RvD2 Limits Senescent Cell Accumulation in Atherosclerotic Plaques

Abstract

Atherosclerosis is a non-resolving inflammatory disease, and mechanisms to promote inflammation resolution, reduce vascular injury and promote repair in atherosclerosis are unmet needs. Specialized pro-resolving mediators (SPMs), like Resolvins, in part, mediate inflammation resolution and limit atherosclerosis progression. Uncovering processes associated with their protective actions are of interest. Senescent cells are maladaptive in atherosclerosis, and their accumulation promotes necrotic core formation in plaques. The SPM Resolvin D2 (RvD2) reduces plaque necrosis in part through its G-protein coupled receptor (GPCR), called GPR18. Here, we show how RvD2 can limit senescent cell accumulation in vivo and in vitro. Loss of myeloid GPR18 in Ldlr^−/− mice led to increased accumulation of senescent cells, and RvD2 treatment in Ldlr^/− mice led to decreased accumulation of senescent cells in plaques. We found that senescent macrophages are not readily efferocytozed due to elevated “don’t eat me” signals called CD24 and CD47. Knockdown or blockade of these signals improved senescent macrophage clearance, but not as efficient as efferocytosis of apoptotic cells in vitro. RvD2 treatment to senescent macrophages in vitro increased Cleaved Caspase-3 (an apoptosis marker) but did not impact the levels of CD24 or CD47. RvD2 enhanced the clearance of senescent macrophages but knockdown or blockade of CD24 and CD47 were also required for efficient clearance. Our work provides a cellular mechanism in which RvD2 treatment may limit plaque necrosis through decreasing senescent macrophages in plaques.

1. Introduction

Atherosclerosis is a non-resolving inflammatory disease and mechanisms to limit atherosclerosis progression and promote repair in the arterial wall are of significant clinical interest[1–3]. Resolution of inflammation (or inflammation resolution) is an active process that is regulated in part through the biosynthesis of specialized pro-resolving mediators (SPMs) like resolvins[4]. The SPM Resolvin D2 (or RvD2), binds and signals through a specific G-protein coupled receptor called GPR18[5], which is protective in atherosclerosis models[6–8], yet mechanisms associated with these actions are not known.

Efferocytosis, or the clearance of dead cells by macrophages, is a critical cellular program of inflammation resolution[9, 10]. Indeed, poor clearance of dead or aberrant cells is a major driver of necrotic core development in atherosclerosis[11] and the upregulation of “don’t eat me signals” are one of several mechanisms that derange efferocytosis and impair resolution in atherosclerosis [12, 13]. Accumulation of senescent cells (SCs) has emerged as a driver of necrotic cores and atherosclerosis progression[14–16] and we found that senescent macrophages (SC-macs) are poor efferocytes[15]. There is limited knowledge on how senescent cells are cleared endogenously[14, 17]. Recent work suggests that the “don’t eat me” signals CD24 and CD47 on senescent epithelial cells limit their clearance[18], but additional mechanisms are needed to understand how to efficiently clear senescent cells.

Here, we observed that the loss of myeloid GPR18 led to increased SC-macs accumulation in atherosclerotic plaques. In addition, Ldlr^−/− mice treated with RvD2 showed reduced SC-mac accumulation in plaques. We also observed that SC-macs had increased levels of CD24 and CD47, which contributed to their poor clearance in vitro. Blockade of these signals on SC-macs improved their clearance by bone marrow derived macrophages (BMDMs), but not to the same degree as apoptotic cells. RvD2 treatment to SC-macs in vitro increased cleaved caspase-3 (an apoptosis marker) but did not impact the levels of CD24 or CD47. RvD2 improved the clearance of SC-macs, but knockdown of CD24 and blockade of CD47 were also required for efficient clearance. Together we found “don’t eat me” signals limit SC clearance in vivo and in vitro, and showed that RvD2 can enhance this clearance, suggesting a possible role as a new therapy in limiting plaque progression.

2. Material and Methods

2.2. Murine atherosclerosis.

All mice were socially housed in standard cages at 22°C under a 12-hr. light and 12-hr. dark cycle at the Albany Medical College animal facility. Mice were fed Western Diet (TD.88137) Adjusted calories diet (42% from fat, Inotiv, West Lafayette, IN). All procedures were performed according to the animal protocols approved by the Albany Medical College Institutional Animal Care and Use Committee. All experiments related to fl/fl→Ldlr^−/− mice and mKO→Ldlr^−/− → mice and Veh vs. RvD2 treatment to Ldlr^−/− mice were performed as in Lipscomb et al[7].

2.3. Atherosclerotic plaque immunofluorescence

2.3.1. aSMC, Mac2, p21 Staining:

Frozen aortic root sections were fixed with acetone (179124-1L Acetone, Sigma-Aldrich, St. Louis, MO) for 10 minutes at −20°C. Sections were permeabilized with 0.05% Triton X-100 (Triton^™ X-100, Cat #9036-19-5, Sigma Aldrich Inc. St. Louis, MO) for 10 minutes. Sections were incubated with blocking buffer (1% Bovine Serum Albumin (BSA) + 10% Goat Serum) for 1 hour at room temperature. Sections were then incubated with rabbit anti-mouse p21 (Cat# 28248-1-AP, Proteintech. Rosemont, IL) at a 1:200 dilution, rat anti-mouse Mac2 (Cat# CL8940AP, Cedarlane. Burlington, NC) at a 1:10,000 dilution[19], and mouse anti-mouse Alpha smooth muscle actin (Cat# 67735-1-Ig, Proteintech. Rosemont, IL) at a 1:200 dilution, followed by a drop of M.O.M (Mouse on Mouse blocking reagent, Cat# MKB-2213-1. Vector Labs, Burlingame, CA) overnight at 4°C in 1% BSA. On the following day, sections were washed 3 times with 1xPBS and incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific, Cat# A-11001. Waltham, MA), Alexa Fluor 594 goat anti-rat secondary antibody (Thermo Fisher Scientific, Cat# A-11007. Waltham, MA), and Alexa Fluor 647 goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Cat# A-21245. Waltham, MA) at 1:200 dilution in 1% BSA + 5% Goat Serum for 2 hours at room temperature. Sections were then washed with 1x PBS. Nuclei were stained with Hoechst (Cat# H3570, Invitrogen by Thermo Fisher Scientific. Waltham, MA) for 10 mins, and images were acquired immediately on a Leica Thunder confocal microscope at 20x magnification and analyzed with Image J software (doi:10.1038/nmeth.2019). Area of Mac2+ plaque was obtained by doing a threshold in the region of interest (ROI), αSMC+ was subtracted from the ROI after creating an inverse ROI of the αSMC+ expression. With a plaque ROI that was positive for Mac2 and negative for αSMC, we measured the expression percentage of p21 in that area.

2.3.2. SMC, Mac2, p16 Staining:

Frozen aortic root sections were fixed with acetone (179124-1L Acetone, Sigma-Aldrich, St. Louis, MO) for 10 minutes at −20°C. Sections were permeabilized with 0.05% Triton X-100 (Triton^™ X-100, Cat #9036-19-5, Sigma Aldrich Inc. St. Louis, MO) for 10 minutes. Sections were incubated with blocking buffer (1% Bovine Serum Albumin (BSA) + 10% Goat Serum) for 1 hour at room temperature. Sections were then incubated with rabbit anti-mouse p16INK4a/CDKN2A (Cat# PA5-20379, ThermoFisher. Waltham, MA) at a 1:100 dilution, rat anti-mouse Mac2 (Cat# CL8940AP, Cedarlane. Burlington, NC) at a 1:10,000 dilution[19], and mouse anti-mouse Alpha smooth muscle actin (Cat# 67735-1-Ig, Proteintech. Rosemont, IL) at a 1:200 dilution, followed by a drop of M.O.M (Mouse on Mouse blocking reagent, Cat# MKB-2213-1. Vector Labs, Burlingame, CA) overnight at 4°C in 1% BSA. On the following day, sections were washed 3 times with 1xPBS and incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific, Cat# A-11001. Waltham, MA), Alexa Fluor 594 goat anti-rat secondary antibody (Thermo Fisher Scientific, Cat# A-11007. Waltham, MA), and Alexa Fluor 647 goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Cat# A-21245. Waltham, MA) at 1:200 dilution in 1% BSA + 5% Goat Serum for 2 hours at room temperature. Sections were then washed with 1x PBS. Nuclei were stained with Hoechst (Cat# H3570, Invitrogen by Thermo Fisher Scientific. Waltham, MA) for 10 mins, and images were acquired immediately on a Leica Thunder confocal microscope at 20x magnification and analyzed with Image J software (doi:10.1038/nmeth.2019). Same method was used for analyzing the p16 area percentage as in p21.

2.3.3. αSMC, Mac2, IL-1 beta Staining:

Frozen aortic root sections were fixed with acetone (179124-1L Acetone, Sigma-Aldrich, St. Louis, MO) for 10 minutes at −20°C. Sections were permeabilized with 0.05% Triton X-100 (Triton^™ X-100, Cat #9036-19-5, Sigma Aldrich Inc. St. Louis, MO) for 10 minutes. Sections were incubated with blocking buffer (1% Bovine Serum Albumin (BSA) + 10% Goat Serum) for 1 hour at room temperature. Sections were then incubated with rabbit anti-mouse IL-1 beta (Cat# 26048-1-AP, Proteintech. Rosemont, IL) at a 1:500 dilution, rat anti-mouse Mac2 (Cat# CL8940AP, Cedarlane. Burlington, NC) at a 1:10,000 dilution[19], and mouse anti-mouse Alpha smooth muscle actin (Cat# 67735-1-Ig, Proteintech. Rosemont, IL) at a 1:200 dilution, followed by a drop of M.O.M (Mouse on Mouse blocking reagent, Cat# MKB-2213-1. Vector Labs, Burlingame, CA) overnight at 4°C in 1% BSA. On the following day, sections were washed 3 times with 1xPBS and incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific, Cat# A-11001. Waltham, MA), Alexa Fluor 594 goat anti-rat secondary antibody (Thermo Fisher Scientific, Cat# A-11007. Waltham, MA), and Alexa Fluor 647 goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Cat# A-21245. Waltham, MA) at 1:200 dilution in 1% BSA + 5% Goat Serum for 2 hours at room temperature. Sections were then washed with 1x PBS. Nuclei were stained with Hoechst (Cat# H3570, Invitrogen by Thermo Fisher Scientific. Waltham, MA) for 10 mins, and images were acquired immediately on a Leica Thunder confocal microscope at 20x magnification and analyzed with Image J software (doi:10.1038/nmeth.2019). We measured the percentage area of IL-1B in the plaque.

2.3.4. CD24, CD47 and CD68 Staining:

Frozen aortic root sections were fixed with 100% methanol for 10 min at −20°C. Fixed sections were incubated with blocking buffer (1% Bovine Serum Albumin (BSA) + 10% Goat Serum) for 1 hr. at room temperature. Sections were then incubated with rat anti-mouse CD24 (Cat# 101802 Biolegend. San Diego, CA) or rat anti-mouse CD47 (Cat# 127502 Biolegend. San Diego, CA) at a 1:100 dilution, with rabbit anti-mouse CD68 (Cat# 97778S Cell Signaling Technologies. Danvers, MA) at a 1:400 dilution[19] overnight at 4°C in 1% BSA. On the following day, sections were then washed 3x with 1x PBS and incubated with Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific, Cat# A-11008. Waltham, MA)and Alexa Fluor 647 goat anti-rat (Thermo Fisher Scientific, Cat# A-21247. Waltham, MA) at 1:200 dilution in 1% BSA + 5% Goat Serum for 2 hrs. at room temperature. Sections were then washed with 1 time with PBS. Nuclei were stained with Hoechst (Cat# H3570, Invitrogen by Thermo Fisher Scientific. Waltham, MA) for 10 mins. Images were acquired immediately on a Leica SPE confocal microscope at 40x magnification and analyzed with Image J software (doi:10.1038/nmeth.2019). CD24⁺ and CD68⁺ cells or CD47⁺ and CD68⁺ were counted and expressed as a percentage of total lesion cells.

2.4. Peritoneal Cell Collection for in vitro Senescent Cells

Senescent macrophages (SC-macs) were generated as in Sadhu et al[15]. Briefly, 8-week-old C57BL/6 mice received peritoneal injections of 1mg/mL of Zymosan (Z4250, Zymosan A from Saccharomyces cerevisiae. Sigma-Aldrich, St. Louis, MO) to induce inflammatory responses in the peritoneum over 72 hrs. [13]. After 72 hrs., mice were lavaged to obtain peritoneal macrophages using sterile 5mL Phosphate-buffered saline (21-040-CM, Corning^® Phosphate-Buffered Saline, 1X without calcium and magnesium, pH 7.4 ± 0.1, Corning, Corning, NY). After collection, tubes were centrifuged at 400rpm for 5 minutes. Cell pellet was resuspended in DMEM supplemented with L-cell media (20% L-cell media, 10% FBS, 1% Penicillin-streptomycin in Dulbecco’s Modified Eagle’s Medium). Cells were plated in a non-treated 6-well plate, each well containing 2x10^6 cells. Cells were allowed to adhere to the plate overnight. Cells were then irradiated at 5Gy using a low-dose rate irradiator (Gammacell 40 Exactor, Best Theratronics, Ottawa, ON, Canada). After irradiation cells were left at 37C for 72 hrs. to allow for senescence.

2.5. Efferocytosis in vitro

Experiments were done as in Hosseini et al[20]. Briefly, bone marrow derived macrophages (BMDMs) from C57BL/6 mice were enumerated and then stained with PKH26 (Cat# PKH26GL, Sigma Aldrich Inc. St. Louis, MO) according to the manufacturer’s instructions. The red-labeled BMDMs were plated on 8 well coverslips (Cat#155411, Lab-Tek, ThermoFisher Scientific, Waltham, MA), with 125,000 cells per well the day prior to the experiment. Apoptotic Cells were obtained from BMDMs after treating with 1uM staurosporine (Cat# ALX-380-014-C250, Enzo Life Sciences) for 48 hrs. Control, Apoptotic, and SCs were then stained green with PKH67 (Cat# PKH67GL, Sigma Aldrich Inc. St. Louis, MO) as per manufacturer’s instructions. Cells were then cocultured with red-labeled BMDMs at a 2:1 ratio for 1 hr. at 37.0 C. Coverslips were washed 3 times with PBS (21-040-CM, Corning^® Phosphate-Buffered Saline, 1X without calcium and magnesium, pH 7.4 ± 0.1, Corning, Corning, NY), fixed with 4% PFA (Paraformaldehyde Solution, 4% in PBS, Thermo Scientific Chemicals. Waltham, MA) for 15 minutes, and nuclei were then stained with Hoechst (Cat# H3570, Invitrogen by Thermo Fisher Scientific. Waltham, MA) for 5 minutes. Cells were then subjected to fluorescence imaging with a Leica Confocal Microscope at 40x magnification. Ten different fields were acquired per well/group and an efferocytic event was defined as a macrophage containing green apoptotic cells. Results were expressed as the percentage of efferocytosis per total macrophages.

2.5.1. siCD24 transfection:

We generated SCs by incubating peritoneal macrophages for 72 hrs. following 5.0 Gy of irradiation as in Sadhu et al [13] in 6-wells at 2x10^6 cells per well. 42 hrs. after irradiation Control or SCs were incubated with Opti-MEM medium (31985062, Gibco, ThermoFisher Scientific, Waltham, MA) containing 600ng of non-targeted scrambled RNA (AllStars Negative Control siRNA, 1027280, Qiagen. Germantown, MD) or CD24 siRNA (FlexiTube GeneSolution GS12484 for Cd24a, GS12484, Qiagen. Germantown, MD) per well, and HiPerFect Transfection Reagent (301704, Qiagen. Germantown, MD) in a 200:1 ratio. Each well received 1200uL of its corresponding treatment. After 4 hrs., 800uL of fresh L-cell DMEM (20% L-Cell, 10% FBS and 5% Pen-Strep in DMEM) was added to each well with the transfection medium. Following 16 hrs. overnight, the now diluted transfection medium was replaced with fresh L-cell DMEM. Cells were incubated at 37C for 72 hrs. to allow for protein silencing prior to use in experiments. CD24 silencing was assessed with Quantitative PCR CD24 forward, reverse, and flow cytometry with anti-mouse CD24-PE antibody (Cat# 101807. Biolegend. San Diego, CA).

2.5.2. CD47 blockade:

Cells were treated with anti-CD47 (Cat# BE0283, MIAP410, BioXCell, Lebanon, NH) at a 10ug/mL concentration for 30 minutes at 37C[13]. Cells receiving the control treatment received IgG (Cat# BE0083, MOPC-2, BioXCell, Lebanon, NH) at a 10ug/mL concentration, with the same incubation time. After treatment, cells were suspended back in L-cell media.

2.5.3. RvD2 treatment:

Cells were treated with 10nM Resolvin D2 (RvD2) (10007279, Cayman Chemical, Ann Arbor, MI) in L-cell DMEM media. Treatment lasted 24 hrs.

2.6. CD24 and CD47 Surface levels Flow Cytometry

Flow cytometry was used to assess anti-mouse CD24-PE (Cat# 101807, Biolegend. San Diego, CA) and anti-mouse CD47-APC (Cat# 127526, Biolegend. San Diego, CA) on control cells and SCs. The mean fluorescence intensity of CD24 and CD47 were analyzed with FlowJo software. For Annexin V experiments we stimulated peritoneal macrophages with 800nM staurosporine or STS (Cat# ALX-380-014-C250Enzo Life Sciences,), in serum-free media, for 48 hrs. at 37 °C to stimulate apoptosis [13] and generate apoptotic cells for use in experiments. Flow cytometry was used to assess Annexin V (Cat# 640945, Biolegend. San Diego, CA) in Control, SC and Apoptotic cells. The percentage of Annexin V positive cells were analyzed with FlowJo software.

2.7. Immunofluorescence staining of Cleaved Caspase 3, CD24, and CD47 in SCs and Apoptotic Cells.

Peritoneal macrophages were plated in 8-well u-slides (Cat# 80827, Ibidi GmbH, Gräfelfing, Germany), with around 50,000 cells in each plate. Cells were fixed with 4% PFA at 37C for 15 minutes, followed by three PBS washes. Permeabilization was done using 0.2% Triton X-100 for 10 minutes at room temperature. Cells were then blocked for 1 hr. at room temperature on a slow shaker, using 200 uL per well of blocking buffer (5% Fish Skin Gelatin (G7041, Sigma Aldrich Inc. St. Louis, MO), 0.5g Bovine Serum Albumin, 500uL of 10% Triton-X 100, in 50mL of 1X PBS). After blocking, cells were incubated with CD24 rat monoclonal primary antibody (Cat# MAB8547, R&D Systems by biotechne, Minneapolis, MN) at a 1:500 dilution, CD47 mouse monoclonal primary antibody (Cat# 66304-1-Ig, Proteintech. Rosemont, IL) at a 1:500 dilution, Cleaved Caspase 3 (Asp175) rabbit polyclonal antibody (Cat# 9661, Cell Signaling Technologies. Danvers, MA) at a 1:400 dilution, and a drop of M.O.M (Mouse on Mouse blocking reagent, Cat# MKB-2213-1. Vector Labs, Burlingame, CA) in blocking buffer overnight at 4C. The next day cells were washed 3 times with 1XPBS, followed by incubation with Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific, Cat# A-32723. Waltham, MA), Alexa Fluor 594 goat anti-rat secondary antibody (Thermo Fisher Scientific, Cat# A-11007. Waltham, MA), and Alexa Fluor 647 goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Cat# A-21245. Waltham, MA) at 1:1000 dilution in blocking buffer for 1 hr. at room temperature. Afterward cells were washed 3 times with PBS, nuclei were stained with Hoechst (Cat# H3570, Invitrogen by Thermo Fisher Scientific. Waltham, MA) for 10 mins at room temperature, followed by 5 minutes of post fixation with 4% PFA at room temperature, finalizing with washing 3 times with PBS. Cells were immediately acquired on a Leica LSM880 Confocal Airyscan with z-stacks of 15 slices at 63x magnification, processed through Imaris software (v10.2.0 Oxford Instruments, RRID:SCR_007370) to create maximum intensity projections (MIPs) of each cell, and analyzed with Image J software (doi:10.1038/nmeth.2019). Each cell was traced, and the mean fluorescence intensity (MFI) of each channel was recorded for at least 10 cells per group.

2.8. Statistical analysis

For all in vivo studies, mice were randomly assigned to their respective groups. Results are represented as mean ± SEM. Prism (GraphPad, La Jolla, CA) software was used for statistical analysis, and statistical differences were determined using the student’s t test, Kruskal Wallis with a Dunn’s multiple comparison test, or one-way ANOVA with a Sidak’s multiple comparisons depending on the contexts. Details regarding statistical tests can be found in the figure legends. Data were considered statistically significant with a p-value less than than 0.05.

3. Results

3.1. RvD2-GPR18 regulates senescent macrophages in plaques

We have previously shown that loss of GPR18 on myeloid cells promotes plaque necrosis in mice[7], therefore we questioned whether this athero-progressive phenotype was associated with increased senescent cells. To test this, we transplanted wild type (fl/fl) or GPR18 myeloid knockout (mKO) bone marrow into Ldlr^−/− mice and placed them on Western diet (WD) for 12 weeks to allow for atherosclerosis progression[7]. We interrogated plaques for the expression of p21 (a marker associated with SC-macs [15]) shown in cyan, Mac2 (a marker for macrophages) shown in red, and α smooth muscle cell actin (a marker for smooth muscle cells) show in green (Figure 1A). Gpr18 mKO→Ldlr^−/− transplanted mice had an increased percent area of p21⁺Mac2⁺αSMC⁻ cells compared with fl/fl→Ldlr^−/− controls (Figure 1A,B). There were no differences in the percent area of p21⁺Mac2⁻αSMC⁺ cells (Figure 1A,C), suggesting that the loss of myeloid GPR18 is associated with an accumulation of SC-macs. Another senescence marker is p16 and we observed that there were no significant differences in the percentage of p16⁺Mac2⁺αSMC⁻ cells or p16⁺Mac2⁻αSMC⁺ cells between the groups (S. Figure 1A–C), suggesting that these p21 expressing SC-macs may be a subset of senescent cells that are regulated by myeloid GPR18 [15]. Lastly, we performed immunofluorescence to determine whether the loss of myeloid GPR18 impacts IL-1B levels in plaques and found no differences between the groups (S. Figure 2).

Fig. 1. Loss of the Gpr18 receptor on myeloid cells increased and treatment with RvD2 to Ldlr^−/− decreased p21⁺Mac2⁺aSMC⁻ cells in atherosclerotic plaques.

(A-C) fl/fl or mKO bone marrow was transferred into Ldlr^−/− mice and fed a WD for 11 wks. Aortic roots were subjected to immunofluorescence staining of and DAPI in blue, alpha smooth muscle actin (aSMC) in green, Mac2 in red, p21 in cyan, and imaged with a Thunder confocal microscope. Images were quantified using Image J. The scale is bar is 250 um. The percentage of p21⁺Mac2⁺aSMC⁻ or p21⁺Mac2⁻aSMC⁺ plaque areas are shown on the right. (D-F) Ldlr^−/− mice were fed WD diet for 14 weeks, after which mice were then treated with Vehicle (PBS) or RvD2 (50 ng/mouse) for an additional 3 weeks while on WD. Imaging and quantification were performed as in panels A-C. All results were analyzed using Student’s t test and are represented as mean ± SEM with each dot representing an individual mouse. *p<0.05, ***p<0.001.

We and others published that RvD2 limits plaque necrosis in atherosclerosis[6–8] and therefore questioned whether RvD2 treatment to Ldlr^−/− mice could limit SC-macs in atherosclerotic plaques. To achieve this, Ldlr^−/−mice were fed WD for 12 weeks, after which mice received intraperitoneal injections of either Veh or RvD2 (50ng) for an additional 3 weeks while still on WD[7]. As above, we performed immunofluorescence staining of p21, Mac2 and, αSMC actin on aortic roots and observed that treatment with RvD2 significantly decreased the percentage of p21⁺Mac2⁺αSMC⁻ cells (Figure 1D,E). There were no differences in the percent area of p21⁺Mac2⁻αSMC⁺ cells (Figure 1D,F), suggesting that RvD2 treatment limits SC-macs in plaques. Lastly, we observed that there were no significant differences in the percentage of p16⁺Mac2⁺αSMC⁻ cells or p16⁺Mac2⁻αSMC⁺ cells between the Veh and RvD2-treated groups (S. Figure 1D–F), suggesting that these p21 expressing SC-macs may be a subset of senescent cells that are regulated by RvD2. Collectively, these results propose that the RvD2-GPR18 axis is associated with the accumulation of SC-macs in advanced atherosclerotic plaques. Next, we questioned why senescent cells are not readily cleared, and whether treatment with RvD2 could promote their clearance.

3.2. Senescent cells are not readily cleared and express CD24 and CD47

SCs accumulate in atherosclerotic plaques[14, 15, 21], which suggests that their clearance may be impaired. We recently developed an in vitro method to study SC-macs [15] and performed efferocytosis assays to determine if SC-macs were readily cleared. For these experiments, we tested whether live healthy control macrophages, SC-macs, or apoptotic macrophages were able to be taken up by otherwise healthy bone marrow-derived macrophage efferocytes. First, we observed that apoptotic cells were readily efferocytosed (Figure 2A), which contrasted with SC-macs which were not taken up (Figure 2A). As expected, healthy macrophages were not efferocytosed either (Figure 2A).

Figure 2. SC-macs are not readily efferocytosed and have elevated levels of the “don’t eat me” signals CD24 and CD47.

(A) Efferocytosis assays were performed as described in the methods section. The data are presented as percent efferocytosis (% of senescent cells engulfed by BMDMs). (B) Percent Annexin V positive cells (expressed as percent out of total cells) were measured using flow cytometry. (C) Surface levels of CD24 (D) and CD47 in unstained (“US”, light grey), control (“Ctrl”, dark grey), and SC (“SC”, blue) macrophages as measured by flow cytometry (left) and the relative expression of CD24 or CD47 in Ctrl versus SC macs quantified (right). All results are presented as mean ± SEM and each dot represents an individual experiment. One-way ANOVA with Sidak’s multiple comparisons test was used for (A&B), Students T-test was used for (C&D). *p<0.05, **p<0.01, ***p<0.001, ****<0.0001.

The balance between “eat me” and “don’t eat me” signals is critical for efficient efferocytosis. To further investigate why SCs were not ingested, we first examined the surface levels of Annexin V which detects the well-known “eat me” signal phosphatidylserine (PS) by flow cytometry (Figure 2B). Compared to apoptotic controls (AC), SC-macs had significantly lower Annexin V as measured as a percent of total cells (Figure 2B). Indeed, Annexin V in SC-macs was comparable to that of healthy controls (Figure 2B).

An increase in the “don’t eat me” signals CD24 and CD47 have been shown to limit cell clearance in a variety of contexts, including atherosclerosis[12, 13, 22]. Therefore, we next investigated the expression of these markers on SC-macs by flow cytometry and found that SC-macs had significantly higher levels of CD24 (Figure 2C), and CD47 (Figure 2D), compared with healthy control macrophages. Collectively, these data suggest that SC-macs have increased levels of “don’t eat me” signals without a concomitant increase in “eat me” signals.

3.3. “Don’t eat me” signals CD24 and CD47 limit the clearance of SCs in vitro

Since we observed that SC-macs had increased levels of CD24 and CD47 relative to control cells, we next questioned whether knock down or blockade of these receptors was sufficient to promote clearance. We first targeted CD24 with siRNA (siCD24) and were able to successfully knock down mRNA (S. Figure 3A) and protein (S. Figure 3B), as validated via qPCR and flow cytometry respectively. To ensure we were evaluating the role of increased expression of CD24 as a “don’t eat me” signal, siRNA knockdown of CD24 decreased expression of CD24 comparable to the levels observed in healthy cells (S. Figure 3A, 3B). We performed in vitro efferocytosis assays with control and SC-macs transfected with either scrambled or CD24 siRNA and found that SC-macs transfected with CD24 siRNA were taken up significantly more as compared to SC-macs transfected with scrambled siRNA and control cells (Figure 3B). The knockdown of CD24 did not promote clearance of otherwise healthy macrophages (Figure 3B). These data suggest that knockdown of CD24 increases clearance of SC-macs.

Figure 3. Knockdown of CD24 or blockade of CD47 on SC-macrophages enhances their clearance.

(A) Representative images of in vitro efferocytosis assays with BMDMs (red), and senescent cell (SC) “food” (green), and cell nuclei (blue) as stained with DAPI. Arrows (white) indicate cells that have efferocytosed with ingested material presenting as yellow or green. Scale=45μm. The number of cells with ingested material per total cells were quantified using ImageJ, where an efferocytosing cell was defined as any BMDM (red) containing ingested material (yellow/green) and “total cells” defined as total red BMDMs. (B) Percent efferocytosis of apoptotic cells in control (Ctrl) or senescent (SC) BMDMs in the presence or absence of CD24 knockdown, (C) IgG or anti-CD47 (D), or combination of both CD24 knockdown and anti-CD47 were analyzed as above. All results are presented as mean ± SEM and each dot represents an individual experiment. One-way ANOVA with Sidak’s multiple comparisons test was used. **p<0.01, ***p<0.001, ****p<0.0001.

To next address a potential role for CD47 in regulating clearance of SC-macs, we performed in vitro efferocytosis assays on control and SC-macs incubated with IgG or anti-CD47 blocking antibody (aCD47). SC-macs incubated with aCD47 were taken up significantly more than IgG incubated control SC-macs (Figure 3C). Additional efferocytosis assays on SC-macs following transfection with siCD24 in conjunction with aCD47 treatment showed that there was not an additive effect of combined treatments in the percent efferocytosis of SC-macs in vitro. (Figure 3D). Importantly, while knockdown of CD24, treatment of aCD47, or a combination of both were able to significantly increase clearance, neither treatment was as efficient as apoptotic cell uptake (Figure 3D). These data suggest that while blocking or silencing “don’t eat me signals” improves clearance, this alone is not sufficient for efficient efferocytosis, therefore other mechanisms such as those involved with promoting cell death and “eat me” signals may be involved.

3.4. RvD2 increases clearance of senescent macrophages

Because we observed that RvD2 treatment of Ldlr^−/− mice resulted in reduced p21⁺Mac2⁺ cells in the plaque (Figure 1B), and because knockdown or blockade of “don’t eat me signals” was not sufficient to promote efficient SC-macs clearance in vitro, we next questioned whether RvD2 modulated the levels of CD24 and CD47 to promote clearance. Immunofluorescence and confocal imaging analysis showed that RvD2 treatment did not significantly modulate the levels of CD24 (red) or CD47 (green) on SC-macs (Figure 4A). Because efficient clearance is associated with apoptotic cell death, we examined if RvD2 increased apoptosis. We treated SC-macs with either Veh or RvD2 (10 nM) for 24 hrs. and evaluated the levels of cleaved caspase-3 using immunofluorescence staining and confocal imaging as above. RvD2 treatment significantly increased levels of cleaved caspase-3 (shown in pink) compared with control SC-macs, but not to the levels observed in apoptotic cell controls (Figure 4A). These results suggest that RvD2 increases apoptotic markers.

Figure 4. RvD2 increases cleaved caspase 3 and clearance of senescent macrophages.

(A) Senescent or apoptotic macrophages treated with either Veh or RvD2 (10nM, 24 hrs., 37C) were subjected to immunofluorescence staining and confocal imaging of CD24 (red), CD47 (green) and cleaved caspase-3 (pink). All cells were counterstained with DAPI (blue). Imaris software was used to analyze the mean fluorescence intensity (MFI) of CD24, CD47, and Cleaved Caspase 3. (B) Efferocytosis assays were conducted as described in the methods. Images were taken with a Leica SPE confocal microscope at 40x magnification and quantified using Image J. Senescent macrophages (green), macrophage efferocytes (red), DAPI (blue), and ingested material (yellow/green) are shown and the scale bar= 45μm. All results are mean ± SEM and each dot represents an individual experiment. The data are presented as percent efferocytosis. One-way ANOVA with Sidak’s multiple comparisons test was used. *p<0.05, **p<0.01.

Based on these findings, we sought to further investigate whether a combination treatment including RvD2, knockdown of CD24 and blockade of CD47 would lead to efficient efferocytosis. First, we observed that RvD2 alone increased the clearance of SC-macs (green) above vehicle in a manner similar to siCD24 or aCD47 treatments (Figure 4B). However, a combination of RvD2, CD24 knockdown, and CD47 blockage resulted in significantly increased efferocytosis of SC-macs, which was comparable to that observed with apoptotic cells (Figure 4B). These data suggest that multiple mechanisms involving the initiation of apoptosis and the blockade of “don’t eat me signals” are critical for efficient clearance of SC-macs.

3.5. The RvD2-GPR18 axis modulated levels of CD24 and CD47 on plaque macrophages.

Because SC-macs expressed elevated levels of CD24 and CD47 in vitro, we further questioned whether the RvD2-GPR18 axis would impact the number of cells that express CD24 and CD47 in atherosclerotic plaques. To examine this, we performed immunofluorescence co-staining of CD24 or CD47 with CD68 (a macrophage marker) in aortic roots. First, we found that loss of the GPR18 receptor on myeloid cells significantly increased the percentage of CD68⁺CD24⁺ cells (Figure 5A) and CD68⁺CD47⁺ cells (Figure 5B). Loss of GPR18 significantly increased the percentage of CD68⁺ cells in atherosclerotic plaques (Figure 5C). Next, we observed that RvD2 treatment of Ldlr^/− mice as in Figure 1. led to a significant decrease in the percent of cells positive for “don’t eat me” signals CD24 (Figure 5D) and CD47 (Figure 5E). In addition, our findings show that RvD2 treatment significantly reduced the percentage of cells positive for CD68 in plaques compared to those from Veh treated mice (Figure 5F). Collectively, these data suggest that the RvD2-GPR18 axis regulates the percentage CD68⁺CD24⁺ and CD68⁺CD47⁺ in plaques.

Figure 5. Loss of the Gpr18 receptor on myeloid cells results in increased CD24⁺ and CD47⁺ macrophages and treatment with RvD2 to Ldlr^−/− mice decreased the percentage of these cells in atherosclerotic plaques.

(A-C) Gpr18 fl/fl or mKO bone marrow was transferred into Ldlr^−/− mice fed WD for 11 wks. (D-F) Ldlr^−/− mice were fed WD diet for 14 weeks, after which mice were then treated with Vehicle (PBS) or RvD2 (50 ng/mouse) for an additional 3 weeks while on WD. Aortic roots were subjected to immunofluorescence staining of CD24, CD47, or CD68 and images were taken with a Leica confocal microscope and quantified using Image J. The percent of CD24⁺CD68⁺ cells (A,D), CD47⁺CD68⁺ cells (B,E) and CD68⁺ cells (C,F) are shown. All results are mean ± SEM and each dot represents an individual mouse. Student’s t-test was used. *p<0.05, ***p<0.001.

4. Discussion

Herein, our data suggests that RvD2 may be an important therapeutic to limit SC-macs in atherosclerotic plaques in vivo. Moreover, we observed that SC-macs have elevated levels of CD24 and CD47, and that efficient efferocytosis involved the knockdown and blocking of these signals, as well as RvD2 treatment, which initiated apoptosis of SC-macs.

Our work with SC-macs is in agreement with recent studies in which senescent fibroblasts and epithelial cells also had increased levels of CD47 and CD24[18]. These findings suggested that senescent cells were not only impaired in their ability to be cleared, by also limited a macrophage’s ability to clear apoptotic cells[18]. An interesting corollary is our previous work with necroptotic cells[13]. Necroptotic cells are dead which makes them fundamentally distinct from senescent cells. But, some similarities between necroptotic and senescent cells include elevated levels of CD47 and the release of pro-inflammatory cytokines/chemokines[23] and lipid mediators[20]. The increased levels of CD47 in necroptotic cells promote a delayed eating response, compared with apoptotic cells that resembles “nibbling” as opposed to whole cell engulfment that occurs with apoptotic cell clearance[13]. The observed impairment in SC clearance could also be due to due to a similar “nibbling” mechanism but more studies are needed to fully dissect this type of clearance. Another possibility is that senescent cells have a “senescent cell associated secretory phenotype” (SASP) that in several contexts also is associated with increased pro inflammatory cytokines/chemokines[24]. The SASP could also impact the clearance of senescence cells by acting in an autocrine manner that releases factors that upregulate “don’t eat me” signals and by acting in a paracrine fashion by released factors that impar efferocytosis. While outside of the aims of this project, additional studies regarding the role of SASP and efferocytosis would be of interest to better understand how to efficiently clear senescent cells.

Additionally, there have been various other studies demonstrating the importance of signals like CD24 and CD47 for limiting aberrant cell clearance in cancers [25–27], atherosclerosis[12, 13] and vascular inflammation in humans [28]. However, CD24 is not only a “don’t eat me” signal but is also an established marker for B-Cells[29] B-1 and marginal zone B cells that produce IgM may be pro-resolving and limit atherosclerosis[30, 31], therefore, directly targeting CD24 in the context of atherosclerosis may not be an ideal target. Importantly, blockade of CD47 has been shown to reduce vascular inflammation in humans[32] and limit atheroprogression in mice[12, 13], in part through the clearance of dead cells. Therefore, combination therapies to improve dead and senescent cell clearance in plaques would be beneficial.

In this regard, RvD2 levels were decreased as atherosclerosis progressed to the advanced stage and addition of RvD2 limited necrosis and promoted a pro-resolving macrophage phenotype in plaques[8]. Loss of GPR18 on myeloid cells resulted in increased plaque necrosis. Collectively, these data suggest that RvD2-GPR18 axis plays a causal role in limiting atherosclerosis progression. Work herein provides a novel cellular mechanism that suggests that RvD2 treatment may limit plaque necrosis through decreases in plaque SC-macs.

Figure 6. RvD2 Increases the Clearance of Senescence Macrophages.

Clearance of senescent macrophages (SC-macs) is hindered due to elevated surface levels of CD24 and CD47. Blockade of CD47, knockdown of CD24, or RvD2 treatment improves efferocytosis. RvD2 enhances cleaved caspase 3 in SC-macs, without impacting the levels of CD47 and CD24 and so a combination of all 3 approaches significantly increasing clearance of SC-macs that is commensurate to the level of clearance associated with apoptotic cells.

Data availability

The data that support the findings of this study are available upon reasonable request from the corresponding author.