
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00296-9
10.1016/j.redox.2024.103318
103318
Research Paper
ALDH2 deficiency augments atherosclerosis through the USP14-cGAS-dependent polarization of proinflammatory macrophages
Rui Haiying abcde1
Yu Huaxiang abcde1
Chi Kai abcde
Han Ziqi abcde
Zhu Wenyong f
Zhang Jian abcde
Guo Haipeng g
Zou Wenyi abcde
Wang Fengxin abcde
Xu Ping abcde
Zou Dan abcde
Song Xiaoshuai abcde
Liu Lulu abcde
Wu Xuting abcde
Wu Wenxiao abcde
Qin Dandan abcde
Cao Yihai h
Xu Feng xufengsdu@126.com
abcde⁎⁎
Xue Li xueli_sdu@126.com
abcde⁎
Chen Yuguo chen919085@sdu.edu.cn
abcde⁎⁎⁎
a Department of Emergency Medicine, Qilu Hospital of Shandong University, Jinan, 250012, China
b Shandong Provincial Clinical Research Center for Emergency and Critical Care Medicine, Institute of Emergency and Critical Care Medicine of Shandong University, Chest Pain Center, Qilu Hospital of Shandong University, Jinan, 250012, China
c Key Laboratory of Emergency and Critical Care Medicine of Shandong Province, Key Laboratory of Cardiopulmonary-Cerebral Resuscitation Research of Shandong Province, Shandong Provincial Engineering Laboratory for Emergency and Critical Care Medicine, Qilu Hospital of Shandong University, Jinan, 250012, China
d Shandong Key Laboratory, Magnetic Field-free Medicine & Functional Imaging (MF), Qilu Hospital of Shandong University, Jinan, 250012, China
e NMPA Key Laboratory for Clinical Research and Evaluation of Innovative Drug, Qilu Hospital of Shandong University, Jinan, 250012, China
f Department of Thoracic Surgery, Qilu Hospital of Shandong University, Qingdao, 266035, China
g Department of Critical Care Medicine, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China
h Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, 17165, Sweden
⁎ Corresponding author. Qilu Hospital of Shandong University, No.107, Wen Hua Xi Road, Jinan, Shandong 250012, China. xueli_sdu@126.com
⁎⁎ Corresponding author. Qilu Hospital of Shandong University, No.107, Wen Hua Xi Road, Jinan, Shandong 250012, China. xufengsdu@126.com
⁎⁎⁎ Corresponding author. Qilu Hospital of Shandong University, No.107, Wen Hua Xi Road, Jinan, Shandong 250012, China. chen919085@sdu.edu.cn
1 Both authors contributed equally to this work.

17 8 2024
10 2024
17 8 2024
76 10331826 6 2024
15 8 2024
16 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism commonly exists in the East Asian populations and is associated with high risks of cardiovascular disease (CVD). However, the cellular and molecular mechanisms that underlie the ALDH2 rs671 mutant-linked high CVD remain elusive. Here, we show that macrophages derived from human ALDH2 rs671 carriers and ALDH2 knockout mice exhibited an enhanced pro-inflammatory macrophage phenotype and an impaired anti-inflammatory macrophage phenotype. Transplanting bone marrow from ALDH2−/−ApoE−/− to ApoE−/− mice significantly increased atherosclerotic plaque growth and pro-inflammatory macrophage polarization in vivo. Mechanistically, ALDH2 inhibited activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in macrophages. Pharmacological inhibition of cGAS by RU.521 completely neutralized ALDH2-deficiency-induced macrophage polarization. In-depth mechanistic investigation showed that ALDH2 accelerated cGAS K48-linked polyubiquitination degradation at lysine 282 in macrophages by reducing the interaction between ubiquitin-specific protease 14 (USP14) and cGAS, mainly through its enzymatic role in mitigating 4-hydroxy-2-nonenal (4-HNE) accumulation. Consistently, USP14 knockdown in bone marrow cells alleviated proinflammatory responses in macrophages and protected against atherosclerosis. Our findings provide new mechanistic insights of ALDH2 deficiency-associated proinflammation and atherosclerosis and new therapeutic and preventive paradigms for treatment of atherosclerosis-associated CVD.

Highlights

• ALDH2 rs671 mutation and ALDH2 deficiency enhances M1 macrophage polarization and inhibits M2 polarization in vivo and in vitro.

• ALDH2 regulates macrophage polarization and inflammation via the cGAS-STING signaling pathway.

• ALDH2 promoted cGAS degradation due to increasing the K48-linked polyubiquitination of lysine 282 in a USP14-dependent manner, which was related to the enzymatic activity of ALDH2 to detoxicate 4-HNE.

• Transplantation of bone marrow cells with lentiviral USP14 knockdown into atherosclerotic mice alleviated macrophage inflammatory responses and exerted protective effects against the progression of atherosclerosis.

Keywords

Atherosclerosis
Macrophage polarization
ALDH2
cGAS deubiquitination
USP14
4-Hydroxy-2-nonenal
==== Body
pmc1 Introduction

Atherosclerosis is a maladaptive inflammatory response caused by the retention of cholesterol-rich lipoproteins in the susceptible areas of the arterial vascular system [1]. Inflammation is a crucial factor in the initiation and progression of atherosclerotic lesions. Macrophages, the main components that respond to cholesterol-rich lipoproteins in plaque lesions, play an important role in promoting the pathogenesis of atherosclerosis because of impaired macrophage polarization and subsequent inflammatory responses [2,3]. Pro-inflammatory macrophages initiate and maintain inflammation, whereas anti-inflammatory macrophages suppress inflammation [4]. Therefore, modulating macrophage polarization and altering the macrophage phenotype proportion within plaques is a promising therapeutic strategy for atherosclerosis [2].

Aldehyde dehydrogenase 2 (ALDH2) is a crucial endogenous health-promoting enzyme. ALDH2 rs671 single nucleotide polymorphism (Glu504Lys, G to A mutation) reduces its enzymatic activity by more than 50 %, leading to a higher risk of cardiovascular disease (CVD), particularly among 30–50 % of East Asians [[5], [6], [7]]. ALDH2 exerts protective effects against atherosclerosis through its non-enzymatic and enzymatic roles. ALDH2 enzymatic function in the setting of atherosclerosis has been attributed to its detoxification of 4-hydroxy-2-nonenal (4-HNE) derived from impaired redox homeostasis in plaques [8]. ALDH2 upregulation in the macrophages promotes the clearance of 4-HNE, subsequently inhibiting oxidized low-density lipoprotein (oxLDL)-induced NLRP3 inflammasome activation [9] and macrophage apoptosis [10]. Moreover, ALDH2 detoxifies 4-HNE and mitigates the progression of atherosclerosis by inhibiting endoplasmic reticulum stress in smooth muscle cells [11] and senescence in endothelial cells [12]. In addition, ALDH2 exerts non-enzymatic effects by interacting directly with low-density lipoprotein receptor and AMP-activated protein kinase in macrophages to block foam cell formation and prevent atherosclerotic disease [13]. Additionally, ALDH2 directly interacts with RAC2, and the rs671 mutation increases the ubiquitination and degradation of RAC2, thereby inhibiting macrophage efferocytosis [14]. However, whether ALDH2 influences macrophage polarization and inflammation remains unclear.

Damage to the artery, especially the impaired redox homeostasis, triggers the release of a variety of substances into the cytoplasm known as danger-associated molecular patterns, including DNA and mtDNA [15]. Cytosolic DNA and mtDNA sensing by cyclic GMP-AMP synthase (cGAS) has been demonstrated to mediate macrophage polarization and promote the development of atherosclerosis [[15], [16], [17], [18], [19]]. cGAS generates cyclic GMP-AMP (cGAMP) to activate stimulator of interferon genes (STING), leading to the activation of TANK binding kinase 1 (TBK1)-interferon regulatory factor 3 (IRF3) and IκB kinase (IKK)-nuclear factor-κB (NF-κB), thereby resulting in the production of type I interferons (IFNs) and inflammatory cytokines, respectively [15]. The cGAS-STING pathway is involved in the onset and progression of atherosclerosis through the regulation of smooth muscle cell senescence [15], endothelial cell pyroptosis [19], and macrophage inflammation [16,17]. In the macrophages derived from human and mouse atherosclerotic lesions, the cGAS-STING pathway is activated by oxLDL to accelerate inflammatory molecule expression and atherosclerosis development [16,17]. Interestingly, ALDH2 has been reported to be an important regulator for the myocyte cGAS-STING pathway, under conditions of lipopolysaccharide exposure or amyloid precursor protein/presenilin 1 overexpression [20,21]. However, the molecular mechanisms of ALDH2 effect on cGAS-STING and its involvement in atherosclerosis remain unknown.

Therefore, this study aimed to investigate the role of ALDH2 in regulating macrophage polarization and inflammation, with a specific focus on the involvement of the cGAS-STING pathway, and to identify potential therapeutic targets for atherosclerosis, especially in populations with the ALDH2 rs671 mutation.

2 Materials and methods

2.1 Human specimens

Healthy human (n = 3) and atheromatous (n = 3) coronary arterial samples with no therapeutic uses for the heart were collected from deceased donors for organ transplantation at Shandong University Qilu Hospital. Written informed consent was obtained from all the included patients. This study was approved by the Institutional Medical Ethics Committee of Qilu Hospital of Shandong University (KYLL-2017-519), following the principles outlined in the Declaration of Helsinki. After formalin fixation and paraffin embedding, human coronary arteries were serially sectioned at 5 μm thickness.

2.2 Human participants

This study included healthy individuals with ALDH2 rs671 single-nucleotide polymorphism (categorized into the GA group: mutant heterozygotes or AA group: mutant homozygotes, n = 16) and controls (GG group: wild-type allele, n = 12) to isolate peripheral blood monocytes (PBMCs) using a Ficoll density gradient (catalog 17-1440-02, BD Biosciences) according to the manufacturer's protocol. The PBMCs were treated with differentiation medium (RPMI-1640, 10 % FBS, 25 ng/mL human macrophage colony-stimulating factor [hM-CSF]) for 10 days for differentiation into macrophages, which were then treated with oxLDL or interleukin (IL)-4/IL-13 for inducing macrophage polarization. The characteristics of the volunteers recruited for the clinical studies are presented in Supplementary Table S1. The exclusion criteria were as follows: (1) age <18 years, (2) pregnancy, (3) body mass index >32 kg/m2, (4) rheumatic or immune diseases, and (5) active acute infection. This study was approved by the Medical Institutional Ethics Committee of the Qilu Hospital of Shandong University (KYLL-202402-020-1). All the participants provided written informed consent. All procedures that involved human blood samples were conducted in accordance with the principles outlined in the Declaration of Helsinki.

2.3 Animals

ALDH2 knockout (ALDH2-KO) transgenic mice were provided by the University of Occupational and Environmental Health (Fukuoka, Japan). C57BL/6 wild-type (WT) and ApoE−/− mice (8–10 weeks old) were purchased from Huafukang Company (Beijing, China). We crossbred ALDH2−/− mice with ApoE−/− mice to obtain ALDH2+/−ApoE+/− heterozygous mice. ALDH2−/−ApoE−/− mice were obtained by inbreeding ALDH2+/−ApoE+/− heterozygous mice. To avoid the influence of estrogen in female mice, only male mice were used in our study. All procedures involving mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All the experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Shandong University. This study was approved by the Animal Ethics Committee of the Qilu Hospital of Shandong University (DWLL-2024−037).

2.4 Bone marrow transplantation

Eight-week-old ApoE−/− mice were used as the recipient mice. The recipient ApoE−/− mice were given sterile water containing gentamicin (320,000 U/L) and erythromycin (250 mg/L) 1 week before lethal irradiation (8.5 Gy with X-rays). After being lethally irradiated, the recipient ApoE−/− mice were implanted with 100 μL of bone marrow cell suspension containing 2 × 106 cells via the tail vein. During the recovery phase of 2 weeks, recipient ApoE−/− mice were fed with sterile water. After recovery, mice were fed either normal chow (NC) or a high-fat diet (HFD) for 16 weeks.

Eight-week-old ApoE−/− and ALDH2−/−ApoE−/− mice were used as bone marrow donors. The tibias and femurs of all four limbs of each donor were collected after euthanasia. The bone cavity was rinsed with sterile phosphate-buffered saline (PBS) using a 1 mL syringe. Primary bone marrow cells were collected by centrifugation after filtering through a 70-mesh filter.

Eight-week-old WT C57BL/6 mice were used as bone marrow donors. Primary bone marrow cells from WT mice were divided into four aliquots. Cells were transduced with empty viral vectors or ubiquitin-specific protease 14 (USP14)-shRNA viral vectors at a multiplicity of infection of 20. The shRNA sequences are listed in Supplementary Table S2. After a 10-min incubation period, the cells were centrifuged at 800 g for 10 min and resuspended in sterile PBS.

2.5 Isolation and culture of mouse peritoneal macrophages

Mice were injected intraperitoneally with 2 mL of 4 % sterile thioglycolate medium. After 3 days, the mice were euthanized, and the abdominal cavity was rinsed with PBS repeatedly with a syringe to collect primary peritoneal macrophages. The peritoneal macrophages were centrifuged for 5 min (800 rpm) and resuspended in Dulbecco's modified Eagle's medium containing 10 % FBS. Peritoneal macrophages were then seeded into well plates for subsequent experiments.

2.6 Isolation and culture of mouse bone marrow-derived macrophages (BMDMs)

After obtaining primary mouse bone marrow cells from WT or ALDH2-KO mice, 50 ng/mL of M-CSF (BioLegend, 576406) was added to the medium to proliferate and differentiate the committed myeloid precursors into BMDMs.

2.7 Cell culture and treatment

BMDMs and RAW264.7 cells were treated with oxLDL (80 μg/mL, Yiyuan Biotech, YB-002), IL-4/IL-13 (20 ng/mL, BioLegend, 574302/575902), or 4-HNE (10 μM, Abcam, ab141502) for 24 h. The ALDH2 agonist alda-1 (20 μM, Sigma, 126920) or the inhibitor daidzin (60 μM, Sigma, 30408) was added to the media 30 min before treatment with oxLDL. N-acetylcysteine (20 μM, Sigma, A7250), USP14 inhibitor IU1 (50 μM, Selleck, S7134), or cGAS inhibitor RU.521 (5 μM, Selleck, S6841) was added to the media before treatment with oxLDL, IL-4/IL-13, or 4-HNE for 24 h.

2.8 Immunofluorescence staining

After fixation with 4 % paraformaldehyde at room temperature for 30 min, RAW264.7 cells were washed three times with PBS, permeabilized with 0.1 % Triton X-100 for 30 min, blocked with 5 % bovine serum albumin for 30 min, and incubated with a primary antibody against cGAS (Thermo Fisher Scientific, PA5-141097) and USP14 (Proteintech, 67746-1-Ig) overnight at 4 °C. The cells were then incubated with secondary antibodies at room temperature for 1 h. Nuclear counterstaining was performed using 4′,6-diamidino-2-phenylindole fluorescent stain. The fluorescence intensities were observed using a Leica TCS SP8 confocal microscope.

2.9 ELISA analysis

IL-1β (SEA563Mu, Cloud-Clone Corp.), IFN-β (SEA222Mu, Cloud-Clone Corp.), and cGAMP (501700, Cayman Chemical) concentrations in the samples were measured using ELISA kits.

2.10 DCFH-DA staining

DCFH-DA staining was performed according to the manufacturer's instructions. RAW264.7 cells were stained with DCFH-DA (10 μM, Beyotime, S0033) at 37 °C for 30 min. The fluorescence was visualized using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

2.11 RNA isolation and quantitative real-time polymerase chain reaction (RT-qPCR)

After extraction with TRIzol reagent (Life Technologies, 15596018), the total mRNA from the whole aorta and macrophages was collected and depurated using DNase. The cDNAs were obtained using HiScript II Q RT SuperMix for qPCR (Vazyme, R223-01). Inflammatory cytokines (TNF-α, IL-6, and IL-1β), pro-inflammatory macrophage markers (iNOS, CXCL-10), anti-inflammatory macrophage markers (TGF-β, IL-10), and cGAS were identified by using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) in accordance with the manufacturer's instructions. The quantified transcripts from the samples were normalized against β-actin gene expression. RT-qPCR primers are listed in Supplementary Table S3.

2.12 Western blotting analysis

Tissue and macrophage lysates with the same protein content (determined using the BCA method; Boster AR0146) were prepared. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a 0.2 μm PVDF membrane (Millipore). After being blocked for 1.5 h in 5 % nonfat milk, the bands were incubated overnight at 4 °C with primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (1:5000 or 1:10000) for 2 h at room temperature. The bands were scanned and detected using a chemiluminescence instrument (General Electric Company, AI600RGB) equipped with a chemiluminescent HRP substrate (Millipore, WBKLS0100). ImageJ software was used to quantify the band intensity. The following primary antibodies were used: anti-GAPDH (Abcam, ab181602), anti-β-actin (Abcam, ab179467), anti-cGAS (CST, 31659S), anti-STING (CST, 50494S), anti-TBK1 (CST, 3504S), anti-p-TBK1 (Ser172, CST, 5483S), anti-IRF3 (CST, 4302S), anti-p-IRF3 (Ser396, CST, 29047S), anti-ALDH2 (Abcam, ab227021), anti-iNOS (Abcam, ab49999), anti-Arg1 (Abcam, ab239731), anti-4-HNE-protein adducts (Abcam, ab46545, ab48506), anti-K48-Ub (CST, 8081S), anti-USP29 (Proteintech, 27522-1-AP), anti-USP27x (Thermo Fisher Scientific, PA5-70389), anti-USP14 (CST, 11931S), anti-HA (CST, 3724S), anti-Myc (CST, 2276S), and anti-Flag (CST, 14793S).

2.13 Histological analysis

The atherosclerotic burden was quantified using en face preparations of the whole aorta stained with Oil red O, and serially sectioned aortic root specimens (5 μm) were stained with hematoxylin and eosin. The serially sectioned aortic root and coronary arterial specimens (5 μm) were then subjected to immunofluorescence or immunohistochemistry. Immunohistochemical staining was performed using a commercial kit (Boster, SA1022), and the primary antibody was cGAS (Thermo Fisher Scientific, PA5-141097), 4-HNE (Abcam, ab48506), or CD206 (Abcam, ab64693). Immunofluorescence staining was performed according to the manufacturer's protocol to detect cGAS (Thermo Fisher Scientific, PA5-141097), p-TBK1 (Ser172, CST, 5483S), CD68 (Abcam, ab303565, ab53444; Servicebio, GB14043), iNOS (Abcam, ab49999), Arg1 (Abcam, ab239731), or α-SMA (CST, 19245S). Images were captured using a slideview microscope (Olympus, VS200).

2.14 Flow cytometric analysis of macrophage differentiation

For in vivo analysis, the spleens or whole aorta of mice were dissected, the ground cells were filtered through a 100 μm cell strainer, and red blood cells were lysed with lysis solution (BL503B, Biosharp). Cells from the spleens or whole aorta were incubated at 4 °C for 30 min with the following labeled antibodies: Brilliant Violet 510™ anti-mouse CD45 (BioLegend, 103137), FITC anti-mouse Ly6G (BioLegend, 127605), PerCP/Cyanine5.5 anti-mouse/human CD11b (BioLegend, 101227), BV421-conjugated anti-mouse F4/80 (BD Horizon™, 565411; BioLegend, 123131), PE-conjugated anti-mouse CD86 (BD Pharmingen™, 561963; BioLegend, 159204), and APC anti-mouse CD163 (BioLegend, 155306), according to the manufacturer's instructions. Subsequently, the cells were washed three times with PBS and analyzed using the Beckman CytoFLEX cytometer.

For in vitro analysis of macrophage differentiation, macrophages were collected and incubated at 4 °C for 30 min with the following labeled antibodies according to the manufacturer's instructions: PE anti-mouse F4/80 (BioLegend, 123110), BV421-conjugated anti-mouse F4/80 (BD Horizon™, 565411), APC anti-mouse CD86 (BioLegend, 105114), PE-conjugated anti-mouse CD86 (BD Pharmingen™, 561963), Brilliant Violet 421™ anti-mouse CD163 (BioLegend, 155309), PE anti-human CD68 (BioLegend, 333808), APC anti-human CD86 (BioLegend, 305412), and APC anti-human CD163 (BioLegend, 333609). Subsequently, macrophages were washed three times with PBS and analyzed using the Beckman CytoFLEX cytometer.

2.15 Assessment of cGAS K48-Linkage polyubiquitin

To detect the levels of cGAS K48-linkage polyubiquitin, cGAS was immunoprecipitated by incubating cell lysate with 5 μg anti-cGAS antibody (CST, 31659S) overnight at 4 °C, followed by incubation with 20 μL PureProteome Protein A/G mix magnetic bead system (Merck, LSKMAGAG10) for 3 h at 4 °C. Immunoprecipitates were washed, resuspended in 1 × sample buffer, boiled for 10 min, and analyzed for K48-Ub expression by western blotting.

2.16 Plasmid constructs

Flag-tagged human cGAS (WT), Flag-tagged human cGAS (K173R; K275R; K282R; K315R; K414R; K285R; K479R), Flag-tagged human cGAS (1–157 aa; 158–522 aa), HA-tagged human USP14 (WT), HA-tagged human USP14 (C114A), HA-tagged human USP14 (1–104 aa; 105–494 aa), Myc-tagged human K48-Ub, Myc-tagged human K63-Ub, Myc-tagged human ALDH2 (WT), and Myc-tagged human ALDH2 (rs671) were inserted into a pcDNA3.0 vector. Clones were selected and amplified by sequencing to verify the results.

2.17 Coimmunoprecipitation (Co-IP)

Macrophage target proteins were precipitated with the respective antibodies and protein A/G beads (Merck, LSKMAGAG10). 293T cells were transfected with a specifically tagged plasmid using Lipofectamine 2000 (Thermo Fisher, USA), followed by Co-IP with Flag antibodies and western blotting with HA, Flag, and Myc antibodies.

2.18 Glutathione S-transferase (GST)-Pull down assay

Purified GST-cGAS and His-USP14 fusion proteins were expressed in Escherichia coli. For GST pull-down, the 0.15 mg GST-cGAS fusion protein was mixed with 30 μL of GST beads and incubated at 4 °C overnight with gentle rocking motion. Subsequently, 0.15 mg of purified USP14 protein was added to the immobilized GST-cGAS and GST beads with or without 4-HNE treatment (100 μM for 0.15 mg protein at room temperature for 2 h) and then incubated at 4 °C with gentle rotation. The beads were washed two to three times with lysis buffer, and 100 μL elution buffer was used to elute the beads. Finally, loading buffer was added and boiled, and the samples were analyzed by western blotting.

2.19 Surface plasmon resonance (SPR)

Recombinant cGAS and USP14 proteins were subjected to SPR analysis using a Biacore T200 instrument. The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The mixture was injected into the Fc3 and Fc4 sample channels at a flow rate of 10 μL/min for 800 s, followed by the injection of 50 μg/mL of USP14 in 10 mM NaAc (pH 4.0) for 900 s into the Fc4 sample channel at a flow rate of 10 μL/min, with the immobilization level determined to be approximately 10390 RU. The chip was deactivated by 1 M ethanolamine hydrochloride at a flow rate of 10 μL/min for 800 s into the Fc3 and Fc4 sample channels. cGAS was diluted with running buffer to obtain seven concentrations (10000, 5000, 2500, 1250, 625, 312.5, 156.3, and 0 nM). The cGAS is injected into the Fc3-Fc4 of channel at a flow rate of 30 μL/min for an association phase of 90 s, followed by 180 s of dissociation. The association and dissociation processes were handled in the running buffer.

2.20 High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS)

For MS analysis, anti-Flag (cGAS) immunoprecipitation (IP) was performed using whole-cell lysates derived from HEK293 cells transfected with Flag-tagged human cGAS and Myc-tagged human K48-Ub. The IP proteins were resolved using SDS-PAGE and identified using Coomassie staining. cGAS was extracted by cutting the gel, and cGAS ubiquitination was measured using HPLC-MS/MS. To confirm that USP14 binds to cGAS, anti-cGAS IP was performed using whole-cell lysates derived from BMDMs with or without 4-HNE stimulation. The IP proteins were resolved by SDS-PAGE and identified using Coomassie staining. cGAS and USP14 protein-unique peptides were measured using HPLC-MS/MS.

Trypsin was used to hydrolyse the proteins in the gel. Nanoflow LC-MS/MS analysis of tryptic peptides was conducted on a quadrupole Orbitrap mass spectrometer (Q Exactive HF-X, Thermo Fisher Scientific, Bremen, Germany) coupled to an EASY nLC 1200 ultra-high-pressure system (Thermo Fisher Scientific) via a nano-electrospray ion source. 500 ng of peptides were loaded on a 25 cm column (150 μm inner diameter, packed using ReproSil-Pur C18-AQ 1.9-μm silica beads; Beijing Qinglian Biotech Co., Ltd, Beijing, China). Peptides were separated using a gradient from 8 to 12 % B in 5 min, then12 % to 30 % B in 33 min and stepped up to 40 % in 7 min followed by a 15 min wash at 95 % B at 600 nl per minute where solvent A was 0.1 % formic acid in water and solvent B was 80 % ACN and 0.1 % formic acid in water. The total duration of the run was 60 min. Column temperature was kept at 60 °C using an in-house-developed oven. Briefly, the mass spectrometer was operated in “top-40” data-dependent mode, collecting MS spectra in the Orbitrap mass analyzer (120,000 resolution, 350–1500 m/z range) with an automatic gain control (AGC) target of 3E6 and a maximum ion injection time of 80 ms. The most intense ions from the full scan were isolated with an isolation width of 1.6 m/z. Following higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27, MS/MS spectra were collected in the Orbitrap (15,000 resolution) with an AGC target of 5E4 and a maximum ion injection time of 45 ms. Precursor dynamic exclusion was enabled with a duration of 16 s. All RAW files were analyzed using the Proteome Discoverer suite (version 2.4, Thermo Fisher Scientific). MS2 spectra were searched against the UniProtKB human proteome database containing both Swiss-Prot and TrEMBL human reference protein sequences.

2.21 Statistical analysis

The results are presented as the mean ± SD from at least three independent experiments using GraphPad Prism 9 (La Jolla, CA, USA). The normality of the distribution of continuous variables was confirmed using the Shapiro-Wilk normality test and visualized using a Q-Q plot. Homoscedasticity was confirmed using the F-test (two groups) and the Brown-Forsythe and Bartlett's tests (≥ three groups). Unpaired two-tailed Student's t-test (two groups) and One-way ANOVA followed by Tukey post hoc test (≥ three groups) were used for continuous variables with a normal distribution and similar variances. Unpaired two-tailed Student's t-test with Welch's correction (two groups) and Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test (≥ three groups) were utilized for continuous variables with a normal distribution and heterogenous variances. For non-normal distribution, Mann-Whitney U test (two groups) and Kruskal-Wallis test followed by Dunn's post hoc test (≥ three groups) were used. Any P value < 0.05 was considered statistically significant.

3 Results

3.1 ALDH2 rs671 mutation in human macrophages enhances pro-inflammatory macrophage polarization and inhibits anti-inflammatory polarization

To study the effect of the ALDH2 rs671 mutation on macrophage polarization, peripheral blood monocytes (PBMCs) were isolated from individuals with ALDH2 rs671 mutation and controls. After the PBMCs differentiated into macrophages, the macrophages were treated with oxLDL. The number of CD68+CD86+ macrophages was higher in the AA group than in the GG group (Fig. 1A and B). IL-4/IL-13 stimulation activates STAT6 through IL-4 receptor signaling for anti-inflammatory polarization [22]. Moreover, during anti-inflammatory polarization, the number of CD68+CD163+ macrophages in the AA group was significantly lower than that in the GG group (Fig. 1C and D). Notably, the GA group exhibited enhanced pro-inflammatory macrophage polarization and reduced anti-inflammatory polarization; however, this effect was less than that observed in the AA group (Fig. 1E–H). In agreement with these results, the mRNA and protein levels of characteristic genes of pro-inflammatory macrophages (iNOS, CXCL-10, TNF-α, IL-6, and IL-1β) and anti-inflammatory macrophages (TGF-β, IL-10, and Arg1) were also regulated by the ALDH2 rs671 polymorphism (Fig. 1I–L). However, the results revealed that the ALDH2 rs671 mutation did not alter macrophage polarization at baseline (Fig. 1I–L).Fig. 1 ALDH2 rs671 mutation in human macrophages enhances pro-inflammatory macrophage polarization and inhibits anti-inflammatory polarization. A, B Human peripheral blood monocytes (PBMCs) from healthy volunteers with ALDH2 rs671 mutation (AA) and controls (GG) were incubated with oxLDL and underwent flow cytometry. Pro-inflammatory macrophages (CD68+CD86+) were measured by flow cytometry (n = 4). C, D Human PBMCs from the AA and GG groups were incubated with IL-4/IL-13 and underwent flow cytometry. Anti-inflammatory macrophages (CD68+CD163+) were measured by flow cytometry (n = 4). E, F Human PBMCs from the GA/AA and GG groups were incubated with oxLDL, and flow cytometry was performed. Pro-inflammatory macrophages (CD68+CD86+) were measured by flow cytometry (n = 3). G, H Human PBMCs from the GA/AA and GG groups were incubated with IL-4/IL-13, and flow cytometry was performed. Anti-inflammatory macrophages (CD68+CD163+) were measured by flow cytometry (n = 3). I Human PBMCs were incubated with or without oxLDL. RT-qPCR of iNOS, CXCL-10, TNF-α, IL-6, and IL-1β in human PBMCs (n = 4). J Human PBMCs were incubated with or without IL-4/IL-13. RT-qPCR of TGF-β and IL-10 in human PBMCs (n = 6). K Human PBMCs were incubated with or without oxLDL. Western blot of iNOS in human PBMCs (n = 4). L Human PBMCs were treated with or without IL-4/IL-13. Western blot of Arg1 in human PBMCs (n = 4). Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (F, H-J). Unpaired two-tailed Student's t-test was used for the analysis in (B, D). Western blot bands were detected under the same conditions (K, L). P < 0.05 was considered significant.

Fig. 1

3.2 ALDH2 regulates macrophage polarization and inflammation in vitro

The effects of ALDH2 on macrophage polarization and inflammation were examined using bone marrow-derived macrophages (BMDMs) from ALDH2-KO and WT mice. The protein expression level of ALDH2 was examined using western blotting (Supplementary Fig. S1A). The regulatory effect of ALDH2 on oxLDL-induced pro-inflammatory macrophage phenotype was analyzed, which indicated that the formation of pro-inflammatory macrophages (F4/80+CD86+ subtype cells) in ALDH2-KO BMDMs was higher than that in WT BMDMs (Fig. 2A and B). The number of anti-inflammatory macrophages (F4/80+CD163+ subtype cells) in BMDMs from ALDH2-KO and WT mice after treatment with IL-4 and IL-13 was measured. Interestingly, anti-inflammatory macrophage polarization was lower in ALDH2-KO BMDMs than in WT BMDMs (Fig. 2C and D). However, ALDH2 deletion did not alter the macrophage phenotype at baseline (Fig. 2A–D). Moreover, the expression levels of macrophage polarization genes (iNOS, CXCL-10, TNF-α, IL-6, IL-1β, TGF-β, IL-10, Arg1) were detected and the results confirmed that ALDH2 deletion impaired IL-4/IL-13-induced anti-inflammatory macrophage polarization, and promoted oxLDL-triggered pro-inflammatory macrophage activation (Fig. 2E–M).Fig. 2 Effects of ALDH2 on macrophage polarization and inflammation in vitro. A, B Bone marrow-derived macrophages (BMDMs) from ALDH2-KO and WT mice were incubated with or without oxLDL. Pro-inflammatory macrophages (F4/80+CD86+) were measured by flow cytometry (n = 3). C, D BMDMs from ALDH2-KO and WT mice were incubated with or without IL-4/IL-13. Anti-inflammatory macrophages (F4/80+CD163+) were measured by flow cytometry (n = 3). E-I RT-qPCR of iNOS, CXCL-10, IL-6, TNF-α, and IL-1β in BMDMs (n = 4–5). J, K RT-qPCR of TGF-β and IL-10 in BMDMs (n = 4). L ALDH2-KO BMDMs and WT BMDMs were incubated with or without oxLDL. Western blot of iNOS in BMDMs (n = 4). M Western blot of Arg1 in BMDMs (n = 4). Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (B, D-H, J, K). Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test was used for the analysis in (I). Western blot bands were detected under the same conditions (L, M). P < 0.05 was considered significant.

Fig. 2

We next assessed the impact of ALDH2 on macrophage polarization in RAW264.7 cells treated with the ALDH2-specific agonist alda-1 or inhibitor daidzin. Similar to the BMDMs, ALDH2 activity upregulation inhibited pro-inflammatory macrophage polarization primed with oxLDL, whereas ALDH2 activity downregulation increased pro-inflammatory macrophage polarization induced by oxLDL (Supplementary Fig. S1B).

3.3 Bone marrow transplantation from ALDH2−/−ApoE−/− mice into ApoE−/− mice accelerates atherosclerosis and promotes pro-inflammatory macrophage polarization in vivo

To test whether macrophage ALDH2 plays a key role in plaque formation and atherosclerotic macrophage polarization, bone marrow transplantation was performed to generate myeloid-specific ALDH2-deficient mice. ApoE−/− mice were subjected to lethal irradiation, and bone marrow cells from ApoE−/− or ALDH2−/−ApoE−/− mice were transplanted into the irradiated mice. Two types of chimeric mice were generated, ApoE−/− to ApoE−/− (ApoE−/− donors), ALDH2−/−ApoE−/− to ApoE−/− (ALDH2−/−ApoE−/− donors). After 2 weeks of recovery, the deletion of ALDH2 was validated in primary peritoneal macrophages by western blotting (Fig. 3A and B). Chimeric mice were fed high-fat diet (HFD) or normal chow (NC) for an additional 16 weeks to induce atherogenesis.Fig. 3 Bone marrow transplantation from the ALDH2−/−ApoE−/− mice into ApoE−/− mice regulates atherosclerotic macrophage polarization in vivo. A Schematic diagram of bone marrow transplantation. B After the bone marrow transplantation experiment adaptation procedure, primary peritoneal macrophages were extracted from ApoE−/− donors and ALDH2−/−ApoE−/− donors to verify the bone marrow clearance efficiency. C RT-qPCR of TNF-α, IL-1β, IL-6, CXCL-10, iNOS, TGF-β, and IL-10 in the aortas of the ApoE−/− mice transplanted with ApoE−/− bone marrow and ApoE−/− mice transplanted with ALDH2−/−ApoE−/− bone marrow, which were fed with a high-fat diet (HFD) or normal chow (NC) (n = 3–4). D, E CD206 immunohistochemistry staining and quantification in cross sections of the aortic roots of the ApoE−/− mice transplanted with ApoE−/− bone marrow or ALDH2−/−ApoE−/− bone marrow, which were fed with HFD (n = 6). F, G Immunofluorescence staining for CD68 (red) and iNOS (green) in the aortic root sections of the ApoE−/− mice transplanted with ApoE−/− bone marrow or ALDH2−/−ApoE−/− bone marrow, which were fed with HFD and quantification of colocalization (n = 6). H, I Immunofluorescence staining for CD68 (red) and Arg1 (green) in the aortic root sections of the ApoE−/− mice transplanted with ApoE−/− bone marrow or ALDH2−/−ApoE−/− bone marrow, which were fed with HFD, and quantification of colocalization (n = 6). J-M Flow cytometry plots and quantitative analysis of macrophage differentiation in the spleens of ApoE−/− mice (n = 6). N-Q Flow cytometry plots and quantitative analysis of macrophage differentiation in the aortas of ApoE−/− mice (n = 3). Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (C, K). Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test was used for the analysis in (L, M). Unpaired two-tailed Student's t-test was used for the analysis in (E, G, I, P, Q). Unpaired two-tailed Student's t-test with Welch's correction was used for the analysis in (O). Western blot bands were detected under the same conditions (B). P < 0.05 was considered significant.

Fig. 3

An en face analysis of the whole aorta showed that, compared with HFD-fed ApoE−/− donors, HFD-fed ALDH2−/−ApoE−/− donors exhibited significantly enlarged atherosclerotic plaque areas (Supplementary Figs. S2A and B). In addition, the plaque volume at the aortic root was measured using hematoxylin and eosin staining to assess the effect of macrophage ALDH2 on atherosclerosis (Supplementary Figs. S2C and D). However, ALDH2 deletion in macrophages did not alter serum TG (triglyceride), CHO (cholesterol), HDL (high-density lipoprotein), and LDL (low-density lipoprotein) (Supplementary Fig. S2E).

Considering that chronic inflammation is a prominent feature of atherosclerosis, the effects of ALDH2 deficiency on the expression of inflammatory mediators in macrophages were analyzed. Compared with HFD-fed ApoE−/− donors, the mRNA levels of inflammatory genes, such as TNF-α, IL-6, and IL-1β, were increased in the aorta of HFD-fed ALDH2−/−ApoE−/− donors (Fig. 3C). Subsequently, the regulatory effect of ALDH2 on the proportion of intraplaque macrophage polarization was determined. As shown in Fig. 3C, the expression of iNOS and CXCL-10 in aortic plaques was significantly higher in ALDH2−/−ApoE−/− donors than in ApoE−/− donors. Conversely, the expression of anti-inflammatory genes (IL-10 and TGF-β) was significantly lower in the aorta of ALDH2−/−ApoE−/− donors than in ApoE−/− donors. Moreover, the expression of the anti-inflammatory gene CD206 in the plaques was lower in the HFD-fed ALDH2−/−ApoE−/− donor group (Fig. 3D and E). Furthermore, iNOS increasingly colocalized with CD68+ cells in the aortic sinus sections of HFD-fed ALDH2−/−ApoE−/− donors after immunofluorescent analysis (Fig. 3F and G). Immunofluorescence staining demonstrated that ALDH2 deficiency significantly decreased Arg1 expression in CD68+ macrophages in plaques (Fig. 3H and I). These results were consistent with the flow cytometric analysis of the proportion of macrophages in the spleens of mice (Fig. 3J–M). To further analyze the effects of ALDH2 on aortic macrophage polarization, the proportion of aortic macrophages was measured using flow cytometry. Compared with the HFD-fed ApoE−/− donor group, the ratios of aortic pro-inflammatory macrophages (CD45+Ly6G−CD11b+F4/80+CD86+CD163−) were increased in HFD-fed ALDH2−/−ApoE−/− donors group. However, the proportions of anti-inflammatory macrophages (CD45+Ly6G−CD11b+F4/80+CD86−CD163+) were lower after ALDH2 deletion (Fig. 3N–Q).

3.4 The cGAS-STING pathway is activated in human and mouse atheromatous lesions

Compared with NC-fed ApoE−/− donors, the levels of cGAS, STING, p-TBK1 (Ser172), and p-IRF3 (Ser396) were significantly higher in the atherosclerotic plaques of HFD-fed ApoE−/− donors (Fig. 4A). Similar results were obtained via cGAS immunohistochemical staining in coronary atherosclerotic plaques and in atherosclerotic lesions from the aortic roots of HFD-fed ApoE−/− donors (Fig. 4B and Supplementary Figs. S3A and B). Notably, double immunoﬂuorescence staining revealed strong immunoreactivity of cGAS and p-TBK1 in the macrophages of human and mouse atheromatous lesions (Fig. 4C, D and Supplementary Figs. S3C–H).Fig. 4 Effects of ALDH2 on HFD/oxLDL-induced activation of the cGAS-STING pathway in vivo and vitro. A Representative western blotting of the cGAS-STING pathway in the aortas of ApoE−/− mice (n = 8). B cGAS immunohistochemistry staining in cross sections of the aortic roots of ApoE−/− mice transplanted with ApoE−/− bone marrow and ApoE−/− mice transplanted with ALDH2−/−ApoE−/− bone marrow (n = 4). C Immunofluorescence staining for CD68 (red) and cGAS (green) in the aortic root sections of the ApoE−/− mice transplanted with ApoE−/− bone marrow or ALDH2−/−ApoE−/− bone marrow (n = 4). D Immunofluorescence staining for CD68 (red) and p-TBK1 (green) in the aortic root sections of the ApoE−/− mice transplanted with ApoE−/− bone marrow or ALDH2−/−ApoE−/− bone marrow (n = 4). E Representative western blotting of the cGAS-STING pathway in human PBMCs (n = 7–8). F Representative western blotting of the cGAS-STING pathway in ALDH2-KO BMDMs and WT BMDMs (n = 7–8). G RT-qPCR of cGAS in the aortas of ApoE−/− mice transplanted with ApoE−/− bone marrow and ApoE−/− mice transplanted with ALDH2−/−ApoE−/− bone marrow (n = 4). H Quantitative analysis of cGAS immunohistochemistry staining in Fig. 4B (n = 4). I RT-qPCR of cGAS in human PBMCs (n = 4). J RT-qPCR of cGAS in ALDH2-KO BMDMs and WT BMDMs (n = 8). Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (G–I). Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test was used for the analysis in (J). Western blot bands were detected under the same conditions (A, E, F). P < 0.05 was considered significant.

Fig. 4

3.5 ALDH2 affects the cGAS-STING pathway activation in vivo and in vitro

Transcriptomic datasets (GSE10000) of WT and ApoE−/− mice in the Gene Expression Omnibus (GEO) datasets were analyzed for differentially expressed genes using the GEO2R tool. Our results revealed that 2187 genes were downregulated in lesions of 78-week-old ApoE−/− mice, encompassing genes that regulate oxidation-reduction processes, metabolic processes, and lipid metabolic processes. In contrast, several genes were upregulated, particularly those enriched for inflammatory responses, immune system processes, and chemotaxis (Supplementary Fig. S4A). In particular, immune system genes, including the STING-encoding gene Tmem173 and Cxcl10, were upregulated, and oxidation-reduction process genes, including Aldh2, were downregulated in atherosclerotic lesions of 78-week-old ApoE−/− mice (Supplementary Fig. S4B).

To investigate the relationship between ALDH2 and the cGAS-STING immune system pathway in macrophages of atherosclerosis, bone marrow transplantation from ALDH2−/−ApoE−/− mice into ApoE−/− mice was performed to specifically reduce the ALDH2 level of macrophages in vivo. Interestingly, compared with HFD-fed ApoE−/− mice transplanted with ApoE−/− bone marrow, protein expression of cGAS, STING, p-TBK1, and p-IRF3 was significantly higher in the aorta of HFD-fed ApoE−/− mice transplanted with ALDH2−/−ApoE−/− bone marrow (Fig. 4A and Supplementary Fig. S5A). Similar results were obtained using cGAS immunohistochemical staining of atherosclerotic lesions (Fig. 4B, H). Moreover, double immunoﬂuorescence staining demonstrated that ALDH2 deficiency further increased cGAS and p-TBK1 expression in macrophages of HFD-induced atheromatous plaques (Fig. 4C, D and Supplementary Figs. S3G and H).

To assess our experimental findings in vivo, we investigated whether ALDH2 influences the oxLDL-induced activation of the cGAS-STING pathway in macrophages. The results revealed that the ALDH2 rs671 mutation increased the baseline cGAS protein levels (Fig. 4E and Supplementary Fig. S5B). The expression of cGAS in PBMCs from ALDH2 WT populations is upregulated after stimulation with oxLDL. After oxLDL stimulation, PBMCs from ALDH2 rs671 mutation populations expressed higher levels of cGAS protein than those from ALDH2 WT populations (Fig. 4E and Supplementary Fig. S5B). Similar changes were observed for the cGAS downstream molecules, STING, p-TBK1, and p-IRF3 (Fig. 4E and Supplementary Fig. S5B). Consistently, baseline cGAS-STING pathway activation was higher in ALDH2-KO BMDMs than in WT BMDMs (Fig. 4F and Supplementary Fig. S5C). Moreover, following oxLDL stimulation, the cGAS-STING pathway activation was enhanced in ALDH2-KO BMDMs than in WT BMDMs (Fig. 4F and Supplementary Fig. S5C). In addition, cGAS transcription increased after HFD/oxLDL treatment, whereas ALDH2 had no effect on the transcription of cGAS (Fig. 4G, I, J).

Consistent with the results in PBMCs and BMDMs, ALDH2 also served as a strong regulator of cGAMP and IFN-β under oxLDL stimulation in RAW264.7 cells (Supplementary Figs. S5D and E). Moreover, the mRNA level of cGAS was not regulated by ALDH2 in RAW264.7 cells (Supplementary Fig. S5F).

Taken together, these data indicate that ALDH2 functions as an upstream regulator of the cGAS-STING pathway during atherosclerosis progression.

3.6 The regulatory effects of ALDH2 on macrophage polarization is dependent on the cGAS-STING pathway

PBMCs from individuals with ALDH2 rs671 mutation and controls were stimulated with oxLDL or IL-4/IL-13, the cGAS-STING pathway was inhibited with the cGAS inhibitor RU.521, and the ratio of macrophage subtypes was observed by flow cytometry. RU.521 impaired oxLDL-induced pro-inflammatory macrophage polarization, whereas it promoted IL-4/IL-13-triggered anti-inflammatory macrophage activation in PBMCs from ALDH2 rs671 mutant and ALDH2 WT populations (Fig. 5A–D). Furthermore, flow cytometry indicated that RU.521 decreased oxLDL-induced pro-inflammatory macrophage polarization, and promoted IL-4/IL-13-triggered anti-inflammatory macrophage activation in ALDH2-KO and WT BMDMs (Fig. 5E–H). These results were validated at the protein and mRNA levels using western blotting analysis and RT-qPCR, respectively. Consistently, RU.521 decreased the expression of pro-inflammatory genes and increased anti-inflammatory genes expression (Fig. 5I–K). Importantly, RU.521 could almost abolish the effects of ALDH2 on macrophage polarization (Fig. 5I and J). In addition, RU.521 had no effect on macrophage polarization at baseline (Supplementary Fig. S6A). Taken together, these results elucidate that ALDH2 regulates macrophage polarization and inflammation via the cGAS-STING signaling pathway.Fig. 5 ALDH2 regulates macrophage polarization and inflammation through the cGAS-STING signaling pathway. A, B Pro-inflammatory macrophages (CD68+CD86+) were measured by flow cytometry in human PBMCs treated with oxLDL with or without RU.521 (n = 3). C, D Anti-inflammatory macrophages (CD68+CD163+) were measured by flow cytometry in human PBMCs treated with IL-4/IL-13 with or without RU.521 (n = 3). E, F Pro-inflammatory macrophages (F4/80+CD86+) were measured by flow cytometry in BMDMs of WT and ALDH2-KO mice treated with oxLDL with or without RU.521 (n = 3). G, H Anti-inflammatory macrophages (F4/80+CD163+) were measured by flow cytometry in BMDMs treated with IL-4/IL-13 with or without RU.521 (n = 3). I ALDH2-KO BMDMs and WT BMDMs treated with oxLDL with or without RU.521. RT-qPCR of iNOS, CXCL-10, IL-6, and TNF-α in BMDMs (n = 3–4). J ALDH2-KO BMDMs and WT BMDMs treated with IL-4/IL-13 with or without RU.521. RT-qPCR of TGF-β (n = 8) and IL-10 (n = 6) in BMDMs. K Western blot of iNOS in ALDH2-KO BMDMs and WT BMDMs (n = 4). Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (B, D, F, H, I). Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test was used for the analysis in (J). Western blot bands were detected under the same conditions (K). P < 0.05 was considered significant.

Fig. 5

3.7 ALDH2 regulates the cGAS-STING pathway by modulating USP14-dependent cGAS ubiquitination

Although ALDH2 significantly altered cGAS protein levels, it did not affect cGAS mRNA levels. These results prompted us to investigate the regulation of cGAS protein degradation by ALDH2. K48-linked ubiquitination of cGAS plays a critical role in regulating cGAS protein levels. Co-IP experiments were initially performed to detect cGAS K48-linked ubiquitination in RAW264.7 cells stimulated by oxLDL under different ALDH2 activity conditions. Notably, the level of endogenous cGAS K48-linked ubiquitination was decreased after oxLDL stimulation, and the ubiquitination phenomenon was significantly reversed by alda-1; daidzin-induced ALDH2 inhibition further decreased cGAS K48-linked ubiquitination (Fig. 6A). The mechanism by which ALDH2 regulates cGAS K48-linked ubiquitination was further explored.Fig. 6 ALDH2 accelerates cGAS K48-linked polyubiquitination degradation by reducing the interaction between USP14 and cGAS. A Co-IP assays were performed to show K48 ubiquitination of cGAS in RAW264.7 cells (n = 4). B Co-IP assays using an anti-cGAS antibody to detect the interaction between cGAS and DUBs and K48 ubiquitination of cGAS in RAW264.7 cells after 10 μM 4-HNE stimulation for 24 h (n = 4). C Flag-cGAS, Myc-K48-Ub, and Myc-K63-Ub were transfected into HEK293T cells treated with or without 4-HNE, and Co-IP assays were performed (n = 3). D Co-IP assays using an anti-cGAS antibody to detect the interaction between cGAS and USP14 in BMDMs after 10 μM 4-HNE stimulation for 24 h (n = 4). E Co-IP assays using an anti-USP14 antibody to detect the interaction between USP14 and cGAS in RAW264.7 cells after 10 μM 4-HNE stimulation for 24 h (n = 4). F USP14 was identified by mass spectrometry (MS) in the protein mixture enriched by anti-cGAS antibody in BMDMs treated with 4-HNE (10 μM, 24 h). The graph represents the unique peptide fragment KQDEWIK, which was specifically referred as the USP14 protein. G Colocalization of cGAS (red) with USP14 (green) in RAW264.7 cells in the presence or absence of 4-HNE treatment (10 μM for 24 h, n = 6). H Co-IP assays were performed in lysates of HEK293T cells expressing Flag-cGAS, Myc-K48-Ub, and HA-USP14 (WT or C114A, n = 3). I Pro-inflammatory macrophages (F4/80+CD86+) were measured by flow cytometry in BMDMs treated with oxLDL with or without IU1 (n = 3). J Anti-inflammatory macrophages (F4/80+CD163+) were measured by flow cytometry in BMDMs treated with IL-4/IL-13 with or without IU1 (n = 3). K Pro-inflammatory macrophages (F4/80+CD86+) in the BMDMs of WT mice were measured by flow cytometry. BMDMs were pretreated with IU1 and then incubated with 10 μM 4-HNE for 24 h (n = 3). Western blot bands were detected under the same conditions (A-E, H).

Fig. 6

ALDH2 is a key enzyme that metabolizes and detoxicates 4-HNE, a lipid aldehyde product of lipid peroxidation (secondary to elevated oxidative stress). It is widely accepted that elevated levels of 4-HNE play a substantial role in regulating the inflammatory response and formation of macrophage foam cells [8]. This contributes to the initiation and progression of atherosclerosis by altering protein function via the adduction of nucleophilic amino acid residues [8]. As expected, greater 4-HNE immunostaining was observed in coronary atherosclerotic plaques (Supplementary Figs. S7A and B). Moreover, alda-1 reduced 4-HNE accumulation mediated by oxLDL, whereas downregulation of ALDH2 activity exhibited the opposite effect in RAW264.7 cells (Supplementary Fig. S7C). In addition, oxLDL-mediated accumulation of 4-HNE was higher in ALDH2-KO BMDMs and the ALDH2-rs671-mutant PBMCs (Supplementary Figs. S7D and E). Notably, 4-HNE levels were significantly elevated in the aortas of HFD-fed ApoE−/− mice transplanted with ALDH2−/−ApoE−/− bone marrow (Supplementary Figs. S7F and G). Meanwhile, upregulation of ALDH2 activity significantly reduced H2O2-mediated cGAS-STING pathway activation, with the downregulation of ALDH2 activity exhibiting opposite effects (Supplementary Figs. S8A and B). Interestingly, ALDH2 is also an important antioxidant within macrophages under oxLDL stimulation (Supplementary Fig. S8C). Importantly, the reactive oxygen species scavenger N-acetylcysteine (NAC) served as a negative regulator of the cGAS-STING pathway and decreased the pro-inflammatory macrophage phenotype (Supplementary Figs. S8D–G). These results also indicate that NAC abolished the effects of ALDH2 on the cGAS-STING pathway and macrophage polarization (Supplementary Figs. S8D–H).

Therefore, we hypothesized that 4-HNE regulates deubiquitinating enzymes (DUBs) to cleave K48-linked polyubiquitin chains of cGAS, leading to the stabilization of cGAS proteins. To support this hypothesis, we first stimulated RAW264.7 cells with 4-HNE (10 μM) for 24 h. As expected, the abundance of cGAS mRNA did not change after 4-HNE stimulation (Supplementary Fig. S9A). Moreover, cGAMP and IFN-β levels were measured, which indicated that 4-HNE facilitated cGAMP and IFN-β production (Supplementary Figs. S9B and C). To further confirm that 4-HNE regulates cGAS degradation and stability, RAW264.7 macrophages were stimulated with cycloheximide (CHX) and 4-HNE for the indicated intervals and found that cGAS was rapidly degraded in RAW264.7 cells treated with CHX, an inhibitor of eukaryotic translational elongation and protein synthesis, while the levels of cGAS remained stable in the presence of 4-HNE (Supplementary Figs. S9D and E). Subsequently, K48-linked polyubiquitination of cGAS was detected, and less ubiquitination was co-immunoprecipitated with cGAS (IP), and increased cGAS protein levels (Input) were detected after 4-HNE stimulation in RAW264.7 cells (Fig. 6B). Consistent with this observation, 4-HNE treatment in HEK293T cells decreased the ubiquitination of cGAS on the K48-linked ubiquitin chain but not on the K63-linked ubiquitin chain (Fig. 6C).

Moreover, screening of cGAS-related DUBs by Co-IP assays demonstrated that endogenous cGAS and USP14 formed a complex; notably, this interaction was enhanced after 4-HNE stimulation in RAW264.7 cells and BMDMs (Fig. 6B, D). Similarly, Co-IP with USP14 Abs and western blotting with cGAS Abs further demonstrated the endogenous interaction between cGAS and USP14 in RAW264.7 cells (Fig. 6E). In addition, the interaction of cGAS and USP14 was determined by mass spectrometry (Fig. 6F and Supplementary Table S4), and the colocalization of cGAS and USP14 in RAW264.7 cells was verified by immunofluorescence (Fig. 6G).

To validate the effect of USP14 on cGAS ubiquitination after 4-HNE stimulation, HEK293T cells were co-transfected with plasmids encoding Flag-tagged cGAS, Myc-tagged K48-Ub, and HA-tagged USP14 (either WT or a catalytically inactive mutant, C114A). As expected, the overexpression of USP14 (WT) significantly inhibited cGAS K48-linked ubiquitination and increased cGAS levels after 4-HNE stimulation, whereas this effect was reversed by USP14 (C114A; Fig. 6H). Furthermore, RAW264.7 macrophages were treated with 4-HNE in the presence or absence of the USP14 inhibitor IU1. IFN-β and cGAMP levels in the medium were detected, and the results indicated that IU1 eliminated 4-HNE-induced IFN-β and cGAMP production (Supplementary Figs. S9F and G).

Subsequently, we analyzed whether the effects of ALDH2/4-HNE on macrophage polarization were dependent on USP14. Flow cytometry results demonstrated that IU1 decreased oxLDL-induced pro-inflammatory macrophage polarization and promoted IL-4/IL-13-triggered anti-inflammatory macrophage activation in ALDH2-KO and WT BMDMs (Fig. 6I, J and Supplementary Figs. S9H and I). In addition, ALDH2 regulated macrophage polarization, whereas it was not detectable in IU1-treated BMDMs (Fig. 6I, J and Supplementary Figs. S9H and I). Furthermore, the number of pro-inflammatory macrophages (F4/80+CD86+ subtype cells) increased in 4-HNE-treated BMDMs, whereas IU1 inhibited the formation of pro-inflammatory macrophages (Fig. 6K and Supplementary Fig. S9J).

To identify the domain(s) of cGAS or USP14 responsible for their interaction, we generated cGAS or USP14 truncation mutants and found that the interaction between cGAS and USP14 depended on the C terminus of cGAS and the catalytic domain of USP14 (Fig. 7A and B). Moreover, a cartoon representation of the complex structures with the predicted interaction interface of cGAS-USP14 was performed using the Hdock tool (Fig. 7C). The direct interaction between cGAS and USP14 was further confirmed by the GST pull-down assay (Fig. 7D) and surface plasmon resonance (SPR; Fig. 7E). To identify the specific ubiquitination sites of cGAS that are USP14 modulated, K414R, K285R, and K479R cGAS were constructed and co-transfected with or without HA-USP14 and Myc-K48-Ub. The results indicated that USP14 does not regulate K48 ubiquitination sites (K414, K285, and K479), as previously reported (Fig. 7F). To further confirm that the lysine sites on cGAS are involved in USP14 deubiquitination modification, HEK293T cells were cotransfected with plasmids encoding Flag-tagged human cGAS and Myc-tagged human K48-Ub. Anti-Flag (cGAS) immunoprecipitation (IP) was performed using whole-cell lysates derived from HEK293 cells. HPLC-MS/MS was used to identify the ubiquitination sites on the cGAS protein; K173, K275, K282, and K315 were identified. K173, K275, K282, and K315 mutant fragments of cGAS were constructed, which identified K282 as the key site for USP14 deubiquitination in cGAS (Fig. 7G and H).Fig. 7 USP14 directly interacts with cGAS and regulates the ubiquitination of cGAS at K282. A HEK293T cells transfected with the indicated constructs and Flag-cGAS were subjected to Co-IP assays with anti-Flag antibody (n = 3). B HEK293T cells transfected with the indicated constructs and HA-USP14 were subjected to Co-IP assays with anti-Flag antibody (n = 3). C Visualization of USP14-cGAS interacting interface prediction using the Hdock tool. The cartoon shows the complex structures with the predicted interaction residues. D Recombinant His-USP14 was incubated with GST or GST-cGAS in the presence or absence of 4-HNE before GST pull-down assay with GST beads (n = 3). E Representative dose-response surface plasmon resonance (SPR) sensorgrams of the binding of an increasing amount of cGAS to USP14 ligand captured on a CM5 chip. F HEK293T cells were transfected with Myc-K48-Ub and Flag-cGAS or its single site mutation, with or without HA-USP14. Cell lysates were collected, and Co-IP assays were performed (n = 3). G Mass spectrometry (MS) of cGAS ubiquitination sites at K282. H HEK293T cells were transfected with Myc-K48-Ub and Flag-cGAS or its single site mutation, with or without HA-USP14. Cell lysates were collected, and Co-IP assays were performed (n = 3). Western blot bands were detected under the same conditions (A, B, F, H).

Fig. 7

Taken together, these data suggest that 4-HNE prevents K48 polyubiquitination-dependent protein degradation of cGAS by enhancing the USP14-cGAS interaction, which may be an important mechanism by which ALDH2 regulates the cGAS-STING signaling pathway and macrophage polarization.

As shown in Fig. 4E and F, ALDH2-KO or ALDH2 rs671 mutations upregulated basal cGAS protein levels. These observations prompted us to hypothesize that ALDH2 may exert a non-enzymatic function, presumably by binding to cGAS and facilitating its degradation. To test this hypothesis, Co-IP experiments were initially performed in HEK293 cells transfected with Myc-ALDH2 (WT; rs671) and Flag-cGAS. As shown in Supplementary Fig. S10A, cGAS and ALDH2 physically bind together, and the ALDH2 rs671 mutation attenuated binding with cGAS (Supplementary Fig. S10A). Endogenous ALDH2 and cGAS interacted in RAW 264.7 macrophages, which was enhanced after oxLDL stimulation (Supplementary Fig. S10B). These findings may represent a novel molecular mechanism by which ALDH2 interacts directly with cGAS.

3.8 USP14-specific knockdown in macrophages promotes the anti-inflammatory macrophage phenotype and alleviates the atherosclerotic development

To identify whether macrophage-specific USP14 knockdown inhibits the activation of pro-inflammatory macrophages and atherosclerosis development, bone marrow transplantation was performed to knock down USP14 expression in macrophages of ApoE−/− mice (Fig. 8A). After a 2-week recovery period, the USP14 deletion was validated in primary peritoneal macrophages by western blotting (Fig. 8B). The effects of USP14 on macrophage polarization in vivo were measured, which indicated a decrease in pro-inflammatory macrophage markers (iNOS, CXCL-10, IL-6, IL-1β, and TNF-α) in the aortic tissue of macrophage-specific USP14 knockdown mice (Fig. 8C). In addition, USP14 deficiency in macrophages promoted an anti-inflammatory macrophage phenotype (Fig. 8C). To further characterize the macrophage population, cells from the spleens of ApoE−/− mice were isolated, and flow cytometric analysis was performed. Total macrophages were defined as CD45+Ly6G−CD11b+F4/80+ cells. CD45+Ly6G−CD11b+F4/80+CD86+CD163− represented pro-inflammatory macrophages, and CD45+Ly6G−CD11b+F4/80+CD86−CD163+ represented anti-inflammatory macrophages. The results showed that anti-inflammatory macrophages were more prominent and pro-inflammatory macrophages were less prominent in the USP14-deficiency groups (Fig. 8D–G).Fig. 8 USP14-specific knockdown in macrophages promotes an anti-inflammatory macrophage phenotype. A Schematic diagram of bone marrow transplantation. B Primary peritoneal macrophages were isolated to verify the bone marrow clearance efficiency. C RT-qPCR of iNOS, CXCL-10, TNF-α, IL-1β, IL-6, TGF-β, and IL-10 in the aortas of ApoE−/− mice (n = 6–7). D-G Flow cytometry plots and quantitative analysis of macrophage differentiation in the spleens of ApoE−/− mice (n = 7). H, I Representative photographs of Oil red O staining and their statistic graphs (n = 7). J, K Representative photographs of hematoxylin and eosin staining and their statistic graphs (n = 7). L, M Representative photographs of CD68 immunofluorescence staining and their statistic graphs (n = 7). N, O Representative photographs of Masson staining and their statistic graphs (n = 7). P, Q Representative photographs of α-SMA immunofluorescence staining and their statistic graphs (n = 7). R Changes of lipid profiles in the mouse serum (n = 6–7). TG: triglyceride; CHO: cholesterol; HDL: high-density lipoprotein; LDL: low-density lipoprotein. Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (E, F, I, K, M, O, Q, R). One-way ANOVA was used for the analysis in C: iNOS, CXCL-10, TNF-α, IL-6, TGF-β, IL-10. Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test was used for the analysis in C: IL-1β. Kruskal-Wallis test followed by Dunn's post hoc test was used for the analysis in (G). Western blot bands were detected under the same conditions (B). P < 0.05 was considered significant.

Fig. 8

To examine the effect of macrophage USP14 on atherosclerotic development, sections of the aortic root were stained with Oil red O and hematoxylin and eosin. Transplantation of USP14-knockdown bone marrow cells significantly alleviated atherosclerotic plaque formation (Fig. 8H–K). The macrophage infiltration was significantly decreased in the ApoE−/− mice transplanted with USP14-knockdown bone marrow cells (Fig. 8L and M). In contrast, the collagen contents and smooth muscle cell components in plaques were markedly increased in the macrophage-specific USP14 knockdown ApoE−/− mice (Fig. 8N–Q). However, USP14 knockdown in bone marrow cells did not alter the serum concentrations of TG (triglyceride), CHO (cholesterol), HDL (high-density lipoprotein), and LDL (low-density lipoprotein) in ApoE−/− mice (Fig. 8R). Taken together, these results suggested that USP14-specific knockdown in macrophages alleviated the progression of atherosclerosis and inflammation.

4 Discussion

The relationship between ALDH2 and macrophage polarization in atherosclerosis remains poorly understood. This study was the first to provide conclusive evidence on the protective role of ALDH2 in atherosclerosis progression through inhibiting the pro-inflammatory macrophage polarization. Mechanistically, ALDH2 promoted the degradation of 4-HNE, resulting in the inhibition of the cGAS-STING signaling pathway by suppressing USP14-dependent cGAS deubiquitination and stability. Our findings identified the translational potential of targeting the ALDH2-USP14-cGAS pathway for atherosclerosis prevention.

Recent animal studies and human data have reported the enzymatic and non-enzymatic roles of ALDH2 in protecting against CVD [8]. The non-enzymatic roles of ALDH2 in regulating cholesterol metabolic processes have also been reported [23,24]. ALDH2 interacts with PARP1 and reduces the nuclear translocation of PARP1, which results in depressed LXRα, elevated levels of ABCA1, and increased HDL biogenesis [23]. Conversely, ALDH2 rs671 mutation or ALDH2 deficiency increases PARP1 nuclear translocation and decreases HDL biogenesis [23]. ALDH2 promotes the formation of the Insig1/gp78–3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) complex after translocation from the mitochondria to the endoplasmic reticulum [24]. This results in the ubiquitination and degradation of HMGCR, thereby limiting cholesterol synthesis [24]. In contrast, the presence of the ALDH2 rs671 mutation increases cholesterol levels by stabilizing HMGCR [24]. In addition to its non-enzymatic roles, ALDH2 functions as an enzyme that detoxifies 4-HNE. Impaired redox homeostasis-induced lipid peroxidation and its product 4-HNE promotes the pathological remodeling of atherosclerosis [8]. 4-HNE can easily cross the membrane and adducts with cellular macromolecules such as proteins and nucleic acids for regulating signaling pathways [25]. A moderate concentration of 4-HNE (1–10 μM) in the monocytes and macrophages elicits inflammatory signaling through the TLR4/NF-κB-dependent pathway [26] and stimulates the formation of macrophage foam cells, possibly by increasing the expression of CD36 [27]. High concentrations of 4-HNE (100 μM) promotes macrophage apoptosis [10]. 4-HNE (1 μM) activates p38 MAPK and ERK, which in turn activates the 5-LO-LTB4-BLT receptor pathway, resulting in the production of MMP-2 in smooth muscle cells, consequently leading to atherosclerotic plaque instability [28]. Cys-73 modification of Trx1 by 4-HNE is sufficient to stimulate monocyte adhesion to endothelial cells, which in turn contributes to the early events of atherosclerosis [29]. The foam cell-released 4-HNE activates PPARδ in vascular endothelial cells, leading to increased TXNIP expression and consequently to senescence [30]. The formation of 4-HNE-SIRT1 protein adducts and the subsequent decrease in p53 deacetylation are mediated by SIRT1 and eventually promote p53-dependent vascular endothelial cell senescence [12,31]. Our findings emphasize that ALDH2 protects against atherosclerosis by regulating macrophage polarization and inflammation, further contributing to our understanding of the underlying mechanism. Because daidzin is the most widely accepted tool for ALDH2 inhibition [32,33], this drug was used in our study to suppress ALDH2 and increase pro-inflammatory macrophage polarization. However, daidzin is not a specific ALDH2 inhibitor. Therefore, ALDH2-KO mouse primary macrophages were simultaneously used in our study, and our findings demonstrated that ALDH2 deficiency enhanced pro-inflammatory macrophage phenotype and impaired anti-inflammatory macrophage phenotype, which is consistent with the results of daidzin. Notably, our study explored the specific molecular mechanism by which ALDH2 regulates macrophage polarization to discover other potential therapeutic approaches for atherosclerosis.

The cGAS-STING signaling pathway, a component of innate immunity has gained considerable attention for its crucial role in non-infectious inflammatory diseases in multiple systems, in addition to its role in resistance to pathogenic infections [34]. Recently, the pivotal role of the cGAS-STING pathway in shaping immune surveillance by macrophages has been recognized, particularly in cardiovascular diseases [35]. For example, Cao et al. reported that cGAS gene deletion significantly reduced the number of M1-type macrophages and increased the number of M2-type macrophages in the infarcted area of a mouse model of acute myocardial infarction in the heart tissue, thereby promoting myocardial tissue repair [35]. Our findings highlighted that the cGAS-STING pathway is upregulated in the macrophages of atheromatous lesions in humans and mice. Moreover, suppression of the cGAS-STING pathway reduced the pro-inflammatory macrophages proportion but increased the anti-inflammatory macrophage proportion. Moreover, our findings demonstrated an association between the cGAS-STING pathway and ALDH2 in the progression of atherosclerosis and elucidated that ALDH2 inhibits the cGAS-STING pathway in atherosclerosis. Notably, ALDH2 regulates macrophage polarization and inflammation through the cGAS-STING signaling pathway.

Defects in cGAS mitigate the progression of atherosclerosis and plaque vulnerability, emphasizing the importance of cGAS protein deficiency in treating atherosclerosis [15]. cGAS activity is regulated by phosphorylation, glutamylation, SUMOylation, and acetylation [36]. However, further research is needed to develop chemicals that can alter the ubiquitination of cGAS to promote protein degradation. For example, cGAS K48-linked ubiquitination degradation prevents high levels of cGAS protein in resting cells, leading to inflammatory response inhibition [37], and this ubiquitination modification can be controlled and directed toward beneficial effects. TRIM14 recruits USP14 to cleave the K48-linked ubiquitination of cGAS at K414, thereby inhibiting cGAS degradation to enhance antiviral immunity [37]. However, the role of USP14 in directly interacting with cGAS for regulating cGAS deubiquitination and stabilizing the cGAS protein remains unclear. The exciting discovery from our current study is that 4-HNE prevented K48-polyubiquitination of cGAS at K282 and stabilized cGAS protein by enhancing the USP14-cGAS interaction. Intriguingly, a recent study using the USP14 inhibitor IU1 demonstrated that IU1 suppressed foam cell formation by promoting CD36 degradation [38]. However, the precise role of USP14 in the regulation of macrophage polarization remains poorly understood. Our study was the first to report that IU1 suppresses oxLDL/4-HNE-induced pro-inflammatory macrophage polarization while promoting IL-4/IL-13-triggered anti-inflammatory macrophage activation by promoting cGAS degradation. However, USP14 has been reported as a pro-atherosclerotic factor by controlling smooth muscle cells proliferation, migration, and phenotypic modulation [39] and as an anti-atherosclerotic factor by controlling endothelial activation and inflammation [40]. The results of the current study clearly showed that USP14 knockdown, specifically in macrophages, significantly increased the anti-inflammatory macrophage phenotype and suppressed macrophage inflammatory responses. Therefore, targeting USP14 in macrophages may be a promising therapeutic option for atherosclerosis.

The shortcoming of this study is the low number of human plaque samples and the corresponding healthy controls. Because human plaques are highly heterogeneous, a larger sample size is required to fully demonstrate the activation of cGAS-STING pathway and the accumulation of 4-HNE in plaques. The expression of the ALDH2 and cGAS-STING pathways in plaques could not be well supported by comparing gene data for 78-week-old ApoE−/− mouse plaques with the corresponding adventitia (GSE10000) due to confounding factors (essentially comparing two different tissue types with different cell populations).

In conclusion, our study reveals the effects of ALDH2 on macrophage polarization in atherosclerosis. ALDH2 promotes the degradation of 4-HNE, resulting in the inhibition of cGAS deubiquitination and stability mediated by USP14 to negatively regulate the cGAS-STING pathway, thereby reducing the pro-inflammatory reprogramming of macrophages and atherosclerotic plaque formation (Fig. 9). This finding describes a novel molecular mechanism contributing to atherosclerosis in individuals with ALDH2 rs671 mutations, implying that the USP14-dependent cGAS ubiquitination may provide potential intervention targets for atherosclerosis.Fig. 9 Working model of ALDH2 in regulating macrophage polarization. In ALDH2-deficient or ALDH2 rs671-mutant macrophages, 4-HNE, a product of impaired redox homeostasis, accumulates and promotes proinflammatory reprogramming of macrophages and atherosclerotic plaque formation by enhancing the interaction of USP14 with cGAS and preventing the K48 polyubiquitination-dependent cGAS protein degradation at K282.

Fig. 9

CRediT authorship contribution statement

Haiying Rui: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Huaxiang Yu: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Kai Chi: Validation, Methodology, Investigation, Formal analysis. Ziqi Han: Validation, Methodology, Investigation, Formal analysis. Wenyong Zhu: Validation, Methodology, Investigation, Formal analysis. Jian Zhang: Validation, Methodology, Investigation, Formal analysis. Haipeng Guo: Validation, Methodology, Investigation, Formal analysis. Wenyi Zou: Validation, Methodology, Investigation, Formal analysis. Fengxin Wang: Validation, Methodology, Investigation, Formal analysis. Ping Xu: Validation, Methodology, Investigation, Formal analysis. Dan Zou: Validation, Methodology, Investigation, Formal analysis. Xiaoshuai Song: Validation, Methodology, Investigation, Formal analysis. Lulu Liu: Validation, Methodology, Investigation, Formal analysis. Xuting Wu: Validation, Methodology, Investigation, Formal analysis. Wenxiao Wu: Validation, Methodology, Investigation, Formal analysis. Dandan Qin: Validation, Methodology, Investigation, Formal analysis. Yihai Cao: Validation, Methodology, Investigation, Formal analysis. Feng Xu: Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation. Li Xue: Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation. Yuguo Chen: Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

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Data availability

Data will be made available on request.

Acknowledgements

This study was supported by the State Key Program of the 10.13039/501100001809 National Natural Science Foundation of China (82030059 ), 10.13039/501100001809 National Natural Science Foundation of China (82172178 , 82072144 , 81873950 , 81873953 , 81300219 , and 81671951 ), National Key R&D Program of China (2020YFC1512700 , 2020YFC1512705 , and 2020YFC1512703 ), National S&T Fundamental Resources Investigation Project (2018FY100600 , 2018FY100602 ), Key R&D Program of Shandong Province (2019GSF108131 , 2021ZLGX02 , 2022ZLGX03 ), Natural Science Foundation of Shandong Province (ZR2022MH078 ), Taishan Young Scholar Program of Shandong Province (tsqn202103173 ), Taishan Pandeng Scholar Program of Shandong Province (tspd20181220 ), Youth Top-Talent Project of National Ten Thousand Talents Plan. Thanks to medical statistician Shuo Wu for supporting the statistical work of this study.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103318.
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