XL092

Immunoproteasome subunit β5i regulates diet-induced atherosclerosis through altering MerTK-mediated efferocytosis in Apoe knockout mice

Jiawei Liao1, Yunpeng Xie1, Qiuyue Lin1, Xiaolei Yang1, Xiangbo An2, Yunlong Xia1, Jie Du3, Feng Wang2, and Hui-Hua Li1

Abstract

The immunoproteasome contains three catalytic subunits (β1i, β2i and β5i) that are important modulators of immune cell homeostasis. A previous study showed a correlation between β5i and human atherosclerotic plaque instability; however, the causative role of β5i in atherosclerosis and the underlying mechanisms remain unknown. Here we explored this issue in apolipoprotein E (Apoe) knockout (KO) mice with genetic deletion or pharmacological inhibition of β5i. We found that β5i expression was upregulated in lesional macrophages after atherogenic diet (ATD) feeding. β5i/Apoe double KO (dKO) mice fed on ATD had a significant decrease in both lesion area and necrotic core area, compared with Apoe KO (eKO) controls. Moreover, dKO mice had less caspase-3+ apoptotic cell accumulation but enhanced efferocytosis of apoptotic cells and increased expression of Mer receptor tyrosine kinase (MERTK). Consistently, similar phenotypes were observed in eKO mice transplanted with dKO bone marrow (BM) or treated with β5i-specific inhibitor PR-957. Mechanistic studies in vitro revealed that β5i deletion reduced IκBα degradation and inhibited NF-κB activation, promoting Mertk transcription and efferocytosis, thereby attenuated apoptotic cell accumulation. In conclusion, we demonstrate that β5i plays an important role in diet-induced atherosclerosis by altering MERTK-mediated efferocytosis. β5i might be a potential pharmaceutical target against atherosclerosis.

Key words: Immunoproteasome; β5i; Atherosclerosis; Mer receptor tyrosine kinase; Efferocytosis; NF-κB

Introduction

Atherosclerotic cardiovascular diseases are currently the leading cause of mortality and morbidity worldwide [1]. Life-threatening events, such as acute coronary syndrome and stroke, are often associated with the presence of advanced vulnerable plaques and subsequent thrombosis, highlighting the significance of plaque stabilization in atherosclerosis [1]. Macrophages are the major cell types in the lesions and play various roles in the disease progression [2]. Besides the well-known functions such as clearance of the oxidized low-density lipoprotein (ox-LDL) and secretion of pro- and anti-inflammatory cytokines, macrophages also engage in the recognition and engulfment of apoptotic debris via ligand-receptor interactions, a process called efferocytosis [2]. Previous studies have demonstrated that impaired efferocytosis is involved in necrosis expansion in advanced plaques [3,4]. Thus, the efferocytotic activity of the macrophage is pivotal to the enlargement and destabilization of atherosclerosis.
The ubiquitin-proteasome system (UPS) is the major non-lysosomal pathway for protein degradation within eukaryotic cells, therefore plays a key role in maintaining the homeostasis of cellular proteins involved in myriad cellular processes, including transcriptional regulation, signal transduction, cell differentiation, and apoptosis [5,6]. Defective UPS has been observed during atherogenesis [7,8] and inactivation of proteasome by its inhibitor bortezomib attenuated the early onset of atherosclerosis [9] but exerted no protection against pre-existing plaques [10]. The 20S proteasome complex is the protein degradation center of the UPS. Normally, the standard 20S proteasome consists of three catalytic subunits, namely, β1 (PSMB6), β2 (PSMB7) and β5 (PSMB5), which have caspase-like, trypsin-like, and chymotrypsin-like protease activity, respectively [5,6]. Upon stimulation with pro-inflammatory cytokines, three inducible catalytic subunits, namely, β1i (PSMB9, LMP2), β2i (PSMB10, MECL-1) and β5i (PSMB8, LMP7), replace the standard subunits to form the so-called immunoproteasome [11,12]. The immunoproteasome has critical functions in the regulation of protein degradation, immune cell homeostasis, oxidative stress and cell apoptosis [13,14]. Recently, we showed that the β2i and β5i subunits are involved in the development of cardiac remodeling, atrial fibrillation and retinopathy, thus extending previous knowledge about the immunoproteasome [15–18]. Notably, subunit β5i was upregulated in the shoulder areas of symptomatic carotid plaques as compared with non-symptomatic plaques, suggesting a correlation between β5i activity and plaque instability [19]. However, the causal role of β5i in the atherogenesis remains unknown. Here we explored this issue in apolipoprotein E (Apoe) knockout (KO) mice with genetic deletion or pharmacological inhibition of β5i.

Materials and methods

Animals, diets and experimental designs

EKO mice (C57BL/6 background) were purchased from Beijing Vital River Laboratory (Beijing, China). β5i KO mice (C57BL/6 background) was purchased from Jackson Laboratories (Bar Harbor, ME, USA), and crossbred with EKO mice to generate β5i/Apoe double knockout (dKO) mice. β5i-specific inhibitor PR-957 was purchased from Selleck (Houston, TX, USA) and administrated at a dosage of 5 mg/kg and 10 mg/kg by subcutaneous injection twice per week. Mice were housed under specific-pathogen-free conditions on a 12 h light/12 h dark cycle and fed with normal rodent chow diet until 8–12 weeks of age, then subjected to an atherogenic diet (ATD) containing 0.5% cholesterol (Amresco, Solon, OH, USA) and 20% fat, as described previously [20], for the next 8 weeks to induce rapid atherosclerosis. Only male mice were included in the experiments. All experimental procedures were in accordance with the guidelines for the care and use of laboratory animals of the National Institute of Health and approved by the Animal Care and Use Committee of Dalian Medical University.

Histopathological analysis

Mice were sacrificed and flushed with phosphate buffered saline. The entire aorta and heart were removed and fixed in 4% paraformaldehyde solution. The aortas were cleaned of fatty tissues and cut open longitudinally. Hearts were embedded in OCT compound (Sakura Finetek, Tokyo, Japan), snap-frozen in liquid nitrogen and cross-sectioned serially at 7 μm thick throughout the aortic root. Atherosclerotic lesions in the en face aortas and the aortic root sections were visualized by staining with oil-red O (Sigma, St. Louis, MI, USA). Lesional macrophages and smooth muscle cells (SMCs) were visualized by immunohistochemical staining using anti-CD68 (MCA1957, 1:200 dilution, Bio-Rad, Hercules, CA, USA) and anti-SM22α (ab14106, 1:200 dilution, Abcam, Cambridge, MA, USA) antibodies, respectively. Lesional collagen was visualized by Picro-Sirius Red staining. Lesional necrotic core was visualized by H&E staining and designated as eosin-negative acellular areas. Lesional apoptosis and in situ efferocytosis were visualized by immunofluorescent staining with anti-cleaved caspase-3 (#9661, 1:200 dilution, CST, Danvers, MA, USA) and anti-F4/80 (ab6640, 1:200 dilution, Abcam) antibodies. Lesional apoptosis was designated as total cleaved caspase-3 mean fluorescence intensity (MFI), and in situ efferocytosis was designated as the ratio of cleaved caspase-3 MFI associated with F4/80 fluorescence to the total cleaved caspase-3 MFI, both normalized to the controls. Lesional β5i expression was visualized by immunohistochemical staining using anti-β5i (ab97584, 1:200 dilution, Abcam) antibody and immunofluorescent staining using anti-β5i (ab97584, 1:200 dilution, Abcam) and anti-CD68 (MCA1957, 1:200 dilution, Bio-Rad) antibodies. All quantifications were performed using ImageJ software (NIH, Bethesda, MD, USA).

RNA isolation and RT-qPCR analysis

Total RNA was extracted, and reverse transcribed as described previously [21]. RT-qPCR was performed using SYBR Green PCR reagents (Molecular Probes, Eugene, OR, USA) and normalized to Gapdh. The primer sequences used are listed in supplementary material, Table S1.

Western blotting analysis

Protein samples were extracted as described previously [22], and using the primary antibodies described below: β5i (ab3329, 1:500 dilution, Abcam); NF-κB p65 (C22B4) (#4764, 1:500 dilution, CST) and phospho–p65 (Ser536) (93H1, #3033, 1:500 dilution, CST); IκB-α (#9242, 1:1000 dilution, CST) and phospho–IκBα (Ser32/36) (5A5, #9246, 1:1000 dilution, CST); MERTK (BAF591, 1:1000 dilution, R&D, Minneapolis, MN, USA) and GAPDH (60004-1-lg, 1:1000 dilution, Proteintech, Beijing, China). Quantification was performed using ImageJ software.

Bone marrow (BM) transplantation

Recipient mice, at 2 months of age, were lethally irradiated by 8.5 Gy dose from a cobalt source. BM was extracted by flushing the femurs and tibiae from donor mice (10 weeks of age) with RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 2% fetal bovine serum (Gibco) and heparin (5 U/ml). Each recipient mouse was injected with 5ⅹ106 BM cells by tail vein injection. Then the recipient mice were fed with acidified, antibiotic water and chow diet for 4 weeks before ATD feeding.

Macrophage (Mφ) culture, adenoviral infection and foam cell induction

Mice were injected with 4% sterile thioglycollate medium (Sigma) and sacrificed 72 h later. Peritoneal Mφs were isolated, washed and cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum. Twenty four hours after plating, cells were treated with ox-LDL (50 μg/ml, Unionbiol, Beijing, China) for 24 h to induce foam cell formation (Mφs treated with ox-LDL-free medium were used as control). Then cell samples were harvested for protein extraction or fixed with 4% paraformaldehyde solution for histological analysis. For adenoviral infection experiments, cells were pre-treated with recombinant adenoviruses encoding green fluorescent protein alone as a negative control (Ad-NC, 50 MOI), NF-κB p65 (Ad-p65, 50 MOI) or si p65 (Ad-si p65, 50 MOI) for 24 h before ox-LDL induction. Recombinant adenoviruses were all obtained from GenePharma (Shanghai, China).

In vitro efferocytosis assay

Peritoneal Mφs were collected, incubated with ox-LDL and/or adenovirus as described above. Jurkat cells were labeled using Calcein AM (C3099, Invitrogen, Carlsbad, CA, USA), irradiated with UV light to induce apoptosis and then incubated with peritoneal Mφs for 2 h at a ratio of 3:1. Cultures were then washed vigorously to remove the free apoptotic Jurkat cells that were not engulfed by Mφs. The percentage of Mφs with internalized apoptotic Jurkat cells were quantified by fluorescence microscopy.

In vitro apoptosis assay

An in vitro assessment of apoptosis was performed using TUNEL staining, with an in situ cell death detection kit (Roche, South San Francisco, CA, USA). Quantification of TUNEL-positive cells was determined as the percentage of TUNEL-positive nuclei per field.

Chromatin immunoprecipitation (ChIP) assay

Cells were fixed with 1% formaldehyde and sonicated on ice to shear the DNA to a length of <500 bp.; one third of the total cell lysate was used as the DNA input control. The remainder was subjected to immunoprecipitation with anti-p65 antibody (D14E12, #8242, 1:50 dilution, CST) or non-immune rabbit IgG. The DNA was subjected to PCR to amplify a 274 bp region of the Mertk promoter using the primers: forward, 5’-GAG TTG CAT CCG TCG CTC TC-3’; reverse, 5’-GGG TCA ACT CCA GCA GGT TA-3’. The PCR products were resolved electrophoretically on a 1% agarose gel and visualized by Gel Red staining. Plasmids and dual-luciferase reporter assay Mertk promotor full-length and segment DNAs were cloned into the vector pGL-3, and cDNA for NF-κB p65 was cloned into the vector pEX-4, using a PCR-based method. For transfection, HEK293T cells were seeded in triplicate in 24-well plates and cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin. A mixture of the luciferase reporter plasmids, pRL-TK-renilla luciferase plasmid and Lipofectamine 2000 reagent (Invitrogen) were added to the medium. Cells were lysed 24 h post transfection in passive lysis buffer, and luciferase activity was measured using a Dual-Luciferase Reporter Assay kit (E1910, Promega, Madison, WA, USA), according to the manufacturer’s protocols. Renilla luciferase activity was used as a control for the transfection. All plasmids were obtained from GenePharma. Data were normalized for transfection efficiency by the division of firefly luciferase activity with that of Renilla luciferase activity. Statistical analyses Data are shown as mean ± SEM. A normality test (Shapiro–Wilk) was first performed to determine whether the data were normally distributed. For comparison between 2 groups, normally distributed data were evaluated using Student’s t-test. Nonparametric data were evaluated using the Mann–Whitney test. For comparison among 3 groups, statistical significance was evaluated by one-way ANOVA; for comparison among 4 groups, statistical significance was evaluated by two-way ANOVA. All evaluations were performed using Prism software (GraphPad Inc., San Diego, CA, USA) with P value < 0.05 considered significant. Results Upregulation of β5i in diet-induced atherosclerotic plaques in Apoe knockout (eKO) mice To determine the roles of the immunoproteasome in atherogenesis, we first examined the levels of transcripts encoding the catalytic subunits of the proteasome and immunoproteasome, namely, β1, β2, β5, β1i, β2i and β5i, in the atherosclerotic plaques of eKO mice. As shown by RT-q PCR analysis, the mRNA level of β5i was significantly upregulated in the aortic atherosclerotic plaques of eKO mice fed an atherogenic diet (ATD) compared with those fed a normal chow diet (Figure 1A). Increased β5i protein expression (2-fold) was verified by immunoblotting analysis (Figure 2B). Moreover, immunohistochemical staining confirmed the expression of β5i in the aortic atherosclerotic lesions of eKO mice fed the ATD (Figure 1C). As the macrophage is the major cell type in atherosclerotic plaques, we performed co-staining for β5i and CD68, a macrophage marker. Our data showed that β5i was largely localized to macrophages in the plaques of eKO mice fed the ATD (Figure 1D). Smooth muscle cells, however, expressed much less β5i, as shown by co-staining of β5i with smooth muscle cell marker α-smooth muscle actin (α-SMA) (supplementary material, Figure S1). Overall, these results suggest that increased β5i expression in macrophages may be involved in diet-induced atherogenesis. Ablation of β5i attenuates diet-induced atherosclerosis To investigate the roles of β5i in atherogenesis, we generated β5i/Apoe dKO mice and fed them and eKO controls with an ATD for 8 weeks. We found that the mRNA levels for proteasome (β1, β2, and β5) and other immunoproteasome (β1i, β2i) subunits in the dKO mice were not significantly changed (supplementary material, Figure S2), as compared with those in the eKO controls. So, as the plasma lipid levels and lipoprotein profiles of the dKO mice (supplementary material, Figure S3). Interestingly, the atherosclerosis burden in the aortic inner surface (Figure 2A) and the aortic root (Figure 2B) of the dKO mice both decreased more than 40%. Further histological analysis showed that in the lesions of the dKO mice, the necrotic core area decreased 37% (Figure 2C), while no significant change in the accumulation of macrophages, smooth muscle cells or collagen in the lesions was observed, as compared with those of the eKO control mice (Figure 2D–F). Moreover, no difference in the infiltration of neutrophils and T cells in the lesions of the dKO mice were detected, as shown by flow cytometry (supplementary material, Figure S4). Thus, our data indicate that deletion of β5i suppresses necrotic core formation and inhibits plaque development in diet-induced atherogenesis in eKO mice. Selective knockout of β5i in BM-derived cells reduces diet-induced atherosclerosis To explore whether β5i expressed in BM-derived cells contributed to the development of atherosclerosis, we generated chimeric mice by transplanting BM-derived cells from dKO or eKO donors into lethally irradiated eKO recipients. After 4 weeks’ recovery from the transplantation, these mice were fed the ATD for 8 weeks. As expected, inactivation of β5i in BM-derived cells did not affect the plasma lipid levels (supplementary material, Figure S5), but led to a roughly 40% and 30% decrease of the atherosclerosis burden in the aorta and the aortic root (Figure 3A,B), respectively, alongside with a 30% decrease of the necrotic core area (Figure 3C) yet no significant change in the infiltration of CD68+ macrophages or SM22α+ SMCs or deposition of collagen in the lesions (Figure 3D–F). Therefore, these results demonstrate that β5i expressed in BM-derived cells contributes to diet-induced formation of necrotic core and atherosclerosis development. Knockout of β5i upregulates MERTK expression and efferocytosis in macrophages To explore how β5i deficiency inhibits the formation of necrotic core in the atherosclerotic lesions, we evaluated the effects of β5i on efferocytosis. Immunofluorescent staining showed that the apoptotic cell accumulation was markedly reduced, while macrophage efferocytosis was increased, in the lesions of dKO mice, as compared with eKO controls (Figure 4A). Using RTqPCR to analyze the mRNA levels for efferocytosis-related signaling molecules, including Mertk, Gas6, C1qa, Tg2, Cd47 and Fas, we found that for Mertk was significantly upregulated in dKO mice (Figure 4B). Further, using immunofluorescence staining with an anti-MERTK antibody, we confirmed the increased expression of MERTK in the atherosclerotic lesions of dKO mice (Figure 4C). Consistent with this, increased MERTK expression (supplementary material, Figure S6A) and enhanced efferocytosis with less accumulation of apoptotic cells (supplementary material, Figure S6B) were observed in the eKO mice reconstituted with the dKO BM, as compared with those received the eKO BM. Overall, these results indicate that β5i deletion increases MERTK expression and efferocytosis, therefore suppresses apoptotic cell accumulation and atherosclerosis development. Deletion of β5i in macrophages regulates MERTK expression via the IκBα/NF-κB pathway in vitro To elucidate the molecular mechanisms by which β5i regulates MERTK expression and efferocytosis, we isolated peritoneal macrophages from β5i KO and wild-type (WT) mice and treated these cells with ox-LDL to induce foam cell formation in vitro. We first confirmed in the peritoneal macrophages that β5i inactivation increased MERTK expression (Figure 5A), improved efferocytosis and decreased cellular apoptosis (Figure 5B) after ox-LDL overload, in consistent to phenotypes observed during diet-induced atherosclerosis in eKO mice with β5i deletion. Recently, we demonstrated that NF-κB and its inhibitory protein IκBα are downstream targets of the immunoproteasome in the regulation of cardiac inflammation during hypertrophic remodeling and atrial fibrillation [15,16,18]. This led us to explore whether β5i is involved in the regulation of the IκBα/NF-κB pathway in macrophage activation during atherosclerosis. Immunoblotting showed that the ox-LDL-stimulated degradation of IκBα and activation of NFκB p65 were significantly reversed in β5i KO macrophages, as compared with WT macrophages (Figure 5C), suggesting that β5i contributes to foam cell formation at least via IκBα/NF-κB pathway. We next explored whether NF-κB regulates MERTK expression directly. Chromatin immunoprecipitation analysis showed that NF-κB p65 bound to the promoter region of the Mertk gene (Figure 5D). Further, we predicted its potential targets using the bioinformatics program TargetScan 6.2 and found that there were two canonical/conserved binding sites (BSs) for NF-κB in the promoter region of Mertk (Figure 5E). To test whether Mertk is a direct target of NF-κB, we constructed plasmids carrying full-length or deletion mutants (BS1 or BS2) of the Mertk promoter and performed a luciferase assay in 293T cells. We found that NF-κB p65 overexpression decreased luciferase activity, while deletion of the BS2 region, but not the BS1 region, fully abolished the inhibitory effects of NF-κB p65 (Figure 5F), suggesting that NF-κB regulates MERTK expression via BS2. In addition, knockdown of NF-κB p65 attenuated the oxLDL-induced suppression of MERTK expression (Figure 5G), while overexpression of NF-κB p65 enhanced this effect (Figure 5H). We further explored whether NF-κB regulates efferocytosis in macrophages. Knockdown of NF-κB p65 attenuated the ox-LDL-induced inhibition of efferocytosis and increase of TUNEL+ apoptotic cells (Figure 5I). Conversely, NF-κB p65 overexpression in macrophages led to the opposite results (Figure 5J), suggesting that NF-κB p65 inhibits MERTK-mediated efferocytosis. Taken together, our data suggest that β5i regulates MERTK-mediated efferocytosis at least in part through IκBɑ/NF-κB signaling. Pharmacological inhibition of β5i suppresses diet-induced atherosclerosis To further explore whether β5i could be a potential therapeutic target for atherosclerosis, we treated eKO mice with the β5i-specific inhibitor PR-957 and fed them with the ATD for 8 weeks. We confirmed that PR-957 administration did not affect plasma lipid levels (supplementary material, Figure S7A,B), but attenuated the atherosclerotic lesion burden and necrotic core formation in the aortic root in a dose-dependent manner (Figure 6A,B), without altering the accumulation of CD68+ macrophages (Figure 6C) and SM22α+ SMCs and deposition of collagen (supplementary material, Figure S7C,D). Moreover, PR-957 treatment also increased MERTK expression (Figure 6D) and efferocytosis and reduced the accumulation of apoptotic cells in the lesions (Figure 6E). Taken together, these results suggest that inhibition of β5i using PR-957 attenuates diet-induced atherosclerosis in the eKO mice. Discussion This study has, for the first time, identified a critical role for immunoproteasome subunit β5i in regulating efferocytosis and diet-induced atherosclerosis in eKO mice. Our data showed that ATD significantly upregulated β5i expression in the atherosclerotic plaques, which promoted IκBα degradation and NF-κB activation, thereby suppressing MERTK-mediated efferocytosis, leading to increased accumulation of apoptotic cells and formation of necrotic cores in the atherosclerotic plaques. Conversely, genetic deletion or pharmacological inhibition of β5i markedly prevented these effects caused by ATD feeding. Atherosclerosis has long been considered as an immuno-inflammatory response to lipid deposition in the artery wall. It is well known that immune and inflammatory cells, including macrophages, T cells and dendritic cells, play an important role in the development and progression of atherosclerosis [23,24]. The immunoproteasome is expressed constitutively in immune cells, and can be induced in immune and non-immune cells upon exposure to proinflammatory stimuli such as viruses, angiotensin (Ang) II, and high salt, thus contributing to the regulation of immuno-inflammatory diseases, including viral myocarditis, hypertrophic remodeling, atrial fibrillation, and vascular cell apoptosis [15,16,18,25–28]. Here, the results presented showed that β5i was upregulated in the atherosclerotic plaques of eKO mice (Figure 1): genetic ablation or inhibition of β5i reduced lesional accumulation of apoptotic cells but enhanced MERTK-mediated efferocytosis. This inhibited necrotic core formation and atherosclerosis progression (Figure 2–4). However, our results are inconsistent with a recent study, which showed that β5i deficiency has no significant effect on the initiation and progression of atherosclerosis in the LDL receptor KO mice fed with ATD [29]. This is not the first time that gene function studies performed in these two mouse models yield totally different results. As pointed out in a recent review, although these two mice are both widely accepted as atherosclerosis disease models, they differ in the precise mechanisms during atherogenesis, such as the distribution of plasma lipoproteins, the initiation of atherosclerosis, the roles of macrophages and lymphocytes, and the gene expression patterns etc., possibly leading to the phenotypic diversity [30]. Regarding the β5i KO mice, the specific reasons for the inconsistent observations in LDL receptor deficient and Apoe deficient background needs further investigation. MERTK, also known as Mer, c-Eyk, and Tyro12, is a receptor tyrosine kinase that is expressed predominantly in phagocytic cells, mostly macrophages, with lower expression detected in other types of cells in atherogenesis, such as endothelial cells and platelets [31–33]. Upon ligand (GAS6, TUB, TULP1, and Protein S) binding, MERTK undergoes autophosphorylation, then induces the activation of several signaling pathways that are important for cell survival, thrombosis, and efferocytosis [31,34,35]. Previous studies have identified a decreased macrophage expression of MERTK in advanced plaques, and defective macrophage MERTK function limited efferocytosis and promoted plaque necrosis [36,37]. However, the role of endothelial MERTK in atherogenesis is currently unknown. ADAM17, as a metalloproteinase, cleaves the ectodomain of MERTK into a soluble MER, which would competitively disrupt the interaction of intact MERTK with ligands and inhibit efferocytosis [38–40]. Interestingly, no significant change of soluble MER was detected in the supernatant collected from β5i-deleted cells, suggesting that β5i possibly did not involve in ADAM17mediated cleavage of MERTK (supplementary material, Figure S8). Besides cleavage, MERTK expression can be inhibited by miR-126 and miR-335, while enhanced by peroxisome proliferator-activated receptor (PPAR) and retinoid X receptor alpha (RXRα) [41,42]. NF-κB is typically characterized as a heterodimer containing a DNA-binding subunit (p50) and a transactivator (p65) subunit, which disaggregates into p50 and p65 monomers upon IKK-mediated phosphorylation and degradation of IκBα by the proteasome. The p65 monomer then translocates to the nucleus and regulates the expression of target genes involved in inflammation, oxidative stress, and cell survival [43]. Recently, we identified IκBα as a target of β5i, and β5i regulates NF-κB activation in atrial fibrillation and retinopathy induced by Ang II [17,18]. The present results extend the mechanisms by which β5i inactivation improves efferocytosis. We found that β5i inactivation reduced IκBα degradation and NF-κB activation, which further blocked the inhibition of MERTK expression and efferocytosis (Figure 5). Thus, these results suggest that β5i is a novel regulator of efferocytosis at least through MERTKmediated efferocytosis in macrophages. Apoptosis occurs as a defense mechanism against a variety of stimuli, including oxidative stress, endoplasmic reticulum (ER) stress, lipid overload and hypoxia, etc. [44]. Accumulation of apoptotic debris by increased apoptosis or defected efferocytosis, attracts immune cells and promotes pro-inflammatory cytokine secretion, leading to secondary necrosis and pro-apoptotic cascade [45]. Previous studies have indicated that the immunoproteasomes play an important role in apoptosis in cancers, partially through regulating oxidative and ER stress [46–48]. Recently, we found that β5i inactivation attenuated SMC apoptosis in angiotensin II–induced aneurysm in eKO mice [49]. Here, we further demonstrated that both genetic deletion and pharmacological inhibition of β5i blunted the apoptotic cells accumulation in diet-induced atherosclerosis through suppression of efferocytosis. Interestingly, besides phagocytosis of the apoptotic debris for degradation, efferocytosis has also been demonstrated to elevate cellular fatty acids and oxygen consumption, therefore fuel mitochondrial respiration and activate an NAD+-dependent signal transduction cascade promoting IL-10 secretion and anti-inflammatory response in post-injury repair [50,51]. Whether β5i inhibition reduces lesional cell apoptosis and promote this cellular pathway in atherosclerosis, however, need further investigation. Ever since the discovery of the UPS, extensive efforts have been devoted to the development of proteasome inhibitors as potent therapeutic approaches for human diseases. Some of the proteasome inhibitors, such as bortezomib and carfilzomib, has already been used against human multiple myeloma, but can cause severe toxic effects due to their broad inhibitory activities [52,53]. Thus, it is important to identify specific proteasome inhibitors. PR-957 (also known as ONX0914) is the first specific compound targeting immunoproteasome β5i subunit. It has been demonstrated to be 20- to 40-fold more selective for the β5i subunit than the constitutive β5 subunit and other immunoproteasome subunits [54]. Increasing studies indicate that inhibition of the β5i subunit by PR-957 is promising for treating inflammatory and autoimmune diseases, with limited side effects [18,54–57]. Here we extended the therapeutic potential of PR-957 and immunoproteasome inhibition in atherosclerosis, , however, this needs further verification in other animal models. 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