two burning cigarettes (3R4F, University of Kentucky) at a rate of 1 cigarette/5 min to a 50 ml centrifuge tube containing 20 ml of RPMI-1640 medium. The pH was adjusted to 7.4. The solution was then filtered through a 0.22-μm filter to eliminate bacteria.

Human-derived THP-1 cells (ATCC^® TIB-202) were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37°C. Cells were treated with phorbol myristate acetate (PMA, 100 nM) for 48 h to induce macrophage differentiation. In transfection experiment, cells were transfected with 50 nM small interfering RNA (siRNA) targeting STIM1 (5′-GTGGTACAGTGGCTGATCA-3′) or negative control sequence (RiboBio, Guangzhou, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. In pharmacological experiment, cells were pretreated with 3 mM NAC (Sigma-Aldrich, St. Louis, MO, United States) for 1 h, with 10 μM SKF-96365 (MedChemExpress, United States) for 2 h, or with 10 μM 2-APB (MedChemExpress, United States) for 2 h before CSE stimulation.

Formalin (10%)-fixed, paraffin-embedded lung tissue sections of healthy non-smokers (HN), smokers without COPD, and smokers with COPD (SC) were deparaffinized using xylene and rehydrated in a graded ethanol series. Heat-induced antigen retrieval was performed using a microwave. After cooling with running tap water, sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity, followed by 1-h incubation in 5% BSA-phosphate-buffered saline (PBS) solution at room temperature to avoid non-specific background. Then, slides were incubated with polyclonal rabbit anti-STIM1 (1:500; Proteintech, United States) antibody at 4°C overnight in a humidified chamber. After washing, sections were incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature. The reactions were developed using a DAB substrate kit with hematoxylin as a counterstain. A Nikon Spot image acquisition and processing system (United States) was used for image assessment.

Cells were fixed with 4% paraformaldehyde for 15 min and stored at −80°C. After washing, slides were incubated in 3% hydrogen peroxide solution in the dark for 10 min. After three washes with PBS for 5 min, slides were blocked in 5% BSA for 20 min. Then, slides were incubated with polyclonal rabbit anti-STIM1 antibody (1:50, Proteintech, United States), monoclonal mouse anti-CD68 antibody (Abcam, United Kingdom), or isotype controls (Becton Dickinson, Franklin Lakes, United States) overnight at 4°C. After washing, slides were incubated with goat anti-rabbit and goat anti-mouse secondary antibodies (Aspen, Wuhan, China) for 50 min at 37°C. DAPI was used to counterstain the nuclei.

Lung tissues (1 g) were obtained from subjects who underwent surgical resection as aforementioned and flash frozen with liquid nitrogen. Samples were thawed, maintained at 2–8°C, and homogenized with 9 mL PBS by a grinder. Then, tissue homogenates were centrifuged at 3,000 rpm for 10 min and the supernatant was collected and examined for concentration. The supernatants were incubated with 1 mmol/L DCFH-DA solution (Elabscience, China) for 30 min at 37°C. The ROS levels were detected by a microplate reader (Molecular Devices, China) at 525 nm.

The levels of ROS in THP-1 cells were detected by a Fluorometric Intracellular Ros Kit (Sigma-Aldrich, Darmstadt, Germany). After pretreated with NAC for 1 h, PMA-differentiated THP-1 cells were treated with CSE for 3 h. Then, cells were washed and incubated with RPMI-1640 containing 1 μM dichlorofluorescein diacetate (H2DCF-DA) for 30 min. After three washes with RPMI-1640 medium for 5 min, cells were digested by trypsin, washed again, and resuspended in 200 μl PBS. Finally, flow cytometry (BD Biosciences, SanJose, CA, United States) was performed to detect the fluorescent signal intensity at 525 nm to indicate the intracellular levels of ROS.

Intracellular Ca²⁺ levels were measured using the Fluo-3, AM Kit (Solarbio, Beijing, China) in accordance with the manufacturer’s protocol. After treated with CSE or other indicated agents, cells were incubated with 5 μM molecular probe Fluo-3, AM for 20 min at 37°C. Next, cells were cultured in HBSS containing 1% fetal bovine serum for 40 min and then resuspended in HBSS buffer saline. The Ca²⁺ levels were determined by flow cytometry (BD Biosciences, SanJose, CA, United States) at 525 nm.

Approximately 5,000 cells were seeded in each well of a 96-well plate (Corning, MA, United States). After 48-h incubation with different concentrations of CSE, Cell Counting Kit-8 (CCK-8; Promoter Biotechnology, Wuhan, China) was used to detect cell viability according to the manufacturer’s instructions. Optical density (OD) values were obtained using a microplate reader (Molecular Devices, China).

The levels of interleukin (IL)-8 and IL-1β in PMA-differentiated THP-1 macrophages were quantified using DuoSet ELISA kit (R&D Systems, Minneapolis, MN, United States) and RayBio ELISA kit (RayBiotech, United States) according to the manufacturers’ instructions. The minimum detectable dose was 31.3 pg/mL and 0.3 pg/ml for IL-8 and IL-1β, respectively.

Total RNA was extracted using Trizol (Takara, Japan) and reverse transcribed to cDNA using PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara, Japan). The mRNA expression was assessed by real-time quantitative polymerase chain reaction (RT-qPCR) using TB Green^® Premix Ex Taq^TM II (Takara, Japan) on BioRad CFX384 (Bio-Rad, CA, United States). The parameters were as follows: 40 cycles at 95°C for 10 s, 59°C for 20 s, and 72°C for 30 s. The relative mRNA expression was determined using the 2^–ΔΔCt method with β-actin as the internal control. The following primers were used in this experiment: β-actin (F: 5′-GCGCGGCTACAGCTTCA-3′; R: 5′-CTTAATGTCACGCACGATTTCC-3′), STIM1 (F: 5′-TTG TCCATGCAGTCCCCTAG-3′; R: 5′-GGTAGTGGTGATGGTG GTGA-3′).

Total protein was extracted using RIPA buffer supplemented with protease inhibitor cocktail (Servicebio, Wuhan, China). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with Tris-buffered saline and tween-20 (TBST) containing 5% non-fat milk for 1 h, the membranes were incubated with rabbit polyclonal anti-STIM1 antibody (1:1,000, Proteintech, Chicago, IL, United States) or rabbit polyclonal anti-β-tubulin antibody (1:4,000, Sungene biotech, Shanghai, China) overnight at 4°C. The membranes were washed with TBST buffer, followed by 1-h incubation with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:4,000; Sigma-Aldrich, Darmstadt, Germany) at room temperature. After washed with TBST, the protein expression was detected using an ECL chemiluminescence detection kit (Advansta, California, United States) and quantified by ImageJ.

All data were normally distributed and expressed as the mean ± SEM. Statistical significance was determined by Student’s t-test or one-way ANOVA with Newman–Keuls multiple comparison test as appropriate. The correlations were analyzed by Pearson correlation. The GraphPad Prism 8 Software (GraphPad Software, San Diego, CA, United States) was used for all statistical analysis and graphic generation. The FlowJo 10 software was used for flow cytometry analysis. P < 0.05 was considered statistically significant.

The clinical characteristics of all subjects are shown in Tables 1, 2. Lung tissues were collected from 12 HN, 12 smokers without COPD (healthy smokers, HS), and 16 SC. BALF samples were obtained from 13 HN, 14 HS, and 12 SC. There was no significant difference in age and gender among these groups. Compare with the HN group, the smoking index of HS and SC was markedly increased. The FEV1% predicted and FEV1/FVC of SC were significantly lower than those of the HN and HS groups.

Immunohistochemistry was performed to detect the expression of STIM1 in the lung tissue of HN, HS, and SC (Figures 1A–C). STIM1 was mainly expressed in alveolar macrophages. Compared with the HN and HS groups, the expression of STIM1 in SC was notably increased. RT-qPCR and Western blot were performed to validate the expression of STIM1. Consistent with the results of immunohistochemistry, intrapulmonary expression of STIM1 in SC was prominently increased compared with the HN and HS group at both mRNA (Figure 1D) and protein (Figures 1E,F) levels. To explore the association between STIM1 expression and pulmonary function, the correlation between STIM1 protein expression and FEV1% predicted in the SC group was analyzed. We found that intrapulmonary STIM1 expression was negatively correlated with the pulmonary function of COPD patients (Figure 1G), indicating the potential role of STIM1 in the development of COPD.

The levels of ROS in the lung tissue supernatants of SC and HS were significantly increased compared with the HN group (Figure 1H). HS showed higher ROS levels than HN, indicating that CS may directly lead to the elevation of ROS levels in the lungs. Moreover, significantly higher levels of ROS were observed in the lung tissues of SC compared with HS, suggesting enhanced oxidative stress in the lungs of SC. To determine the association between STIM1 expression and ROS production, we performed correlation analysis in all subjects. The results showed that pulmonary ROS levels were positively associated with STIM1 expression at both mRNA (Supplementary Figure 1A) and protein (Supplementary Figure 1B) levels.

Previous immunohistochemistry analysis showed that STIM1 was mainly expressed in alveolar macrophages. To further define the location and expression level of STIM1, immunofluorescence staining and Western blot were performed using alveolar macrophages isolated from the BALF samples. Immunofluorescence staining against CD68 (a marker of macrophages), STIM1, and DAPI revealed that STIM1 was predominantly located in the cytoplasm rather than the nucleus of macrophages (Figure 2A). Moreover, alveolar macrophages from different individuals were cultured for 24 h and the protein expression of STIM1 was detected by Western blot. The level of STIM1 in SC was significantly higher than that of the HS and HN groups, and there was no significant difference between the HN and HS groups (Figures 2B,C). These results indicated that STIM1 may play a key role in the development of COPD. We therewith treated human macrophages with 5% CSE to investigate the effect of CSE on STIM1 expression. We found that CSE significantly upregulated the expression of STIM1 in alveolar macrophages in vitro (Supplementary Figures 2A,B).

Cell viability was not affected by CSE at a concentration lower than or equal to 10% (Supplementary Figure 3). To assess the potential effects of CSE and NAC on intracellular ROS production, PMA-differentiated THP-1 cells were incubated with or without CSE at different concentrations for 3 h. Cells were also pretreated with 3 mM NAC or control medium for 1 h before CSE treatment. Flow cytometry analysis showed that 5 and 10% CSE significantly induced ROS production in cells (Figures 3A,B). CSE at a concentration of 5% was used to investigate the effect of NAC on ROS production. Compared with the control groups, cells treated with 5% CSE showed increased intracellular ROS production, while pretreatment with 3 mM NAC markedly reduced CSE-induced ROS production (Figures 3C,D).

To further examine whether the expression of STIM1 was regulated by CSE-induced ROS, we measured the expression of STIM1 in PMA-differentiated THP-1 cells treated with different concentrations of CSE. We found that 5% CSE significantly increased the expression of STIM1 (Figures 3E,F). However, 1-h pretreatment with 3 mM NAC markedly suppressed CSE-induced upregulation of STIM1 in cells (Figures 3G,H). These results support the hypothesis that CSE-induced ROS may be involved in the regulation of STIM1 expression in macrophages.

The above data showed that intracellular ROS levels were upregulated by CSE treatment and NAC alleviated CSE-induced ROS production. CSE-induced upregulation of STIM1 in PMA-differentiated THP-1 cells was also inhibited by NAC. Thus, we speculated that CSE might increase the expression of STIM1 by promoting the production of ROS. To further investigate the effects of CSE-induced ROS on inflammatory cytokines and to determine whether it was subjected to the regulation of STIM1, we examined the effects of STIM1 knockdown and NAC pretreatment on IL-8 and IL-1β, two important cytokines in the pathogenesis of COPD, in PMA-differentiated THP-1 cells (Pauwels et al., 2011). Western blot was used to confirm STIM1 knockdown in cells (Figures 4A,B). NAC pretreatment suppressed CSE-induced IL-8 release in cells transfected with si-NC or si-STIM1. Knockdown of STIM1 further decreased the level of IL-8 in CSE-stimulated cells (Figure 4C). These results indicated a potential regulation of IL-8 production by ROS and STIM1 in CSE-challenged cells. Combined with previous findings that ROS regulated the expression of STIM1, we hypothesized that the regulation of IL-8 by ROS and STIM1 was initiated by CSE. Neither STIM1 knockdown nor NAC pretreatment significantly altered the production of IL-1β (Figure 4D).

To study whether intracellular Ca²⁺ is involved in the regulation of IL-8 by ROS/STIM1 in CSE-stimulated cells, we performed flow cytometry to detect intracellular Ca²⁺ levels. The results showed that pretreatment with NAC suppressed CSE-induced increase in intracellular Ca²⁺ levels, and STIM1 knockdown further decreased intracellular Ca²⁺ levels in CSE-stimulated cells (Figures 5A,B). Moreover, treatment with SKF-96365 and 2-APB, two pharmacological inhibitors targeting Ca²⁺ channels, significantly inhibited CSE-induced increase in IL-8 levels (Figure 5C). Taken together, it could be concluded that CSE promoted the release of IL-8 in PMA-differentiated THP-1 cells possibly through the ROS/STIM1/Ca²⁺ axis.