Animal model

Six-week-old male ApoE−/− mice and wild-type (WT) mice with a C57BL/6J background (identification number: SCXK2016-0006) were procured from Vital River Laboratory Animal Technology (Beijing, China). These mice were housed under specific pathogen-free conditions at the Guangxi Medical University Laboratory Animal Centre. The experiments were conducted in strict accordance with the Experimental Animal Ethics guidelines of Guangxi Medical University. The experimental protocols strictly adhered to the ARRIVE guidelines for transparent and comprehensive reporting of animal research. ApoE−/− mice were divided into CAVS groups and fed a high-fat diet (HFD) for 12, 24 and 36 weeks, respectively; the HFD was obtained from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd., Nanjing, China, as described previously. The WT mice in the control group were fed a standard normal diet.

Adeno-associated virus 9 (AAV9) for in vivo administration and neutralizing antibodies against IFN-γ

To overexpress IFN-γ, recombinant AAV9 expressing IFN-γ and AAV9-GFP were purchased from Shanghai Jikai Co. (Shanghai, China). The titers of the AAV vectors (viral genomes/ml) were determined by quantitative real-time PCR. AAV9-NC, which was packed with empty AAV9 virus, was used as a negative control. Mice were injected with 2 × 1013 VG of AAV9- IFN-γ or AAV9-NC twice: at weeks 0 and 12 of the HFD(Knezevic et al. 2016). The hearts were harvested from all injected mice 3 weeks after the second injection to examine IFN-γ expression by western blotting or immunofluorescence staining. For neutralizing antibody intervention, mice were injected with received the 100 µg of the anti-IFN-γ antibody (Cat. BE0055; BioXcell, USA) intraperitoneally (i.p.) every 3 days from week 0 to week 24 of the HFD. Vehicle mice were injected with InVivoPure pH 7.0 Dilution Buffer (Cat. IP0070; BioXcell, USA) which was used to dilute the solvent. All mice treated with AAV9 or neutralizing IFN-γ antibodies were killed at week 24, and the hearts, spleens and peripheral blood were collected for further experiments.

Echocardiography

Aortic valve function was assessed with a MyLab™ Sat ultrasonic imaging system equipped with a 22-MHz probe (Esaote, Genova, Italy). Cardiac echocardiography was performed at 12, 24, and 36 weeks. During cardiac echocardiography, the mice underwent mild anesthesia through the i.p. injection of 10 µl of 1.25% avertin solution per gram of body weight. The heart rate was maintained at 490 ± 30 beats/minute. Aortic valve flow velocity was assessed by continuous-wave Doppler recorded from a five-chamber view via the apical approach and averaged over 5 beats. M-mode echocardiograms were employed to capture aortic valve systolic dimensions and left ventricular function. The aortic valve shape was observed on transverse sections of the heart, and the mean aortic valve area was recorded and calculated. The data were collected and analyzed by a skilled sonographer following the double-blind principle. To determine the left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV), the Teichholz formula was used.

Histology

To evaluate calcium deposits and collagen deposition in mouse aortic valve tissue, paraffin-embedded hearts were sectioned into 5-µm-thick slices comprising three valve leaflets. These sections were stained with hematoxylin and eosin (HE), von Kossa (Solarbio, Beijing, China), and Masson (Solarbio, Beijing, China) following the manufacturer’s guidelines. Quantitative analysis of the staining was performed using Image-Pro Plus software (Media Cybernetics, Bethesda, USA) to determine the percentage of von Kossa-positive and Masson-positive staining areas.

Immunohistochemistry (IHC)

Tissue sections of the aortic valve were subjected to heat-induced epitope retrieval and then incubated overnight with rabbit anti-NLRP3 (1:250; Abcam, Cambridge, UK) and rabbit anti-STAT1 (1:500; Abcam, Cambridge, UK) antibodies following routine deparaffinization and hydration. The sections were then incubated with a secondary antibody (goat anti-rabbit IgG-HRP) and stained with hematoxylin. Image-Pro Plus software (Media Cybernetics) was used to analyze the percentage area of immunopositive staining and the average optical density in both valvular leaflets and interleaflet triangles. The average optical density values were computed using the following formula: AOD (average optical density) = IOD (integrated optical density)/area.

Immunofluorescence

The paraffin sections were dewaxed and subjected to Tris-EDTA antigen retrieval (pH 8.0). Valve interstitial cells (VICs) cultured in vitro were fixed in 4% paraformaldehyde. The cells were then treated with 0.1% Triton X-100 for 15 min, followed by epitope blocking for one hour in 5% BSA in PBS. Primary antibody staining was conducted overnight at 4 °C in a blocking solution containing mouse anti-CD4 (1:200; Abcam, Cambridge, UK), rabbit anti-IFN-γ (1:100; Abcam, Cambridge, UK), rabbit anti-STAT1 (1:200; Abcam, Cambridge, UK), rabbit anti-NLRP3 (1:200; Abcam, Cambridge, UK), mouse anti-caspase-1 (1:200; Abcam, Cambridge, UK), rabbit anti-RUNX2 (1:200; Abcam, Cambridge, UK), rabbit anti-BMP2 (1:200; Abcam, Cambridge, UK), or rabbit anti-α-SMA (1:200; Abcam, Cambridge, UK). The sections were then stained with goat anti-rabbit IgG-AF488, donkey anti-mouse IgG-AF555 or goat anti-rabbit IgG-AF750 (1:1000; Abcam, Cambridge, UK) secondary antibodies for 1 h at room temperature, followed by DAPI staining (Solaibao, Beijing, China). The sections were imaged at magnifications ranging from 4× to 60×. CD4+ and IFN-γ+ cells were counted manually and normalized to the area of the DAPI mask corresponding to the leaflet. The immunofluorescence intensities of NLRP3, Caspase-1, RUNX2, BMP2 and α-SMA were calculated by ImageJ.

Flow cytometry

Splenic single-cell suspensions were extracted following established protocols. Macrophages cultured in vitro were digested into single-cell suspensions by Accutase (Invitrogen, Carlsbad, USA). T cells were stimulated with PMA (50 ng/mL; Cat. HY-18,739; MCE, USA) and ionomycin (1 µg/mL; Cat. HY-13,434; MCE, USA) for 6 h. BFA (10 µg/mL; Cat. HY-16,592; MCE, USA) was introduced during the last 4 h to induce cytokine secretion. To prevent nonspecific staining, single-cell suspensions were preincubated with anti-CD16/CD32 antibodies (clone 2.4G2; Fc block; BD Biosciences) for 15 min. Then, the cells were stained with anti-CD4 (eBioscience, Carlsbad, USA), anti-CD45 (eBioscience, Carlsbad, USA), anti-F4/80 (eBioscience, Carlsbad, USA), anti-CD11b (eBioscience, Carlsbad, USA), anti-Ly6C (eBioscience, Carlsbad, USA), or anti-Ly6G (eBioscience, Carlsbad, USA) antibodies. For intracellular staining, cells were treated with anti-IFN-γ, anti-NLRP3 (R&D Systems, Minnesota, USA), or anti-caspase-1 (Genetex, San Antonio, USA) antibodies, followed by fixation and permeabilization using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, USA). Macrophages were identified as CD45+CD11b+F4/80+, and monocytes were identified as CD45+CD11b+Ly6G-. The spleen and heart samples were analyzed using a FACSVerse Flow Cytometer and a FACSCanto Flow Cytometer (BD Biosciences). FlowJo V10 software (BD Biosciences) was used for data analysis.

Th1 induction

Spleens from WT C57BL/6J mice were gently crushed, and red blood cells were lysed in erythrocyte lysis buffer (Solarbio, China) and washed thoroughly with PBS. The resulting splenocyte suspensions were filtered through a 70-µm sieve (BD Biosciences, USA). Naive CD4+ T cells were isolated using a mouse naive CD4+ T-cell isolation kit (Cat. 130-104-453; Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The isolated naive T cells were induced to differentiate into Th1 cells by adding 20 ng/ml IL-2 (Proteintech, Chicago, USA) and 10 µg/ml anti-IL-4 (BioXcell, NH, USA).

Coculture of BMDMs and Th1 cells

BMDMs were extracted from WT C57BL/6J mice as previously described and induced with 20 ng/ml M-CSF for 7 days (Dorighello et al. 2022). Subsequently, the BMDMs were stimulated with ox-LDL for 48 h, and the ATP intervention group was pretreated with 3 mM ATP for 30 min. For the CY-09 intervention group, BMDMs were treated with 5 µM CY-09 for 48 h. In vitro coculture assays were conducted using cell culture inserts featuring porous polycarbonate filters with a pore size of 0.4 μm placed within 24-well plates. Th1 cells (5 × 106) were added to the upper chamber of the Transwell chamber, and BMDMs were treated with ox-LDL and ATP in the lower chamber for 2.5 days. Then, BMDMs were collected for testing or cultured with fresh medium for 2 days to obtain conditioned medium for subsequent experiments with VICs.

VICs isolation and culture

Murine VICs were isolated from noncalcified aortic valves of WT C57BL/6J mice via collagenase digestion as previously reported (Zeng et al. 2017). These interstitial cells were cultured in M199 complete medium at 37 °C under 5% CO2. Cells derived from passages 3 to 5 were chosen for experiments. To induce calcification, VICs at a concentration of 5 × 105 cells/mL were cultured in M199 medium supplemented with 5% fetal bovine serum, 50 µg/mL ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphoric acid as previously described(Li et al. 2017). To examine the impact of macrophages on the osteogenic differentiation of VICs, 1 mL of nonconditioned (fresh) medium or conditioned medium obtained from macrophages after intervention as previously described was added to the VICs procalcifying medium. After a 7-day incubation period, the harvested cells were prepared for experiments. To characterize osteoblast-like phenotypes, ALP and alizarin red staining were performed using an ALP detection kit and alizarin red S staining solution (Biyuntian, Shanghai, China), and the OD at 405 nm was used to determine ALP activity according to the manufacturer’s instructions.

ELISA

The cell culture supernatant was centrifuged at 300 × g for 5 min, aliquoted and stored at − 80 °C. The murine cytokines IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Wuhan Huamei Bioengineering Co., Ltd., China).