Yin‐Yang 1/Neural Precursor Cell‐Expressed Developmentally Downregulated 4‐Like Axis Suppresses Mer Tyrosine Kinase‐Mediated Macrophage Efferocytosis to Exacerbate Atherosclerosis Via Triggering Pyroptosis

Abstract

Background

Atherosclerosis is considered as a major contributor for cardiovascular disease with high morbidity and mortality globally. However, the cross‐talk between efferocytosis and inflammation in atherosclerosis remains elusive.

Methods

ApoE (apolipoprotein E)^−/− mice and oxidized low‐density lipoprotein‐induced in vitro atherosclerosis models were established. The histological changes and lipid accumulation in arteries were evaluated by hematoxylin and eosin and Oil red O staining. Efferocytosis was monitored by in situ or in vitro efferocytosis assay. Western blot, immunohistochemistry, and ELISA were employed to detect the expression of target molecules and pyroptosis‐related molecules. Caspase‐1/propidium iodide was used to assess cell pyroptosis in macrophages. The interaction between NEDD4L (neural precursor cell‐expressed developmentally downregulated 4‐like) and MerTK (Mer tyrosine kinase), as well as the ubiquitination of MerTK was examined by co‐immunoprecipitation. Additionally, the interaction between YY1 (Yin Yang 1) and NEDD4L promoter was detected by chromatin immunoprecipitation and luciferase assays.

Results

YY1 and NEDD4L were upregulated, but MerTK was downregulated in the arteries of ApoE^−/− mice. The addition of apoptotic cells deteriorated atherosclerosis through activating NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3)‐mediated inflammation and pyroptosis. Silencing of MerTK exacerbated atherosclerosis via suppressing efferocytosis and activating NLRP3‐mediated inflammation and pyroptosis, whereas NEDD4L knockdown or YY1 silencing exerted opposite effects in the in vitro atherosclerosis model. Mechanistically, NEDD4L was identified as an E3 ligase responsible for MerTK degradation, and YY1 served as a transcriptional activator of NEDD4L.

Conclusions

Our findings demonstrated that YY1 positively regulated NEDD4L to modulate MerTK‐mediated efferocytosis and activate NLRP3‐mediated inflammation and pyroptosis, thus exacerbating atherosclerosis.

Nonstandard Abbreviations and Acronyms

AC

apoptotic cells

MerTK

Mer tyrosine kinase

YY1

Yin‐Yang 1

Research Perspective.

What Is New?

• This study uncovers how YY1 (Yin Yang 1) regulates NEDD4L (neural precursor cell‐expressed developmentally downregulated 4‐like) to modulate MerTK (Mer tyrosine kinase)‐mediated efferocytosis.

• Efferocytosis activates NLRP3 (Nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3)‐mediated inflammation and pyroptosis, thus deteriorating atherosclerosis.

What Question Should Be Addressed Next?

• YY1/NEDD4L/MerTK axis was implicated in the regulation of efferocytosis and NLRP3 inflammasome activation in atherosclerosis, providing novel insights into anti‐inflammatory therapy for atherosclerosis.

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally. ¹ Atherosclerosis (atherosclerosis) is characterized by inflammation, cholesterol, and low‐density lipoprotein (LDL) accumulation in the arteries, and it is considered as a major contributing factor for CVDs, such as stroke and myocardial infarction. ² Current therapies for Atherosclerosis aim to control the risk factors, including antihypertensive, antihyperglycemic, and lipid‐lowering therapeutic strategies. ³ In recent years, novel lipid‐lowering therapies, such as PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors and the hepatic PCSK9 synthesis inhibitor inclisiran, could improve clinical outcomes of atherosclerosis. ⁴ , ⁵ , ⁶ However, inadequate LDL‐cholesterol reduction and failure in the prevention of atherosclerosis progression remain challenges for lipid‐lowering therapies. ⁷ Accumulating evidence supports the pivotal role of inflammation in atherosclerosis development and progression. ⁸ , ⁹ It is well established that increased immune cells, including macrophages, dendritic cells, lymphocytes, foam cells, and other inflammatory cells are observed in the atherosclerotic lesions. These cells contribute to the chronic inflammatory responses in atherosclerosis; therefore, anti‐inflammatory therapy has emerged as a promising approach for atherosclerosis treatment. ¹⁰ , ¹¹ Currently, several anti‐inflammatory agents have offered benefits to patients with atherosclerosis. ¹² , ¹³ Elucidating the mechanism underlying the inflammatory responses in atherosclerosis will provide novel insights into the developments in anti‐inflammatory therapy and improve the clinical outcomes.

In the early stage of atherosclerosis, the dysfunction and death of endothelial cells lead to the release of proinflammatory chemokines and cytokines, as well as the recruitment of monocytes. Monocytes differentiate to macrophages, and lesional macrophages were then take up by oxidized LDLs (ox‐LDL) to form foam cells, thereby triggering necrotic cores formation in atherosclerotic plaques. ² In advanced plaques, macrophages contribute to the clearance of apoptotic cells (ACs), namely efferocytosis, and this process is recognized as a resolution of inflammation. ¹⁴ , ¹⁵ , ¹⁶ Defective efferocytosis in atherosclerosis results in accumulation of ACs within the plaque and inflammatory responses. ¹⁶ More important, a novel programmed cell death pyroptosis is closely related to inflammation. ¹⁷ It is generally accepted that pyroptosis is implicated in the development and progression of atherosclerosis. ¹⁸ , ¹⁹ However, little is known about the cross‐talk between efferocytosis and pyroptosis in atherosclerosis.

MerTK (Mer tyrosine kinase) is a tyrosine kinase receptor in phagocytic cells that mediates efferocytosis of ACs with the help of growth arrest‐specific gene 6, which bridges phosphatidylserine on the plasma membrane of ACs with MerTK. ²⁰ In the mouse model of atherosclerosis, mutation of MerTK impairs efferocytosis and facilitates AC accumulation and plaque necrosis. ²¹ Emerging evidence illustrates that MerTK may serve as a promising regulator in atherosclerosis. ²² , ²³ , ²⁴ , ²⁵ NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3) inflammasome senses the injury and activates caspase‐1, leading to the secretion of IL (interleukin)‐18/IL‐1β and pyroptosis. ²⁶ Compelling evidence supports the critical role of NLRP3 and pyroptosis in CVD, ²⁷ Recent researches and clinical trials have illustrated that either directly targeting IL‐1β (eg, canakinumab) or upstream inhibition via NLRP3 inflammasome improve cardiac function. ²⁸ , ²⁹ , ³⁰ , ³¹ However, the upstream regulatory axis of NLRP3 inflammasome in atherosclerosis remains largely undefined. A recent study has demonstrated that MerTK suppresses NLRP3 inflammasome activation after subarachnoid hemorrhage through inducing autophagy, ³² raising the possibility that dysregulation of MerTK might cause defective efferocytosis to activate NLRP3 inflammasome, thereby facilitating the formation of necrotic cores.

NEDD4L (neural precursor cell‐expressed developmentally downregulated 4‐like), a member of NEDD4 family, belongs to HECT family of E3 ubiquitin ligase, which is responsible for the degradation of multiple membrane proteins. ³³ Recently, a number of studies have demonstrated that NEDD4L‐associated axes act as key players in CVD, such as hypertension, arrhythmia, and atherosclerosis. ³⁴ , ³⁵ For instance, NEDD4L is implicated in miR‐30a‐5p‐attenuated atherosclerosis by modulating M1/M2 ratio of macrophages and ox‐LDL uptake. ³⁵ In addition, neovascularization in atherosclerotic lesions contributes to plaque progression and destabilization. ³⁶ It has reported that NEDD4L also promotes angiogenesis, cell migration, and growth in endothelial cellss, ³⁷ suggesting the multifunctionality of NEDD4L in atherosclerosis. Previous study has illustrated that NEDD4 suppresses NLRP3 activation and pyroptosis in macrophages. ³⁸ However, whether NEDD4L also served as a negative regulator of NLRP3 inflammasome‐mediated pyroptosis remains uninvestigated. Notably, NEDD4L was identified as an E3 ubiquitin ligase of MerTK by UbiBrowser. We hypothesized that NEDD4L might catalyze the ubiquitination of MerTK to regulate efferocytosis in atherosclerosis.

YY1 (Yin‐Yang 1) is a zinc finger transcription factor that is involved in the regulation of lipid metabolism in liver cells. ³⁹ , ⁴⁰ More important, elevation of phosphor‐YY1 at S118 in endothelial cells promotes atherosclerosis through modulating HDM2 (human double minute 2). ⁴¹ Additionally, lack of YY1 ameliorates ox‐LDL‐induced macrophage inflammation and lipid accumulation via PCSK9/LDLR (LDL receptor) pathway. ⁴² Our preliminary bioinformatics analysis predicted that YY1 might serve as a transcription factor of NEDD4L, promoting us to hypothesize the YY1 functions as an upstream regulator of NEDD4L/MerTK axis in atherosclerosis.

In this study, we reported that YY1 and NEDD4L were upregulated, but MerTK was downregulated in the arteries of ApoE^−/− (apolipoprotein E) mice. The addition of ACs deteriorated atherosclerosis through activating NLRP3‐mediated inflammation and pyroptosis. Gain‐ and loss‐of‐function studies revealed that silencing of MerTK exacerbated atherosclerosis via suppressing efferocytosis and activating NLRP3‐mediated inflammation and pyroptosis, whereas NEDD4L knockdown or YY1 silencing exerted opposite effects in the in vitro atherosclerosis model. Mechanistically, NEDD4L mediated the ubiquitin‐proteasomal degradation of MerTK, and YY1 served as a transcriptional activator of NEDD4L. In summary, YY1 regulated NEDD4L to modulate MerTK‐mediated efferocytosis and activate NLRP3‐mediated inflammation and pyroptosis, thus exacerbating atherosclerosis. These findings delineated the cross‐talk between efferocytosis and inflammation in atherosclerosis, and shed light on the anti‐inflammatory therapy for atherosclerosis.

METHODS

Data Availability Statement

The authors declare that all data and supporting materials are available within the article and its supplemental material.

Animal Study

Male C57BL/6J (Sham) mice or apoE^−/− mice (n=6 per group, 6‐week‐old) were from Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed at room temperature with 12‐hour/12‐hour light/dark cycle. C57BL/6J and apoE^−/− mice received a high‐fat diet for 10 weeks as described. ²⁴ All animal studies were approved by the Second Affiliated Hospital of Nanchang University (approval number: 2023‐0077).

Histological Analysis

The aortic tissues were fixed with 4% paraformaldehyde. The sections of aorta were stained with hematoxylin and eosin (Sigma‐Aldrich, St. Louis, MO, USA). To evaluate plaque area and lipid accumulation, the aortic tissues were stained with Oil red O stain solution (Sigma‐Aldrich). Images were acquired under a microscopy (Nikon, Tokyo, Japan).

In Situ Efferocytosis Assay

In situ efferocytosis assay was performed. ⁴³ In brief, sections were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X‐100. DNA fragmentation was assessed using Click‐iT Plus TUNEL Assay Kit, Alexa Fluor 488 (C10617, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Sections were blocked with 5% BSA and incubated with anti‐CD68 (1:100, ab125212, Abcam, Cambridge, UK) and anti‐MerTK (1:100, 14‐5751‐82, Invitrogen) antibodies at 4 °C overnight. Slides were incubated with Alexa Fluor 555‐anti‐rabbit (1:500, A31572, Invitrogen) and Alexa Fluor 488 anti‐rat antibodies (1:500, A11006, Invitrogen). Images were acquired under a confocal microscope (Nikon). Macrophage‐associated ACs were calculated as: CD68⁺TUNEL⁺ cells/total CD68⁺ cells×100%.

Immunofluorescent and Immunohistochemistry Staining

For immunofluorescent staining, sections were deparaffined and rehydrated, and subjected to antigen retrieval. Slides or cells were then blocked and incubated with primary antibodies at 4 °C overnight, followed by the incubation with secondary antibodies. For immunohistochemistry analysis, sections were incubated with primary antibodies at 4 °C overnight and incubated with secondary antibody horseradish peroxidase. Signals were visualized using DAB substrate (P0203, Beyotime, Shanghai, China), and images were detected by microscopy (Nikon). Antibodies used in immunofluorescent or immunohistochemistry analysis: anti‐CD68 (1:100, ab125212, Abcam), anti‐YY1 (1:100, ab109237, Abcam), anti‐NEDD4L (1:200, A302‐513A, Bethyl Laboratories, Montgomery, TX, USA), anti‐MerTK (1:100, ab250715, Abcam), anti‐NLRP3 (1:100, ab263899, Abcam), anti‐ASC (apoptosis‐associated speck‐like protein containing a caspase recruitment domain; 1:500, ab309497, Abcam), anticleaved caspase‐1 (1:100, PA599390, Invitrogen) and anti‐GSDMD‐N (gasdermin D N‐terminal domain) (1:200, PA5115330, Invitrogen) antibodies.

ELISA Assay

Whole mouse blood was harvested and allowed to clot at room temperature. Serum was collected after centrifugation. The serum levels of mouse IL‐18 and IL‐1β were measured using ELISA kits (IL‐18, KMC0181; IL‐1β, BMS6002‐2, Invitrogen) following the instructions. The serum MerTK level was measured using Mouse Mer DuoSet ELISA (DY591, R&D Systems, Minneapolis, MN, USA). A450 was quantified using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Primary Bone Marrow‐Derived Macrophages Isolation, Culture and Treatment

Primary bone marrow‐derived macrophages were isolated as previously described. ⁴³ Briefly, both mouse femurs and tibias were dissected and passed through a 100 μm‐cell strainer. Cells were then spun down and rinsed with PBS and cultured in RPMI1640 containing 10% FBS and 10 ng/mL M‐CSF (Gibco, Grand Island, NY, USA) for 5 days to differentiate into macrophages. For foam cell formation, bone marrow‐derived macrophages were treated with 0, 25, 50, or 100 μg/mL ox‐LDL (L3486, Invitrogen) for 48 hours. ⁴⁴ , ⁴⁵ Cells were then fixed with 4% paraformaldehyde and subjected to Oil red O staining as described. ⁴³ To block ubiquitin‐proteasome pathway, macrophages were incubated with proteasome inhibitor MG132 (20 μM, Sigma‐Aldrich) for 48 hours. For protein stability assay, macrophages were incubated with cycloheximide (50 μg/mL, Sigma‐Aldrich) for 0, 15, 30, 60, 120, or 240 minutes.

Cell Transfection

Lentiviruses containing sh‐MerTK, sh‐NEDD4L, sh‐YY1, or negative control (sh‐NC), were from GenePharma. macrophages were plated into 6‐well plates (8×10⁵ cells/well) with ∼70% confluence were infected with the specific lentiviruses supplemented with 4 μg/mL polybrene at a multiplicity of infection of 20.

In Vitro Efferocytosis Assay

In vitro efferocytosis assay was carried out as described with modifications. ²⁴ , ⁴⁶ macrophages were treated with vehicle control or 50 μg/mL ox‐LDL for 48 hours. Human Jurkat cells were obtained from ATCC (Manassas, VA, USA) and grown in RPMI1640 containing 15% FBS at 37 °C/5% CO₂. Jurkat cells were labeled with phycoerythrin (1:100, 12‐4801‐82, Invitrogen) or PKH26 (PKH26GL, Sigma‐Aldrich), and macrophages were stained with anti‐F4/80 antibody, labeled with CFSE (C1157, Invitrogen) or PKH67 (PKH67GL, Invitrogen). To induce apoptosis, Jurkat cells were exposed to 30 mJ/cm² UV for 15 minutes and incubated under normal condition for 1 hour. After culture, the apoptotic rate of ACs were examined by flow cytometry following Annexin V/propidium iodide (PI) staining, and ~80% of the Jurkat cells were apoptotic (Figure S1A). These cells were then cocultured with bone marrow‐derived macrophages at a 5:1 ratio for 45 minutes. ²⁵ , ⁴⁷ Images were acquired using a confocal microscope (Nikon). The efferocytosis index was calculated as macrophages that engulfed ACs/total macrophages×100%.

Western Blot

Protein lysates were prepared using RIPA lysis buffer (Pierce), quantified using BCA Protein Kit (Pierce) and separated by SDS‐PAGE. Proteins were then transferred onto NC membrane (Beyotime) and blocked with 5% nonfat milk. The blots were incubated with primary antibody at 4 °C overnight, and incubated with secondary antibody for 1 hour. Signals were detected using SuperSignal Pico‐PLUS chemiluminescent substrate (Pierce). Primary antibodies used in western blot: anti‐YY1 (1:2000, ab109237, Abcam), anti‐NEDD4L (1:2000, A302‐513A, Bethyl Laboratories), anti‐MerTK (1:1000, ab250715, Abcam), anti‐NLRP3 (1:1000, ab263899, Abcam), anti‐ASC (1:1000, ab309497, Abcam), anticleaved caspase‐1 (1:1000, PA599390, Invitrogen), anti‐GSDMD‐N (1:1000, PA5115330, Invitrogen), anti‐HA (hemagglutinin) tag (1:2000, 26 183, Invitrogen) and β‐actin (1:2000, ab8226, Abcam) antibodies.

Flow Cytometry

Cell pyroptosis was detected using caspase‐1 and propidium iodide staining. Cells were incubated with the caspase‐1 probe at 37 °C for 1 hour, followed by the PI staining for 10 minutes. The percentage of caspase‐1‐ or/and PI‐positive cells was analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).

CoImmunoprecipitation

Cells were lysed using immunoprecipitation lysis buffer (Pierce), and 1 mg protein lysates were incubated with anti‐NEDD4L (2 μg, A302‐513A, Bethyl Laboratories), anti‐MerTK (2 μg, ab250715, Abcam) antibody, or normal rabbit IgG at 4 °C overnight. The immune complex was then enriched by Protein A/G agarose (Pierce) at 4 °C for 4 hours, followed by elution and detection. The protein levels of NEDD4L, MerTK, and HA‐tagged ubiquitin were assessed by western blot.

Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation assay was conducted using Pierce Magnetic ChIP kit (26157, Pierce). Macrophages were cross‐linked with 1% formaldehyde and lysed with Membrane Extraction Buffer. This was followed by MNase digestion and sonication. The chromatin fragments were incubated with anti‐YY1 antibody (2 μg, ab109237, Abcam) or normal rabbit IgG at 4 °C overnight. The complexes were enriched by Protein A/G magnetic beads, and DNA was then extracted and analyzed by real‐time quantitative polymerase chain reaction, which was performed using SYBR Green PCR MasterMix (ABI, Foster City, CA, USA). The level of NEDD4L BS1 or BS2 was calculated using 2^−ΔΔCT method.

Luciferase Reporter Assay

The promoter region of NEDD4L containing BS1 (−1759/−1748) and BS2 (−1077/−1066) was cloned into pGL3 (Promega, Madison, WI, USA) and designated as wild‐type NEDD4L. The mutated NEDD4L was also cloned luciferase vector. The 293T cells or macrophages were cotransfected with sh‐NC/sh‐YY1 and luciferase construct, and luciferase activity was quantified using Dual Luciferase Reporter System (Promega) at 48 hours post transfection.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 9 software. Each experiment was performed in triplicate and expressed as the mean±SD. Normality was assessed using the Shapiro–Wilk test and all the data meet the assumption of normal distribution. For cellular study, 3 biologically independent replicates (n=3) were conducted, with triplicate technical replicates within each biological replicate. For animal study, 6 animals (n=6) were included in each group. The differences among multiple groups or between 2 groups were analyzed by 1‐way ANOVA or Student’s t test, respectively. P<0.05 was considered to be statistically significant.

RESULTS

YY1 and NEDD4L Are Upregulated, But MerTK Is Downregulated in the Aortic Tissues of ApoE ^−/− Mice

To investigate the role of YY1/NEDD4L/MerTK axis in atherosclerosis, the expression of these molecules in mouse aortic tissues were examined. As presented in Figure 1A and 1B, clear plaque and lipid core were observed in apoE^−/− mice aorta, along with increased lipid accumulation as detected by Oil red O staining. Additionally, MerTK⁺ cells were decreased, whereas CD68⁺ cells were increased, which was accompanied with the reduction of MerTK⁺CD68⁺ cells in the atherosclerosis group (Figure 1C). In situ efferocytosis assay revealed the defective efferocytosis in the aortic plaque of apoE^−/− mice in which the percentage of macrophage‐associated ACs (TUNEL⁺ CD68⁺) were decreased in apoE^−/− mice, compared with that of sham mice (Figure 1C). Immunofluorescent staining showed that YY1 and NEDD4L were elevated, whereas MerTK was decreased in CD68⁺ macrophages of apoE^−/− mice (Figure 1D). This was accompanied with the upregulation of pyroptosis‐related proteins, including NLRP3, ASC, c‐caspase‐1 (cleaved caspase‐1,) and GSDMD‐N, in the aortic tissues of apoE^−/− mice (Figure 1E). In line with these findings, ELISA assay further showed the increased serum levels of IL‐18 and IL‐1β in apoE^−/− mice, whereas serum MerTK level was not significantly elevated (Figure 1F). These data indicate that YY1 and NEDD4L are upregulated, but MerTK is downregulated in apoE^−/− mice aortic tissues, along with the suppression of efferocytosis and enhanced pyroptosis in apoE^−/− mice.

Figure 1. YY1 and NEDD4L are upregulated, but MerTK is downregulated in the aortic tissues of apoE^−/− mice.

Mice were randomly divided into 2 groups (n=6 per group): sham and apoE^−/− groups. A, Histological changes of aorta were evaluated by H&E staining. Scale bar, 50 μm. B, Lipid accumulation was monitored by Oil red O staining. Scale bar, 50 μm. C, The in situ expression of MerTK and CD68 was examined by immunofluorescent staining. The phagocytic activity of macrophages was assessed by in situ efferocytosis assay. Green, TUNEL; red, CD68, blue, DAPI. Scale bar, 50 μm. D, Immunofluorescent staining of YY1, NEDD4L, and MerTK in CD68⁺ macrophages. Green, YY1/MerTK/NEDD4L; red, CD68; blue, DAPI. Scale bar, 50 μm. E, The immunoreactivities of NLRP3, ASC, c‐Caspase‐1, and GSDMD‐N in the aortic tissues was detected by immunohistochemistry analysis. Scale bar, 50 μm. F, The serum levels of IL‐18, IL‐1β, and serum MerTK were detected by ELISA assay. n=6, ***P<0.001. AS indicates atherosclerosis; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD‐N, gasdermin D N‐terminal domain; H&E, hematoxylin and eosin; IL, interleukin; MerTK, Mer tyrosine kinase; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; c‐Caspase‐1, cleaved Caspase‐1; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; and YY1, Yin Yang 2.

The Addition of ACs Exacerbates Atherosclerosis Through Activating NLRP3‐Mediated Inflammation and Pyroptosis

We next sought to verify these findings in vitro. macrophages were treated with ox‐LDL and labeled with PKH67, followed by the coculturing with PKH26‐labeled ACs for 45 minutes. Confocal microscopy revealed that the phagocytic activity of macrophages was suppressed by ox‐LDL, which was consistent with the result of flow cytometry (Figure 2A; Figure S1B). Previous studies have demonstrated that ox‐LDL triggers pyroptosis and induces NLRP3 inflammasome activation in macrophages. ⁴⁸ , ⁴⁹ Macrophages were thus treated with different doses of ox‐LDL for 48 hours. As shown in Figure 2B, lipid droplets were increased by ox‐LDL in a dose‐dependent manner. Additionally, YY1 and NEDD4L were induced, but MerTK was reduced by ox‐LDL dose‐dependently (Figure 2C). Besides, ACs promotes MerTK cleavage and induced soluble MerTK secretion in macrophages, whereas ox‐LDL does not induce the secretion of MerTK, and had no effect on the increase secretion of MerTK induced by ACs (Figure S2A and S2B). Moreover, increased lipid accumulation was found in ox‐LDL‐treated macrophages, and coculturing macrophages and ACs further potentiated ox‐LDL‐induced lipid accumulation. The addition of ACs alone had no effect on lipid accumulation (Figure 2D). Intriguingly, ox‐LDL also triggered pyroptosis in macrophages, and the percentage of pyroptotic cells was remarkably increased in ox‐LDL+ACs group as detected by flow cytometry (Figure 2E). Additionally, immunofluorescent staining and western blot unequivocally showed that caspase‐1⁺ or NLRP3⁺ cells, as well as the expressions of YY1, NEDD4L, and pyroptosis‐related proteins including NLRP3, ASC, c‐caspase‐1, and GSDMD‐N, were induced in macrophages upon ox‐LDL treatment, and these pyroptosis‐related proteins were further elevated in ox‐LDL+ACs group. By contract, the protein level of MerTK exhibited an opposite trend in ox‐LDL‐treated cells (Figure 2F and 2G). It is worth noting that ACs slightly suppressed pyroptosis (Figure 2E through 2G). ACs upregulated MerTK expression, but no change of YY1 or NEDD4L was induced by ACs. In ox‐LDL‐treated macrophages, ox‐LDL offset ACs‐induced MerTK expression to facilitate pyroptosis (Figure 2G). Furthermore, IL‐18 and IL‐1β secretion were induced by ox‐LDL, and the upregulation of these cytokines were further potentiated in ox‐LDL‐treated macrophages‐ACs cocultures (Figure 2H). Collectively, these findings suggest that the addition of ACs exacerbates atherosclerosis through activating NLRP3‐mediated inflammation and pyroptosis in vitro.

Figure 2. The addition of ACs exacerbates atherosclerosis through activating NLRP3‐mediated inflammation and pyroptosis.

Macrophages were treated with 50 μg/mL ox‐LDL for 48 h. PKH67⁺ macrophages were cocultured with PKH26‐labeled ACs for 45 min. A, The phagocytic activity of macrophages was assessed by in vitro efferocytosis assay. Green, PKH67; red, PKH26; blue, DAPI. Scale bar, 25 μm. Macrophages were treated with 0, 10, 25, 50, or 100 μg/mL ox‐LDL for 48 h. B, Lipid accumulation was monitored by Oil red O staining. Scale bar, 100 μm. Macrophages were treated with 0, 25, 50, or 100 μg/mL ox‐LDL for 48 h. C, The protein levels of YY1, NEDD4L, and MerTK were detected by western blot. macrophages were treated with 50 μg/mL ox‐LDL for 48 h, followed by the coculturing with ACs for 45 min. D, Lipid accumulation was monitored by Oil red O staining. Scale bar, 100 μm. E, Pyroptosis was monitored by caspase‐1/PI staining with quantitative analysis. F, The caspase‐1⁺ or NLRP3⁺ cells were detected by immunofluorescent staining. Green, caspase‐1; red, NLRP3; blue, DAPI. Scale bar, 100 μm. G, The protein levels of YY1, NEDD4L, MerTK, NLRP3, ASC, c‐Caspase‐1, and GSDMD‐N were detected by western blot. H, The secretion of IL‐18 and IL‐1β were detected by ELISA assay. n=3, *P<0.05, **P<0.01, ***P<0.001. AC indicates apoptotic cell; GSDMD‐N, gasdermin D N‐terminal domain; IL, interleukin; MerTK, Mer tyrosine kinase; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; c‐Caspase‐1, cleaved Caspase‐1; ox‐LDL, oxidized low‐density lipoprotein; PI, propidium iodide; and YY1, Yin Yang 2.

Silencing of MerTK Exacerbates Atherosclerosis Via Suppressing Efferocytosis and Activating NLRP3‐Mediated Inflammation

To study the biological role of MerTK in atherosclerosis, macrophages were transfected with sh‐NC or sh‐MerTK for 72 hours, followed by the coculturing with ACs for 45 minutes. In vitro efferocytosis assay revealed that lack of MerTK attenuated the phagocytic activity of macrophages, in comparison with control macrophages (Figure 3A). In the in vitro atherosclerosis model, coculturing with ACs increased lipid accumulation. These effects were more prominent in MerTK‐knockdown macrophages in which silencing of MerTK markedly potentiated ox‐LDL‐induced lipid accumulation in the presence of absence of ACs, compared with corresponding controls (Figure 3B). Similar trend of pyroptosis was also detected by flow cytometry (Figure 3C). In addition, western blot confirmed the successful knockdown of MerTK in macrophages (Figure 3D). Silencing of MerTK induced pyroptosis in macrophages, and the addition of ACs upregulated MerTK expression but reversed sh‐MerTK‐increased pyroptosis in the absence of ox‐LDL. In ox‐LDL‐treated macrophages, loss of MerTK or coculturing with ACs upregulated the levels of NLRP3, ASC, c‐caspase‐1, GSDMD‐N, IL‐18, and IL‐1β, and these effects were further potentiated in sh‐MerTK+ACs group (Figure 3D and 3E). It was worth noting that ACs induced MerTK expression and further increased pyroptosis in ox‐LDL+sh‐NC+ACs group, compared with sh‐NC+ACs group (Figure 3D). These data suggest that MerTK plays a pivotal role in the regulation of efferocytosis and NLRP3‐mediated inflammation and pyroptosis in the in vitro atherosclerosis model.

Figure 3. Silencing of MerTK exacerbates atherosclerosis via suppressing efferocytosis and activating NLRP3‐mediated inflammation.

Macrophages were transfected with sh‐NC or sh‐MerTK for 72 h, followed by the coculturing with ACs for 45 min. Macrophages and ACs were labeled with F4/80‐CFSE and phycoerythrin, respectively. A, The phagocytic activity of macrophages was assessed by in vitro efferocytosis assay. Green, CFSE; red, phycoerythrin; blue, DAPI. Scale bar, 25 μm. Transfected macrophages were treated with 50 μg/mL ox‐LDL for 48 h, followed by the coculturing with ACs for 45 min. B, Lipid accumulation was monitored by Oil red O staining. Scale bar, 100 μm. C, Pyroptosis was monitored by caspase‐1/PI staining with quantitative analysis. D, The protein levels of MerTK, NLRP3, ASC, c‐Caspase‐1, and GSDMD‐N were detected by western blot. E, The secretion of IL‐18 and IL‐1β were detected by ELISA assay. n=3, *P<0.05, **P<0.01, ***P<0.001. AC indicates apoptotic cell; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD‐N, gasdermin D N‐terminal domain; IL, interleukin; MerTK, Mer tyrosine kinase; NC, negative control; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; c‐Caspase‐1, cleaved Caspase‐1;ox‐LDL, oxidized low‐density lipoprotein; and PI, propidium iodide.

NEDD4L Knockdown‐Enhanced Efferocytosis Alleviates Atherosclerosis Through Inhibiting NLRP3‐Mediated Inflammation and Pyroptosis

We further investigated the function of NEDD4L in atherosclerosis. As presented in Figure 4A. Knockdown of NEDD4L enhanced efferocytosis in macrophage‐ACs cocultures. Additionally, Oil red O staining and flow cytometry revealed that lack of NEDD4L suppressed ox‐LDL‐induced lipid accumulation and pyroptosis in macrophages, whereas coculturing with ACs exerted opposite effects on lipid accumulation and pyroptosis. By contrast, the negative effects of ACs were partially counteracted by NEDD4L knockdown (Figure 4B and 4C). Furthermore, western blot and ELISA assay showed that transfection of NEDD4L reduced NEDD4L expression in macrophages, and sh‐NEDD4L also downregulated the levels of NLRP3, ASC, c‐caspase‐1, GSDMD‐N, IL‐18, and IL‐1β, but upregulated MerTK protein level in the in vitro atherosclerosis model. ACs exhibited opposite effects on the expression of pyroptosis‐related molecules, whereas silencing of NEDD4L reversed ACs‐mediated changes of pyroptosis‐related molecules (Figure 4D and 4E). Together, these findings indicate that lack of NEDD4L enhanced efferocytosis to ameliorate atherosclerosis by suppressing NLRP3‐mediated inflammation and pyroptosis in vitro.

Figure 4. NEDD4L knockdown‐enhanced efferocytosis alleviates atherosclerosis through suppressing NLRP3‐mediated inflammation and pyroptosis.

Macrophages were transfected with sh‐NC or sh‐NEDD4L for 72 h, followed by the coculturing with ACs for 45 min. Macrophages and ACs were labeled with F4/80‐CFSE and phycoerythrin, respectively. A, The phagocytic activity of macrophages was assessed by in vitro efferocytosis assay. Green, CFSE; red, phycoerythrin; blue, DAPI. Scale bar, 25 μm. Transfected macrophages were treated with 50 μg/mL ox‐LDL for 48 h, followed by the coculturing with ACs for 45 min. B, Lipid accumulation was monitored by Oil red O staining. Scale bar, 100 μm. C, Pyroptosis was monitored by caspase‐1/PI staining with quantitative analysis. D, The protein levels of NEDD4L, MerTK, NLRP3, ASC, c‐Caspase‐1, and GSDMD‐N were detected by western blot. E, The secretion of IL‐18 and IL‐1β were detected by ELISA assay. n=3, *P<0.05, **P<0.01, ***P<0.001. AC indicates apoptotic cell; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD‐N, gasdermin D N‐terminal domain; IL, interleukin; MerTK, Mer tyrosine kinase; NC, negative control; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; c‐Caspase‐1, cleaved caspase‐1; ox‐LDL, oxidized low‐density lipoproteinand PI, propidium iodide.

NEDD4L Mediates the Ubiquitin‐Proteasomal Degradation of MerTK

Bioinformatics analysis based on UbiBrowser (http://ubibrowser.bio‐it.cn/ubibrowser/home/index) predicted that NEDD4L is a potential E3 ubiquitin ligase of MerTK. Western blot revealed that transfection of sh‐NEDD4L led to a remarkable reduction of NEDD4L in macrophages, and ox‐LDL‐upregulated NEDD4L was reversed by sh‐NEDD4L. Conversely, NEDD4L knockdown increased MerTK expression, and it also caused a rebound of MerTK in ox‐LDL‐treated macrophages (Figure 5A). As anticipated, co‐immunoprecipitation revealed a direct interaction between MerTK and NEDD4L in macrophages in which antibody against NEDD4L successfully immunoprecipitated MerTK and vice versa (Figure 5B). Moreover, the proteasome inhibitor MG132 also potentiated sh‐NEDD4L‐increased MerTK in macrophages (Figure 5C), indicating that ubiquitin‐proteasome pathway might be responsible for MerTK degradation. Protein stability assay further showed that loss of NEDD4L slowed down the degradation of MerTK in the presence of protein synthesis inhibitor cycloheximide (Figure 5D). In accordance with these findings, silencing of NEDD4L abrogated the ubiquitination of MerTK in macrophages upon MG132 treatment as detected by co‐immunoprecipitation (Figure 5E). These data suggest that NEDD4L is responsible for the ubiquitin‐proteasomal degradation of MerTK in macrophages.

Figure 5. NEDD4L mediates the ubiquitin‐proteasomal degradation of MerTK.

Macrophages were transfected with sh‐NC or sh‐NEDD4L for 72 h, followed by the treatment of ox‐LDL for 48 h. A, The protein levels of NEDD4L and MerTK were detected by western blot with quantitative analysis. B, The interaction between NEDD4L and MerTK in macrophages were assessed by co‐immunoprecipitation. Whole cell lysates were used as an input control, and normal IgG served as a negative control. Macrophages were transfected with sh‐NC or sh‐NEDD4L for 72 h, followed by the treatment of MG132. C, The protein level of MerTK in macrophages was detected by western blot with quantitative analysis. Macrophages were transfected with sh‐NC or sh‐NEDD4L and treated with cycloheximide for 0, 15, 30, 60, 120, and 240 min. D, The protein level of MerTK in macrophages was detected by western blot with quantitative analysis. Macrophages were transfected with sh‐NC/sh‐NEDD4L or/and hemagglutinin‐ubiquitin (HA‐ubiquitin), followed by the treatment of MG132. E, The ubiquitination of MerTK in macrophages was detected by co‐immunoprecipitation. Whole cell lysates were used as an input control. n=3, *P<0.05, **P<0.01, ***P<0.001. CHX indicates cycloheximide; HA, hemagglutinin; IP, immunoprecipitation; MerTK, Mer tyrosine kinase; NC, negative control; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; and ox‐LDL, oxidized low‐density lipoprotein.

YY1 Silencing‐Enhanced Efferocytosis Ameliorates Atherosclerosis Through Suppressing NLRP3‐Mediated Inflammation and Pyroptosis

Knockdown coupled with functional experiments were next carried out to evaluate the function of YY1 in vitro. In macrophages‐ACs cocultures, silencing of YY1 enhanced the phagocytic capacity of macrophages (Figure 6A). Similar with NEDD4L, knockdown of YY1 exhibited rescue effects on lipid accumulation and pyroptosis in ox‐LDL‐treated macrophages, and it also counteracted ACs‐induced effects on lipid accumulation and pyroptosis (Figure 6B and 6C). As expected, transfection of sh‐YY1 downregulated YY1 protein level in macrophages, and this was accompanied with the downregulation of NEDD4L and pyroptosis‐related proteins, as well as the upregulation of MerTK in macrophages. Moreover, coculturing with AC induced the expression of NLRP3, ASC, c‐caspase‐1, GSDMD‐N, IL‐18, and IL‐1β in the in vitro atherosclerosis model. YY1 knockdown attenuated the effects of ACs on the expression of pyroptosis‐related molecules (Figure 6D and 6E). These findings indicate that YY1 knockdown rescues atherosclerosis via enhancing efferocytosis and inhibiting NLRP3‐mediated inflammation and pyroptosis.

Figure 6. YY1 silencing‐enhanced efferocytosis ameliorates atherosclerosis through suppressing NLRP3‐mediated inflammation and pyroptosis.

Macrophages were transfected with sh‐NC or sh‐YY1 for 72 h, followed by the coculturing with ACs for 45 min. Macrophages and ACs were labeled with F4/80‐CFSE and phycoerythrin, respectively. A, The phagocytic activity of macrophages was assessed by in vitro efferocytosis assay. Green, CFSE; red, phycoerythrin; blue, DAPI. Scale bar, 25 μm. Transfected macrophages were treated with 50 μg/mL ox‐LDL for 48 h, followed by the coculturing with ACs for 45 min. B, Lipid accumulation was monitored by Oil red O staining. Scale bar, 100 μm. C, Pyroptosis was monitored by caspase‐1/PI staining with quantitative analysis. D, The protein levels of YY1, NEDD4L, MerTK, NLRP3, ASC, c‐Cc‐Caspasde‐1, cleaved caspase‐1;aspase‐1, and GSDMD‐N were detected by western blot. E, The secretion of IL‐18 and IL‐1β were detected by ELISA assay. n=3, *P<0.05, **P<0.01, ***P<0.001. AC indicates apoptotic cell; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD‐N, gasdermin D N‐terminal domain; IL, interleukin; MerTK, Mer tyrosine kinase; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; c‐Caspase‐1, cleaved caspase‐1;ox‐LDL, oxidized low‐density lipoprotein; PI, propidium iodide; and YY1, Yin Yang 1.

YY1 Served as a Transcriptional Activator of NEDD4L

We next tested if YY1 regulated NEDD4L at the transcriptional level. Western blot showed that YY1 knockdown resulted in dramatical reduction of YY1 and NEDD4L, whereas the expression of MerTK was elevated in YY1‐knockdown macrophages (Figure 7A). Animal transcription factor database (TFDB) predicted the putative binding sites of YY1, including BS1 (−1759/−1748) and BS2 (−1077/−1066), at the promoter region of NEDD4L (Figure 7B). Chromatin immunoprecipitation assay revealed that anti‐YY1 antibody enriched both BS1 and BS2 fragments in macrophages (Figure 7C). Consistently, luciferase assay further confirmed the direct association between YY1 and NEDD4L promoter. Knockdown of YY1 attenuated the luciferase activity, whereas the mutation of NEDD4L abolished this effect (Figure 7D). Interestingly, ox‐LDL induced the luciferase activity in NEDD4L‐wild‐type‐overexpressing cells, whereas cotransfection of NEDD4L‐wild‐type and sh‐YY1 reduced the luciferase activity in ox‐LDL‐treated cells, in comparison with corresponding control. On the contrary, no similar trend was observed in mutated NEDD4L overexpressing cells (Figure 7E). These data suggest that YY1 positively regulates NEDD4L expression transcriptionally in macrophages.

Figure 7. YY1 served as a transcriptional activator of NEDD4L.

Macrophages were transfected with sh‐NC or sh‐YY1 for 72 h. A, The protein levels of YY1, NEDD4L, and MerTK in macrophages were detected by western blot with quantitative analysis. B, The putative binding sites between YY1 and NEDD4L promoter were predicted by bioinformatics analysis based on AnimalTFDB4 (https://guolab.wchscu.cn/AnimalTFDB4//#/). C, The association between YY1 and BS1 or BS2 fragment of NEDD4L promoter was detected by chromatin immunoprecipitation assay. The total chromatin fragments were used as the input control, and normal IgG was employed as a negative control. D, The interaction between YY1 and NEDD4L promoter was assessed by dual‐luciferase assay. Macrophages were transfected with sh‐NC or sh‐YY1 for 72 h, followed by the treatment of ox‐LDL. E, The interaction between YY1 and NEDD4L promoter was assessed by dual‐luciferase assay. n=3, *P<0.05, **P<0.01, ***P<0.001. MerTK indicates Mer tyrosine kinase; Mut, mutated; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; ox‐LDL, oxidized low‐density lipoprotein; TSS, transcriptional start site; WT, wild type; and YY1, Yin Yang 1.

YY1 Regulates NEDD4L to Modulate MerTK‐Mediated Efferocytosis and Activates NLRP3‐Mediated Inflammation, Thus Exacerbating Atherosclerosis

To further delineate the role of YY1/NEDD4L/MerTK axis in atherosclerosis, gain‐ and loss‐of‐function experiments were carried out. In vitro efferocytosis assay revealed that silencing of YY1 promoted efferocytosis, whereas knockdown of MerTK suppressed the phagocytic activity of macrophages. Cotransfection of sh‐YY1 and sh‐MerTK abolished sh‐YY1‐increased efferocytosis in macrophages‐ACs cocultures (Figure 8A). As expected, coculturing with ACs increased lipid accumulation and pyroptosis, whereas lack of YY1 exerted rescue effects in the in vitro atherosclerosis model. More important, silencing of MerTK attenuated the promoting protective effects of sh‐YY1 on lipid accumulation and pyroptosis (Figure 8B and 8C). Western blot and ELISA assay further showed that compared with sh‐NC‐overexpressing macrophages, YY1 and NEDD4L was increased, but MerTK was decreased in sh‐NC‐overexpressing macrophages, along with the upregulation of pyroptosis‐related molecules. YY1 knockdown counteracted ACs‐mediated changes of pyroptosis‐related molecules, while silencing of MerTK further abrogated the effects of sh‐YY1 in ox‐LDL‐treated macrophages‐ACs cocultures (Figure 8D and 8E). These findings indicate that MerTK acts as a downstream effector of YY1/NEDD4L axis in the in vitro atherosclerosis model.

Figure 8. YY1 regulates NEDD4L to modulate MerTK‐mediated efferocytosis and activate NLRP3‐mediated inflammation, thus exacerbating atherosclerosis.

Macrophages were transfected with sh‐NC, sh‐YY1, or sh‐MerTK for 72 h, followed by the coculturing with ACs for 45 min. Macrophages and ACs were labeled with F4/80‐CFSE and phycoerythrin, respectively. A, The phagocytic activity of macrophages was assessed by in vitro efferocytosis assay. Green, CFSE; red, phycoerythrin; blue, DAPI. Scale bar, 25 μm. Transfected macrophages were treated with 50 μg/mL ox‐LDL for 48 h, followed by the coculturing with ACs for 45 min. B, Lipid accumulation was monitored by Oil Red O staining. Scale bar, 100 μm. C, Pyroptosis was monitored by caspase‐1/PI staining with quantitative analysis. D, The protein levels of YY1, NEDD4L, MerTK, NLRP3, ASC, c‐Caspase‐1, and GSDMD‐N were detected by western blot. E, The secretion of IL‐18 and IL‐1β were detected by ELISA assay. n=3, *P<0.05, **P<0.01, ***P<0.001. AC indicates apoptotic cell; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD‐N, gasdermin D N‐terminal domain; IL, interleukin; MerTK, Mer tyrosine kinase; NC, negative control; NEDD4L, neural precursor cell‐expressed developmentally downregulated 4‐like; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; c‐Caspase‐1, cleaved caspase‐1; ox‐LDL, oxidized low‐density lipoprotein; PI, propidium iodide; and YY1, Yin Yang 1.

DISCUSSION

Efferocytosis maintains tissue homeostasis. Defective efferocytosis and apoptosis of macrophages lead to the formation of necrotic cores. ⁵⁰ Clearance of ACs is essential to avoid uncontrolled inflammatory responses, and previous studies have also illustrated that efferocytosis is linked to different types of cell death, including necrosis, apoptosis and pyroptosis. ⁵¹ In this study, we demonstrated that YY1 positively regulated NEDD4L expression transcriptionally, and elevated NEDD4L facilitated the ubiquitin‐proteasomal degradation of MerTK to impair efferocytosis, leading to the activation of NLRP3‐mediated inflammation and pyroptosis to exacerbate atherosclerosis. These findings improved knowledge for the anti‐inflammatory therapy for atherosclerosis.

It is well established that dysfunctional efferocytosis contribute to AC accumulation and inflammation in the plaque. ¹⁶ Consistently, increased lipid droplets were found in the arteries of apoE^−/− mice, along with the impaired phagocytic activity of macrophages. More important, defective efferocytosis was accompanied with the upregulation of NLRP3 inflammasome molecules and increased release of IL‐18 and IL‐1β. In macrophages, 2 signals, namely priming and activation signals, are involved in the activation of NLRP3 inflammasome. For instance, priming signal is induced by cytokines or NF‐κB (nuclear factor kappa B)‐mediated upgradation of NLRP3 or pro‐IL‐1β. On the other hand, multiple stimuli (eg, ATP, particulate matter, or viral RNA) also activate NLRP3 inflammasome in macrophages. ⁵² , ⁵³ In addition to these upstream signalings, this study demonstrated that inefficient efferocytosis resulted in the activation of NLRP3 inflammasome and pyroptosis in atherosclerosis. In accordance with the earlier in vivo findings, the in vitro knockdown experiments illustrated the essential role of MerTK in efferocytosis in macrophages‐ACs cocultures. ACs upregulated MerTK expression but reversed sh‐MerTK‐enhanced pyroptosis in macrophages, whereas ox‐LDL+sh‐NC+ACs further increased pyroptosis, compared with sh‐NC+ACs group. These findings suggest that ox‐LDL compensates the effects of ACs on the regulation of pyroptosis, and other mechanism(s) might be implicated in MerTK‐regulated pyroptosis. Previous study has reported that MerTK suppresses NLRP3 inflammasome activation in an autophagy‐dependent manner in subarachnoid hemorrhage. ³² Similarly, our findings showed that depletion of MerTK activated NLRP3 inflammasome and triggered pyroptosis in atherosclerosis. If autophagy was also involved in the regulation of this biological process in atherosclerosis merits in‐depth investigation.

Protein UPS (ubiquitin proteasome system) comprises E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase catalyzes covalent bond between ubiquitin and target proteins. ⁵⁴ In addition, E3 ubiquitin ligase specifically recognizes substrates, and recent reports have suggested that E3 ubiquitin ligases have emerged as key regulators of inflammation in CVD. ⁵⁵ NEDD4L is implicated in the ubiquitin‐proteasomal turnover of various membrane proteins. ³³ In line with this report, we found that NEDD4L acted as an E3 ubiquitin ligase of transmembrane protein MerTK. Loss of NEDD4L reduces macrophage polarization from M2 to M1 and the uptake of ox‐LDL through inhibiting PPAR‐γ (peroxisome proliferator‐activated receptor gamma) ubiquitination, Smad1/2 phosphorylation, and modulating ABCA1 (ATP‐binding cassette transporter A1), ABCG1 (ATP‐binding cassette subfamily G member 1), LDLR, and PCSK9. ³⁵ M1 and M2 macrophages are implicated in pro‐ and anti‐inflammatory responses, respectively. ⁵⁶ NEDD4L knockdown decreases M1/M2 ratio, ³⁵ indicating reduced inflammation in macrophages. Consistent with this finding, our results also showed that silencing of NEDD4L inhibited NLRP3 inflammasome activation and pyroptosis and impaired ox‐LDL‐induced lipid uptake in macrophages. In addition to PPAR‐γ, MerTK was identified as a novel substrate of NEDD4L. Subsequent study should focus on the downstream signaling of MerTK‐mediated efferocytosis in atherosclerosis.

YY1 is a transcription factor that is closely associated with lipid metabolism. ⁵⁷ In atherosclerosis, YY1 inhibits ox‐LDL‐induced form cell formation by regulating miR‐155. ⁵⁸ Dysregulated growth and migration of vascular smooth muscle cells (VSMCs) are involved in atherosclerosis progression. ⁵⁹ miR‐147b regulates VSMCs growth and migration via targeting YY1 and Wnt/β‐catenin signaling. ⁶⁰ Moreover, YY1 is identified as a transcription factor of fibulin‐5, and it alleviates aberrant proliferation and migration of VSMCs in response to ox‐LDL, ⁶¹ suggesting the role of YY1 in the regulation of VSMCs in atherosclerosis. Besides the critical function of YY1 in VSMCs, the present study reported that YY1 regulated efferocytosis and NLRP3 inflammasome activation in macrophages‐ACs cocultures through modulating NEDD4L/MerTK axis. Our findings broaden the understanding of YY1 function in atherosclerosis. There is a surge of interest to unravel how YY1 leverages the regulatory axes in macrophages and VMSCs in atherosclerosis.

Conclusions

In conclusion, we reported that YY1/NEDD4L/MerTK axis regulated efferocytosis and NLRP3 inflammasome activation in atherosclerosis. These findings provide growing silver linings for the anti‐inflammatory therapy for atherosclerosis.

Sources of Funding

This work was supported by the National Natural Science Foundation of China (82160093, 82360145).

Disclosures

There is no conflict of interest.

Supporting information

Data S1

Figures S1–S2