Animals

Eight-week-old wild-type male C57BL/6 and ApoE−/− mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All the mice were maintained in a controlled environment with a temperature of 22 ± 2 ℃, 55 ± 5% relative humidity, and a 12-h light/dark cycle. Wild-type C57BL/6 J mice were used for the preparation of bone marrow-derived macrophages (BMDMs). For establishment of a mouse model of AS, ApoE−/− mice were fed a high-fat diet (HD) for three months, and those fed a normal diet (ND) were used as controls. For ISO treatment, after one month of HD feeding, the mice were given daily intraperitoneal injections of 40 mg/kg ISO for two months [30]. Macrophage-specific overexpression of lysine demethylase 4A (KDM4A) and S-phase kinase associated protein 1 (SKP1) in mice was achieved via the use of recombinant adeno-associated virus 9 (AAV9) vectors. AAV9-oe-NC (overexpression-negative control) (AAV-F4/80-empty vector) or the gene overexpression vectors (AAV-F4/80-oe-KDM4A/AAV-F4/80-oe-SKP1) were administered to the mice through the tail vein [31]. These procedures resulted in the following seven groups: ND, HD, HD + ISO, AAV-oe-NC, AAV-oe-KDM4A, AAV-oe-KDM4A + AAV-oe-NC, and AAV-oe-KDM4A + AAV-oe-SKP1 (n = 6 in each group). At the end points, the mice were sacrificed via the intraperitoneal administration of an overdose (150 mg/kg) of sodium pentobarbital. Blood samples were immediately collected, and the hearts and aortas were perfused with saline containing heparin (50 U/mL) and 4% paraformaldehyde. All animal experiments were approved by the Animal Care and Use Committee of Xiangya School of Pharmaceutical Sciences, Central South University (202,410,113), and the procedures were conducted following the Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA).

Analysis of Atherosclerotic Lesions

The whole aorta was isolated and fixed overnight in 4% paraformaldehyde after the surrounding fat was removed. The following day, the aorta was stained with Oil Red O, rinsed with 60% isopropanol, and imaged with the surface facing upward. The lesion areas were assessed. The aortic root was embedded in optimal cutting temperature compound, sectioned, and subjected to Oil Red O and hematoxylin and eosin (HE) staining.

Blood Lipid Measurement

High-density lipoprotein (HDL; F003-1–1), LDL (A113-1–1), triglyceride (TG; A110-1–1), and cholesterol (TC; A111-1–1) assay kits (NanJing JianCheng Bioengineering Institute, Nanjing, China) were used. Mouse blood was centrifuged to obtain mouse serum. HDL, LDL, TG, and TC levels in mouse serum were subsequently determined per the kit instructions.

Isolation and Treatment of BMDMs

C57BL/6 mice were also euthanized by an overdose of sodium pentobarbital, and the tibias and femurs were isolated, from which the bone marrow cells were carefully flushed out and harvested. Following centrifugation, red blood cells were removed utilizing red blood cell lysis buffer (R7757, Sigma‒Aldrich, Merck KGaA, Darmstadt, Germany) [32]. The cells were subsequently induced to differentiate into BMDMs by stimulation with macrophage colony-stimulating factor (M-CSF, 20 ng/mL; Thermo Fisher Scientific, Inc., Waltham, MA, USA) [33, 34].

The lentiviral vectors encapsulating oe-KDM4A, oe-SKP1, or oe-NC used for gene intervention were provided by Hanbio Technologies (Shanghai, China). BMDMs were infected with lentiviral vectors for 2 d to obtain stable overexpression cell lines. For induction of macrophage pyroptosis, BMDMs were challenged with oxidized low-density lipoprotein (ox-LDL, 50 μg/mL) for 24 h. Subsequently, ISO was administered at a dose of 40 μM for 24 h.

Immunofluorescence Staining and Terminal Deoxynucleotidyl Transferase (TdT)-mediated DUTP Nick End Labeling (TUNEL)

Frozen sections of mouse aortic root tissues and cell slides were fixed, permeabilized with proteinase K, and then blocked with bovine serum albumin (BSA). The sections were incubated with antibodies against F4/80 (1:50, ab6640, Abcam, Inc., Cambridge, MA, USA), N-GSDMD (1:100, #DF13758, Affinity Biosciences, OH, USA), cleaved caspase 1 (1:50, HY-P80622, MedChemExpress, Monmouth Junction, NJ, USA), KDM4A (1:100, ab191433, Abcam), SKP1 (1:500, 67,745–1-Ig, Proteintech Group, Inc., Wuhan, Hubei, China), NLRP3 (1:100, 30,109–1-AP, Proteintech), Cullin1 (1:200, 66,978–1-Ig, Proteintech), and ASC (1:100, sc-514414, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Next, the sections were incubated with secondary antibodies (Alexa Fluor 647 goat anti-rat, 1:500, A-21247, Thermo Fisher; Alexa Fluor 488 goat anti-rabbit SFX Kit, 1:500, A31627, Thermo Fisher; and Alexa Fluor 647 goat anti-mouse, 1:500, A-21235, Thermo Fisher) at room temperature for 1 h in the dark. After staining with DAPI, images were captured via a fluorescence microscope.

The degree of cell death in mouse aortic roots was determined via a TUNEL kit (C10617, Thermo Fisher). After being fixed and permeabilized, the prepared sections were incubated for 1 h in TdT reaction solution, followed by the addition of Click-iT Plus TUNEL reaction buffer and incubation at 37 ℃ in the dark for 30 min. After being washed with BSA, the sections were stained with F4/80 antibody as described above.

Enzyme-linked Immunosorbent Assay (ELISA)

Serum samples from each group of mice as well as the culture supernatants of BMDMs were collected and added to 96-well plates. Reagents were added following the protocols of the IL-18 (E-EL-M0730, Elabscience Biotechnology Co., Ltd., Wuhai, Hubei, China) and IL-1β (MLB00C, R&D Systems, Minneapolis, MN, USA) kits. The absorbance at 450 nm was evaluated using a microplate reader to construct a standard curve, and the IL-18 and IL-1β levels for each group were subsequently evaluated.

Immunohistochemistry (IHC)

Mouse aortic root tissue sections were permeabilized and blocked with BSA for 30 min. The sections were subsequently incubated overnight at 4 ℃ with antibodies against KDM4A (1:200, ab191433, Abcam) and SKP1 (1:100, 10,990–2-AP, Proteintech), followed by incubation with goat anti-rabbit IgG H&L (HRP) (1:1000, ab6721, Abcam) for 1 h. Visualization was performed utilizing a 3,3’-diaminobenzidine kit (G1212-200 T, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China). After counterstaining with hematoxylin, the sections were observed and photographed under a microscope.

Hoechst/propidium Iodide (PI) Staining

A Hoechst/PI assay kit (40744ES60, Yeasen Biotechnology Co., Ltd., Shanghai, China) was used to analyze BMDM pyroptosis. The differentially treated BMDMs were incubated in the dark with 5 μL of Hoechst 33,342 and PI at 4 ℃ for 20 min to assess cell membrane integrity.

Cell Counting Kit-8 (CCK-8) Assay

A CCK-8 assay kit (HY-K0301, MCE) was used to analyze the viability of the BMDMs. In brief, the differentially treated BMDMs were seeded in six-well plates. Each well was then loaded with 10 μL of CCK-8 solution for 2 h of incubation. The absorbance at 450 nm was read using a microplate reader.

Lactate Dehydrogenase (LDH) Release Detection

The cell culture supernatants of the differentially expressed BMDMs were collected. LDH release in the supernatants was evaluated following the instruction manual of the LDH detection reagent (C0016, Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). The absorbance was measured at 490 nm after a 15-min incubation in the cell incubator.

Western Blotting (WB) Analysis

RIPA lysis buffer (C500007, Shanghai Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) was used to extract total protein from BMDMs. After determination of the concentration via a bicinchoninic acid kit (PC0020, Solarbio Science & Technology Co., Ltd. (Beijing, China)), equivalent amounts of protein samples were loaded onto polyvinylidene fluoride membranes following SDS‒PAGE separation. After blocking with 3% nonfat milk for 1 h, the membranes were probed with antibodies against NLRP3 (1:1000, 30,109–1-AP, Proteintech), N-GSDMD (1:1000, 10,137, Cell Signaling Technology, Beverly, MA, US), caspase 1 (1:1000, ZRB1233, Sigma‒Aldrich), KDM4A (1:1000, ab191433, Abcam), H3K36me3 (1:2000, ab9050, Abcam), H3K9me3 (1:2000, ab8898, Abcam), SKP1 (1:1000, 10,990–2-AP, Proteintech), and GAPDH (1:5000, ab8245, Abcam) overnight at 4 ℃. The following day, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h. Signal development was achieved with enhanced chemiluminescence (32,209, Thermo Fisher), and ImageJ software was used to evaluate protein levels.

Chromatin Immunoprecipitation (ChIP)-quantitative Polymerase Chain Reaction (qPCR)

A SimpleChIP Enzymatic Chromatin IP Kit (9003, Cell Signaling Technology) was used for the ChIP experiments. Formaldehyde was added to crosslink the cellular protein and DNA in the plates, which was quenched by the addition of glycine. The BMDM lysates were then subjected to enzyme digestion and ultrasonication for chromatin truncation, yielding 100–500 bp genomic DNA fragments. Immunoprecipitation was performed with KDM4A (1:50, ab191433, Abcam), H3K36me3 (1:50, ab9050, Abcam), H3K9me3 (1:50, ab8898, Abcam), and rabbit IgG. The protein‒DNA complexes were harvested with magnetic beads. After decrosslinking with NaCl and proteinase K, the DNA was eluted and purified. qPCR analysis was subsequently performed to detect the enrichment of the SKP1 promoter fragments.

Coimmunoprecipitation (co-IP) Assays

The BMDM lysates were centrifuged to collect supernatants, which were incubated with antibodies against SKP1 (1:50, 10,990–2-AP, Proteintech), NLRP3 (1:50, 30,109–1-AP, Proteintech), and IgG (1:50, ab172730, Abcam) overnight at 4 ℃ to form antibody‒protein complexes. These complexes were then mixed and incubated for 4 h with Protein A/G agarose beads (20,422, Thermo Fisher). For HA-tagged proteins, direct mixing with HA agarose (26,181, Thermo Fisher) was performed for supernatant incubation at 4 ℃ overnight. The antibody‒protein‒agarose bead complexes were collected and boiled in sample buffer, and the target proteins in the precipitated pellets were analyzed by WB.

Ubiquitination Assessment

For analysis of NLRP3 ubiquitination, Flag-tagged SKP1 (Flag-SKP1), HA-tagged NLRP3 (HA-NLRP3), and Myc-tagged Ub (Myc-Ub) plasmids were transfected into BMDMs utilizing Lipofectamine 3000 (L3000001, Thermo Fisher Scientific) for 48 h. Subsequently, immunoprecipitation was carried out with anti-HA agarose beads to detect the enrichment of Myc (1:1000, ab9106, Abcam) and Flag (1:1000, 2368, Cell Signaling Technology).

Statistical Analysis

All the data were analyzed using Prism 8.0 software (GraphPad, La Jolla, CA, USA), and the values are presented as the means ± SDs. Comparisons between two groups were assessed using t tests; for comparisons among three or more groups, one-way or two-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc test. A p value of < 0.05 was considered statistically significant.