CD137 signaling induces macrophage M2 polarization in atherosclerosis through STAT6/PPARδ pathway

Tianxin Geng, Yang Yan, Liangjie Xu, Mengfei Cao, Yu Xu, Jun Pu, Jin Chuan Yan

PII: S0898-6568(20)30105-4
DOI: https://doi.org/10.1016/j.cellsig.2020.109628
Reference: CLS 109628

To appear in: Cellular Signalling

Received date: 20 February 2020
Revised date: 25 March 2020
Accepted date: 31 March 2020

Please cite this article as: T. Geng, Y. Yan, L. Xu, et al., CD137 signaling induces macrophage M2 polarization in atherosclerosis through STAT6/PPARδ pathway, Cellular Signalling (2019), https://doi.org/10.1016/j.cellsig.2020.109628

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CD137 signaling induces macrophage M2 polarization in atherosclerosis through STAT6/PPARδ pathway
Tianxin Geng1*, Yang Yan2*, Liangjie Xu1, Mengfei Cao1, Yu Xu1, Jun Pu2 and Jin Chuan Yan1
1Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang,

Jiangsu Province, 212000, China.

2Department of Cardiology, Ren Ji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200135, China.
*Contributed equally

Correspondence to: Jinchuan Yan, [email protected], and Jun Pu, [email protected].
Key words: atherosclerosis, CD137, macrophage polarization, peroxisome proliferator-activated receptor δ,signal transducers and activators of transcription 6
Running title: Geng Tianxin et al: CD137 induce M2 polarization

Abbreviations: ApoE-/-, apolipoprotein E–deficient; PPARδ, peroxisome proliferator-activated receptor δ; STAT6, signal transducers and activators of
transcription 6; CD137L, CD137 ligand; HUVECs, human umbilical vein endothelial cells; MBVECs, mouse brain microvascular endothelial cells, DMSO, dimethylsulfoxide; PBS, phosphate buffer saline; RT, room temperature; EdU,
5-Ethynyl-2’-deoxyuridine; EC, Endothelial cell; IL, interleukin; ELISA
enzyme- linked immunosorbent assay; iNOS, inducible nitric oxide synthase; FIZZ1, found in inflammatory zone 1; ARG1, Arginase 1; MRC1, mannose receptor 1.

Abstract

CD137 signaling plays an important role in the formation and development of atherosclerotic plaques. The purpose of the present study was to investigate the effects of CD137 signaling on macrophage polarization during atherosclerosis and to explore the underlying mechanisms. The effect of CD137 signaling on macrophage phenotype in atherosclerotic plaques was determined by intraperitoneal injection of agonist-CD137 recombinant protein in apolipoprotein E–deficient ( ApoE-/-) mice, an established in vivo model of atherosclerosis. Murine peritoneal macrophages and RAW 264.7 cells were treated with AS1517499 and siPPARδ (peroxisome proliferator-activated receptor δ) to study the role of STAT6 (signal transducers and activators of transcription 6)/PPARδ signaling in CD137- induced M2 macrophage polarization in vitro. Results from both in vivo and in vitro experiments showed that CD137 signaling can transform macrophages into the M2 phenotype during the process of atherosclerotic plaque formation and regulate the angiogenic features of M2 macrophages. Furthermore, activation of the CD137 signaling pathway induces phosphorylation of STAT6 and enhances the expression of PPARδ. We further found that macrophage M2 polarization is reduced when the STAT6/PPARδ pathway is inhibited. Together, these data show a role for the STAT6/PPARδ signaling pathway in the CD137 signaling- induced M2 macrophage polarization pathway.

Introduction

A common pathological basis for acute cardiovascular events is the rupture of vulnerable atherosclerotic plaques. The development of atherosclerosis plaque is often accompanied by angiogenesis, which accelerates plaque bleeding and rupture [1]. It is now clear that macrophages play a key role in the formation of atherosclerotic plaques and angiogenesis, and the macrophage phenotype is reversible depending on microenvironmental cues. There are two types of macrophage polarization, which have different effects on the initiation and development of plaques. The M1 phenotype is mainly involved in plaque formation, whereas the M2 phenotype mainly contributes to the progression of plaques [2].
PPARδ is a member of the nuclear receptor transcription factor superfamily, and is downstream of STAT6. PPARδ regulates cell metabolism, inflammation, and differentiation [4]. Some studies suggest that activation of the STAT6/PPARδ pathway may induce gene expression in macrophages [5].
CD137 is a co-stimulatory molecule that plays an important role in the development of atherosclerosis [6]. Soluble CD137 is elevated in the blood of patients with acute myocardial infarction. Binding of CDl37 to its ligand (CDl37L) can trigger intercellular molecular signal transduction and produce various biological effects, including inducing T cell activation, releasing chemokines and cytokines, and intensifying the immune response[24]. Bartkowiak T et al. found that CDl37 was highly expressed in some tumor cells, and tumor growth could be inhibited by blocking the activation of CDl37 signal, suggesting that CDl37 signal may also be

involved in tumor formation[25]. Recent studies have shown that CDl37 is highly expressed in atherosclerotic plaques in human body, and activating CDl37 signal can lead to the release of various inflammatory factors, and has a biological effect, which can further affect the activation of downstream transcription factors[26]. Our previous studies found that CD137–CD137L interactions can exacerbate atherosclerosis [7]. In addition, we also demonstrated that CD137 signaling promotes angiogenesis in atherosclerotic plaques [8]. Jetten et al. [3] found that M2 macrophages, but not M1 macrophages, promote angiogenesis in vivo. However, the relationship between CD137 signaling and macrophage phenotype remains undefined and the molecular mechanisms remain largely unknown. Therefore, the present study aimed to explore the role of macrophage polarization in atherosclerosis and to decipher the signaling pathway that mediates the polarization of macrophages.

Materials and Methods

Reagents. Agonist-CD137 recombinant protein was purchased from Sangon Biotech (Shanghai, China). Antibodies against β-actin (3700S) and Arginase-1 (93668) were purchased from Cell Signaling Technology (Boston, USA). Antibodies against p-STAT6 (ab28829), STAT6 (ab32520), PPARδ (ab23673), iNOS (ab49999) and
CD68 (ab31630) were purchased from Abcam (Cambridge, USA). Antibodies for flow cytometry including Alexa Fluor®647-conjugated anti- mouse CD206 (565250), BV421 anti- mouse CD86 (564198), PE-conjugated anti- mouse F4/80 (565410), and PE-conjugated anti- mouse CD137 (558976) were purchased from BD Biosciences

(New Jersey, USA). CD137 (4-1BB) inhibitory monoclonal antibodies (16-1371-85) were purchased from eBioscience (California, USA). STAT6 inhibitor (AS1517499) and PPARδ antagonist (GSK3787) were purchased from MedChemExpress (New Jersey, USA).
Ethics statement and animal procedures. The experimental scheme was approved by the Animal Care and Use Committee of Jiangsu University (Jiangsu, China). Male, 8-week-old C57BL/6J mice were purchased from the animal center of Jiangsu University. Male, 8-week-old ApoE-/- mice were purchased from Vital River Laboratories (Beijing, China). All ApoE-/- mice were fed with a high fat diet and water ad libitum, and were euthanized at the end of each experiment. Thirty ApoE-/- mice were divided into five groups, each with n=6 animals, including the control group (treated with 50 μg/kg/day of isotype antibody), the agonist-CD137 group (treated with 50 μg/kg/day of the CD137 recombinant protein), the anti-CD137 group (treated with 50 μg/kg/day of the CD137 recombinant protein plus 50 μg/kg/day of the CD137 inhibitor), the anti-STAT6 group (50 μg/kg/day of the CD137 recombinant protein plus 10mg/kg/day of AS1517499), and the anti-PPARδ group (treated with 50 μg/kg/day of the CD137 recombinant protein plus 10mg/kg/day of GSK3787). All mice were injected intraperitoneally daily with the reagents listed above for four months.
Isolation of primary murine peritoneal macrophages. C57BL/6J mice were

intraperitoneally injected with 1 mL of 3% thioglycollate (Sigma, St. Louis, MO USA) every day for 3 d, and then were executed by cervical dislocation. After 3 min of

disinfection with 75% ethanol, 5 mL of precooled phosphate buffer saline (PBS) was injected into the abdominal cavity of mice. The abdomen of the mice was gently rubbed for 5 min. A small incision was made in the abdomen using ophthalmic scissors, and the abdominal cavity was flushed twice with a sterile pasteurized transfer pipet to recover about 8 mL of lavage fluid. The obtained lavage solution was centrifuged at 1000 rpm/min for 10 min to obtain a cell pellet, followed by resuspending the cell pellet in RPMI-1640 culture solution containing 10% FBS. Subsequently, the resuspension was inoculated in a petri dish and the unattached cells were washed after incubating for 2–3 h in a 37°C/5% CO2 cell incubator.
Cell culture. RAW264.7 cells, human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (MBVECs) were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. RAW264.7 cells and endothelial cells were cultured in Dulbecco’s modified eagle medium, and primary murine peritoneal macrophages were cultured in RPMI-1640 medium. All media contained 10% FBS and 100 U/mL penicillin/streptomycin. Cells were grown in a 5% CO2 humidified incubator at 37°C. The cells were passaged at the density of 70% to 80%.
Cell treatment. Agonist-CD137 recombinant protein (10 μg/mL) was used to stimulate CD137 signaling for 48 h. Cells were pretreated with the CD137 inhibitory antibody for 30 min to inhibit CD137 before agonist-CD137 recombinant protein stimulation. Cells were pretreated with STAT6 inhibitor AS1517499 (dissolved in DMSO (dimethylsulfoxide) to a final concentration of 10 M) for 30 min to inhibit

STAT6 before agonist-CD137 recombinant protein stimulation. DMSO was used as a negative control for STAT6 inhibitor AS1517499.
Cell transfection. The PPARδ siRNA duplexes were synthesized by Biomics Biotechnologies (Jiangsu, China) (see Table SI for PPARδ siRNA sequence duplexes). The siRNA and Lipofectamine 2000 (Invitrogen, California, USA) were diluted with Opti-MEM (Gibco, MA, USA) prior to mixing. The siRNA and Lipofectamine 2000 mixture was incubated for 20 min at room temperature (RT) and then added to the cell culture. Incubation was continued for 24 h, and the levels of PPARδ protein in each sample was determined using western blot.
Flow cytometry. Macrophages from different mouse treatment groups were collected

and prepared as a single cell suspension and incubated with cell surface fluorescent antibody F4/80 (1:200) and CD86 (1:200) or CD137 (1:200) (IgG antibody was added to the homotypic control group) in the dark for 30 min at 4°C. After washing, the cells were permeabilized and incubated with the intracellular fluorescent antibody CD206 (1:200) for 45 min. For each sample, at least 1×105 cells were analyzed using the BD FACS Calibur cytometer (Becton Dickinson, New Jersey, USA).
Quantitative PCR. Total RNA from RAW264.7 cells and murine peritoneal macrophages was extracted using TRIzol reagent (Invitrogen) per the manufacturer’s instructions. Reverse transcription was performed using Thermo Fisher RT reagents (California, USA), and analyzed by real- time quantitative PCR with SYBR Premix Ex TaqTMa (RR420A, Kyoto, Japan, TaKaRa). All primers were synthesized by Sangon Biotech (see Table SII for primers applied for qRT-PCR).

Western blotting. Macrophages were lysed with RIPA lysis and extraction buffer (89901, Pierce, California, USA), followed by centrifugation at 12000 rpm for 15 min at 4°C. The samples were stored in -20°C. The proteins were separated on an SDS-PAGE gel (PG112, EpiZyme, Shanghai, China), and then transferred to a polyvinylidene fluoride membrane (WJ001, EpiZyme). Membranes were blocked and incubated with primary antibodies followed by secondary antibodies, and bands were detected by enzyme-linked chemiluminescence according to the manufacturer’s protocol.
Immunofluorescence assay. Mouse aortas from each group were fixed in 4% paraformaldehyde for 20 min, cut into 5 μm sections by pathology (Affiliated Hospital of Jiangsu University, Jiangsu, China)?, and the aortal sections were permeabilized with 0.1% Trinton-X100 for 20 min at room temperature. After blocking in 3% BSA for 30 min, the sections were incubated with primary antibodies against CD68 (1:200), Arginase-1(1:50), p-STAT6 (1:100), and PPARδ (1:100)
overnight at 4°C. After washing, the sections were incubated with the corresponding secondary antibodies (1:200) at RT for 1 h in the dark. DAPI solution (1:7000) was then added to stain nuclei and the stained samples were visualized with a fluorescence microscope.
ELISA assay. The concentrations of interleukin-10 (IL-10) and interleukin-12p70 (IL-12p70) in the supernatants of each macrophage culture were measured by enzyme- linked immunosorbent assay (ELISA) kits (MultiSciences, Zhejiang, China) according to the manufacturer’s instructions. Three independent experiments were

performed, and each sample was quantified using three replicates.

5-Ethynyl-2’-deoxyuridine (EdU) proliferation assay. Endothelial cell (EC) proliferation was determined using the BeyoClick™ EdU-555 cell proliferation assay kit (Beyotime, Shanghai, China). The ECs in each group were cultured for 2 h in serum- free medium for synchronization. The 5-Ethynyl-2’-deoxyuridine working solution was added to the final concentration (10 μM) and incubated at 37°C for 6 h. After the incubation, the cells were fixed in 4% paraformaldehyde at RT for 15 min. The cells were washed with 3% BSA for 2 times and then incubated in 0.3% Triton-X-100 (T8200, Solarbio, Beijing, China) at RT for 15 min to permeabilize the cells. The cells were incubated with the reaction system according to the manufacturer’s instructions and DAPI solution (1:500) was added to stain nuclei.
Transwell migration assay. Cell migration assays were performed as described previously [9]. Each group of treated ECs was prepared as a suspension in serum- free medium. Cell suspension (1×106 cells/mL) was placed in the upper section of a Transwell Boyden chamber, and 500 μL medium containing 5% fetal bovine serum was added to the lower chamber. After a 24- hour incubation at 37°C, the cells that remained on the upper side of the filter were wiped with a cotton swab, and the cells on the lower side of the filter were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet.
HUVEC tube formation assay. The tube formation assay was performed as described previously [10]. The ECs (1×105 cells) were seeded on 96-well plates containing 50 μ L Matrigel (Trevigen) per well and incubated at 37°C for 48 h.

Brightfield cell imaging was performed using an Olympus microscope (Kyoto, Japan), and the number and length of tubes were analyzed using Image J. Three independent fields were randomly selected for each observation, and the experiment was repeated three times.
Statistical analysis. Values were expressed as means ± standard deviation (SD). All data were from at least three independent experiments and were analyzed by SPSS version 23.0. LSD-t test. ANOVA was used to compare differences among groups, and P values less than 0.05 were considered significant.

Results

Activation of the CD137 signaling pathway induces M2 macrophage polarization and accelerates the progression of atherosclerosis .
Compared with the control group , the atherosclerot ic lesion area in the agonist-CD137 group was larger, the fiber cap of plaque was thinner, and the number of foam cells was greater. In addition, more cracks were found under the fiber cap, and plaque invading the tunica media caused the elastic plate to break (Fig. 1A–F). Immunofluorescence analysis showed that the expression of iNOS (Fig. 1G–I) in the anti-CD137 group was greater than in the control group, and was sharply reduced after agonist-CD137 recombinant protein administration. Co-staining with Arginase-1 revealed that agonist-CD137 group contained significantly more M2 macrophages than the control group, and the number of M2 macrophages was lower when mice were treated with the CD137 inhibitor (Fig. 1J–L).

To monitor M2 polarization in vitro, we examined the expression of CD86/CD206, iNOS/Arginase-1, and the secretion level of IL-12p70/IL-10, which are established phenotypic markers associated with M1 and M2 macrophages respectively[11]. Macrophages were isolated from the abdominal cavity of mice as described in the Materials and Methods. Immunofluorescence and flow cytometry analysis revealed that the purity of macrophages was more than 95% (Fig. S1A and B). Almost all the peritoneal macrophages expressed CD137 (Fig. S1C). As expected, the mean fluorescence intensity of CD206in the agonist-CD137 group was significantly greater than the control group, but decreased when CD137 signaling was blocked (Fig. 2C, D). Inversely, the mean fluorescence intensity of CD86 in the agonist-CD137 group was obviously weaker than the control group, but increased in the anti-CD137 group (Fig. 2A, B). Consistent with the flow cytometry findings, western blot (Fig. 2E-G) and ELISA (Fig. 2H, I) assays showed that Arginase-1 and IL-10 levels were also increased in the agonist-CD137 group compared with the control and anti-CD137 groups, but iNOS and IL-12p70 levels were decreased in the agonist-CD137 group compared with the control and anti-CD137 groups These findings suggest that CD137 signaling could drive macrophages into M2 phenotype.

CD137 signaling regulates phosphorylation of STAT6 and PPARδ expression in macrophages.
Both STAT6 and PPARδ have profound effects on alternative activation. We investigated whether CD137 signaling is involved in M2 macrophage polarization

through transcription factor and nuclear receptor mediated responses. We treated peritoneal macrophages with recombinant protein (10 μg/mL) to activate CD137 signaling. Western blotting results showed that the phosphorylation level of STAT6 was gradually up-regulated over time, whereas that of PPARδ peaked at 0.5 h (Fig. 3A). Furthermore, immunofluorescence analysis and Western blotting showed that the increased levels of both p-STAT6 and PPARδ induced by CD137 activation could be inhibited using the CD137 inhibitory antibody (Fig. 3B–F and H–J,). These results indicated that STAT6 and PPARδ are two downstream mediators of CD137 signaling in macrophages.

Inhibition of STAT6 attenuated the expression of PPARδ and M2 macrophage polarization induced by CD137 activation.
To investigate the role of the STAT6/PPARδ pathway in macrophage alternative activation induced by CD137, we treated macrophages with the STAT6 inhibitor AS1517499 at different time points and varying concentrations. As shown in Fig. 4A, pretreating macrophages with 200 nM AS1517499 for 30 min was the optimal condition for inhibiting the expression of PPARδ, so we chose this condition for subsequent experiments. Both immunofluorescence analysis and western blot assays showed that both p-STAT6 and the expression of PPARδ induced by CD137 activation were reduced by inhibiting STAT6 in macrophages (Fig. 3G and K and Fig. 4B and C). Flow cytometry results showed that peritoneal M2 macrophage polarization induced by CD137 signaling was blocked by inhibiting STAT6 (Fig. 4D),

but no obvious change in the expression of CD86 (a marker of M1 macrophages) was observed after inhibiting STAT6 in peritoneal macrophages (Fig. S2). Similar results were observed in RAW264.7 (Fig. 4E). Meanwhile, the expression levels of mRNA of M2 phenotype markers (FIZZ1, ARG1, and MRC1) induced by agonist-CD137 were significantly decreased in the anti-STAT6 treatment group (Fig. 4F).

PPARδ silencing inhibited CD137-induced macrophage M2 polarization.

As shown in Fig. 5A, we selected the most suitable small interfering RNA sequence from three siPPARδ RNA sequences and identified its optimal concentration (50 nM). As expected, the siPPARδ group significantly inhibited the protein expression of PPARδ (Fig. 5B). Flow cytometry results showed that transfection of siPPARδ prevented peritoneal macrophages from transforming into the M2 phenotype induced by agonist-CD137 recombinant protein (Fig. 5C); however, transfection of siPPARδ had little effect on CD68 expression (Fig. S3). To confirm this result, the effect of silencing PPARδ on the restraint in macrophage alternative activation was also examined in RAW264.7 cells (Fig. 5D). Consistent with the flow cytometry results, qRT-PCR results showed that the mRNA levels of M2 phenotype marke rs were increased under agonist-CD137 treatment but decreased when transfected with siPPARδ (Fig. 5E). Collectively, these data suggested that siPPARδ may attenuate M2 macrophage polarization induced by CD137 signaling.

CD137 signaling regulates proangiogenic features of M2 macrophages via

STAT6/PPARδ pathway.

M2 macrophages are known to promote angiogenesis in atherosclerosis [3]. To determine whether regulation of M2 polarization by activating CD137 signaling could subsequently affect the biological characteristics of M2 macrophages, the effects of CD137 signaling and the STAT6/PPARδ axis on ECs were measured. Peritoneal macrophages were divided into four groups as shown in Fig. 6A: the control group, the agonist-CD137 group, the anti-STAT6 group, and the siPPARδ group. After treating the cells for 48 h, the supernatant was collected and added to MBVECs and HUVECs.
The effect of CD137 signaling on the proliferation of MBVECs was analyzed by EdU assay. The data show that the number of proliferating cells in the agonist-CD137 group was increased compared with the control group, but declined in the anti-STAT6 group and the siPPARδ group (Fig. 6B). We then tested the impact of CD137 signaling on the migration capacity of MBVECs using the Transwell migration assay. As shown in Fig. 6C, the number of migrating MBVECs in the agonist-CD137 group was significantly increased compared with the control group, but decreased in the anti-STAT6 group and the siPPARδ group. Next, we investigated whether CD137 signaling can promote lumen formation by analyzing the tube formation ability of HUVECs. Our data showed that the total tube length and the number of branch points of the endothelial lumen were significantly increased in the agonist-CD137 group but decreased in the anti-STAT6 group and the siPPARδ group (Fig. 6D). These results demonstrated that CD137 signaling contributes to the proangiogenic features of M2

macrophages through the STAT6/PPARδ pathway.

Discussion

CD137, also known as 4-1BB, is a new member of tumor necrosis factor receptor superfamily. The cross- linking between CD137 and its ligand CD137L has a two-way signal transduction effect, which can regulate the functions of a variety of immune cells. CD137 is highly expressed in human atherosclerotic plaques and the CD137–CD137L interaction can accelerate the inflammation in atherosclerotic plaques. It has been shown that atherosclerotic plaque area was significantly reduced in CD137-/- mice and in mice injected with CD137L blocking monoclonal antibody. Several publications support the hypothesis that the CD137-CD137L axis plays an important role in promoting the formation of atherosclerosis and affects the stability of plaque [12, 13]. Atherosclerosis is a chronic inflammatory process characterized by endothelial injury, lipid deposition, monocyte infiltration, and plaque formation. Angiogenesis can lead to bleeding and rupture in atherosclerotic plaques [14]. Pathological angiogenesis can increase atherosclerotic plaque vulnerability by transporting lipids, inflammatory cells, and activated proteases into atherosclerotic lesions [15]. Pedro et al. found that apolipoproteins A-I and B were localized around neovessels, suggesting the local lipid depositions were derived from the microvasculature [14]. Our group has shown that activation of CD137 signaling can promote angiogenesis in atherosclerosis by modulating the endothelial Smad1/5-NFATc1 pathway [8].

Macrophages play a key role in the initiation and long-term progression of atherosclerosis. Studies from atheroscle rotic lesions of both mouse models and human plaques have shown diverse macrophage phenotypes. The phenotypic transition into M1 and M2 and other intermediate states allows macrophage cells to adapt to the changing environment in a timely manner through the regulation of transcription factors. In response to different stimulating factors macrophages can be induced to polarize into M1, M2, or other types. While Ml and M2 macrophages can undergo reciprocal transformation, the polarization to M4 phenotype may be irreversible [16]. Macrophage polarization is considered the driving force in the development of plaques in atherosclerosis. M1 macrophage-derived inflammatory cytokines are involved in the early stages of plaque formation [17], whereas the role of M2 macrophages in the formation of atherosclerosis remains controversial. It is now clear that M2 macrophages promote angiogenesis in vivo [3]. Therefore, we speculated that CD137 signaling may promote angiogenesis in atherosclerosis by modulating macrophage M2 polarization, thereby enhancing plaque vulnerability.
In the present study, we showed that ApoE-/- mice injected with agonist-CD137 recombinant protein have larger lesion areas, thinner fiber caps, and more CD68+ macrophages infiltrated into the aorta, suggesting that CD137 signaling promotes the progression of atherosclerosis. Furthermore, immunofluorescence staining showed that macrophages in the plaque of the agonist-CD137 group mice expressed more M2 macrophage marker Arginase-1. Similarly, in vitro experiments indicate that the mean fluorescence intensity of CD206 and the level of IL-10 in the agonist-CD137 group

are higher than those in the anti-CD137 group. In general, we verified the effect of CD137 signaling on macrophage M2 polarization from three aspects: the surface markers of macrophages were detected by flow cytometry (M1: CD86, M2: CD206); the intracellular markers in macrophages were detected by western blot (M1: iNOS, M2: Arginase-1); and the secretory markers of macrophages were detected by ELISA (M1: IL-12p70, M2: IL-10). Based on these data, we can conclude that CD137 signaling accelerates atherosclerosis progression and induces macrophage M2 polarization in vivo and in vitro.
To investigate the molecular mechanism of CD137 signaling in regulating macrophage M2 polarization, we treated peritoneal macrophages with agonist-CD137 recombinant protein in vitro. Our results show that CD137 signaling increased the phosphorylation of STAT6 and the expression of PPARδ in peritoneal macrophages, and this increase can be suppressed with a CD137 inhibitor. However, p-STAT6 was still increased after PPARδ began to degrade. We believe that there are several possible explanations for this observation. Firstly, CD137 signaling activates the p-STAT6/PPARδ pathway, PPARδ enters the nucleus and triggers the biological effects. Once PPARδ has transmitted the stimulatory signal from CD137 to its downstream target genes, its presence in the nucleus is no longer necessary and therefore subjects to degradation. Secondly, the biological effects triggered by PPARδ may promote the p-STAT6 expression as a feedback mechanism. Thirdly, the macrophages are under continuous agonist-CD137 stimulation in the whole progress of the experiment, which may further induce the expression of p-STAT6. Our results

also showed that the expression of PPARδ decreased when STAT6 was inhibited, suggesting that PPARδ might be a downstream effector of STAT6. We also showed that STAT6 inhibitor and PPARδ siRNA can reduce CD137-induced proliferation, migration, and even lumen formation of endothelial cells. These findings indicate that CD137 signaling can influence the proangiogenic features of M2 macrophages through the STAT6/PPARδ pathway.
The polarization of macrophages is a complex process that is regulated by a variety of inflammatory cytokines and signaling pathways. Interferon promotes M1 polarization of macrophages via the STAT1 signaling pathway, and IL-10 promotes M2 polarization of macrophages via STAT3 pathway. However, the molecular mechanisms regulating macrophage polarization at the transcription level remains largely unknown [18]. Early studies showed that STAT family members play a key role in the phenotypic transformation of macrophages. Recent studies have shown that STAT6 can regulate M2 macrophage polarization by interacting with cytokines IL-4 and IL-13 [19]. Mishra et al. [20] found that the M2 polarization level of macrophages in STAT6-/- mice was lower than that in wild mice. Here, we demonstrated for the first time that activation of CD137 signaling up-regulated STAT6 phosphorylation.
Although the biological functions of PPARα and PPARγ have been well studied, the role of PPARδ is still unclear due to the lack of PPARδ ligands for clinical use [21]. PPARγ and PPARδ and their downstream molecules of the JAK/STAT pathway are involved in the activation and oxidation metabolism of M2 macrophages. Kang et al. found that PPARδ activation through a STAT6 binding site on its promoter induced

M2 polarization in adipose tissue and Kupffer cells in mice [22]. Interestingly, studies have shown that PPARδ activation can induce endothelial cell proliferation and promote angiogenesis by increasing the expression of vascular endothelial growth factors, suggesting that PPARδ can regulate angiogenesis [23]. Consistent with these reports, our results confirmed that PPARδ acted as a downstream effector of STAT6, and their cooperation regulates the expression of many M2 marker genes (CD206, Arginase-1, FIZZ1, MRC1), indicating that activation of the STAT6/PPARδ pathway is necessary for the mature polarization of macrophages.
In conclusion, we demonstrated that CD137 signaling induces macrophage M2 polarization in vivo and in vitro by activating the STAT6/PPARδ pathway. However, this study has some limitations. First, the effects of CD137 signaling activation on endothelial cells are indirect and mediated by apparently unknown factors secreted by the macrophages, and the specific mechanism needs to be further explored. Second, it is generally known that sterile inflammation promotes the development of atherosclerosis. It appears that M2 macrophages may suppress this local inflammation while promoting angiogenesis, which may enhance plaque rupture. This relationship appears to be considerably complex and requires further study. In any case, whether macrophage phenotypic plasticity is beneficial or harmful in the process of atherosclerosis is controversial. Therefore, future studies exploring molecular mechanisms regulating the plasticity of macrophages will provide new insight on how macrophage polarization contributes to plaque formation and form the basis for the development of new therapeutic strategies for atherosclerosis.

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Funding

This work was supported by the National Natural Science Foundation of China (81970379, 81670405), Natural Foundation of Jiangsu Province (BK20161355), Cardiovascular Disease Clinical Research Center of Zhenjiang (SS2018008), and Graduate Student Reseaech Innovation Granting of Jiangsu Province (KYCX17_1818).
Availability of data and materials

All data generated or analyzed during this study are included in this published article.
Ethics approval and consent to participate

This study was approved by the Ethical Committee of Jiangsu University and conducted in agreement with the institutional guidelines.
Conflicts of interest

The authors declare that there is no conflict of interes.

Author contributions

Tianxin Geng and Jinchuan Yan conceived and designed the study. Tianxin Geng, Yang Yan, and Liangjie Xu performed the experiments. Jinchuan Yan and Jun Pu provided reagents and technical support. Mengfei Cao and Yu Xu searched the literature. Tianxin Geng and Yang Yan wrote the paper.

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Figure 1: CD137 signaling accelerates atherosclerosis progression and induces M2 macrophage polarization in vivo. The aorta sections are from ApoE-/- mice at 16 weeks after feeding with a high fat diet (control group) and injecting with agonist-CD137 recombinant protein (agonist-CD137) or/and CD137 inhibitor (anti-CD137) daily. Hematoxylin staining showing the extent of atherosclerotic lesions in aortas of control and agonist-CD137 group mice, the arrows point to where plaque invades the tunica media. (A-D). The quantitative analysis showed the lesion area (E, F). Co-immunofluorescent staining showing the fluorescence of Arginase-1 (Green, J-L) and iNOS (Red, G-I) positive cells in atherosclerotic plaque area in mice

aortas. DAPI (M-O), Merge (P-R). The quantitative analysis showed the rate of fluorescence intensity of Arginase-1/iNOS expressed by macrophages (S). Scale bars=100 μm. Data are presented as the mean ± SD from independent experiments, n=6. **, P< 0.01; ***, P< 0.001. iNOS: inducible nitric oxide synthase, DAPI: 4',6-diamidino-2-phenylindole.

Figure 2: CD137 signaling induces M2 macrophage polarization in vitro. The mean fluorescence intensity of CD86 (A, B) and CD206 (C, D) expressed by peritoneal macrophages of C57BL/6J mice were determined by flow cytometry. The expressions of iNOS and Arginase-1 at the protein level were detected by Western blotting analysis (E-G). The concentrations of IL-12p70 and IL-10 in the supernatant were analyzed by ELISA (H, I). Data are presented as the mean ± SD from at least three independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001. IL: interleukin.

Figure 3: CD137 signaling regulates phosphorylation of STAT6 and PPARδ expression in macrophage. Activation of CD137 signaling induced phosphorylation of STAT6 and expression of PPARδ in macrophage, and peaked at 24 and 0.5 hours, respectively (A). Data are presented as the mean ± SD from at least three independent experiments. *, P < 0.05; **, P< 0.01; ***, P< 0.001. Levels of p-STAT6 were detected by immunofluorescence (D–G, Green: p-STAT6, Red: CD68, Blue: DAPI, Orange: p-STAT6+CD68+cells) and Western blot (B). Expression levels of PPARδ were investigated by immunofluorescence (H–K, Green: PPARδ, Red: CD68, Blue: DAPI, Orange: PPARδ+CD68+cells) and western blot (C). The quantitative analysis showed the numbers of p-STAT6+CD68+ (L) and PPARδ+CD68+cells (M). Scale bars=50 μm, n=6. PPARδ: peroxisome proliferator-activated receptor δ, p-STAT6: phospho-signal transducers and activators of transcription 6.

Figure 4: Inhibition of STAT6 attenuated the expression of PPARδ and M2 macrophage polarization induced by CD137 signaling. The macrophages were treated with 100 nM, 200 nM, and 400 nM AS1517499 (the specific inhibitor of the STAT6) for 15 min, 30 min, 60 min, and 90 min. The inhibitory effect of AS1517499 on PPARδ protein expression was validated by western blot (A). Western blot was performed to detect STAT6 phosphorylation (B) and PPARδ expression (C). Flow cytometry analysis was performed to analyze the percentage of CD206+ cells in peritoneal macrophages (D) and RAW 264.7 cells (E). mRNA of FIZZ1, ARG1, and MRC1 in macrophages was tested by qRT-PCR (F). Data are presented as the mean ± SD from at least three independent experiments. *, P < 0.05; **, P< 0.01; ***, P<
0.001. DMSO: dimethyl sulfoxide, FIZZ1: found in inflammatory zone 1, ARG1: Arginase 1, MRC1: mannose receptor 1.

Figure 5: PPARδ silencing inhibited CD137- induced M2 macrophage polarization. qRT-PCR was used to confirm the siRNA knockdown of PPARδ (A). Western blot

was performed to detect PPARδ expression (B). Flow cytometry analysis was employed for analyzing the percentage of CD206+ cells in peritoneal macrophages (C) and RAW 264.7 cells (D). mRNA of FIZZ1, ARG1, and MRC1 in macrophages was measured by qRT-PCR (E). Data are presented as the mean ± SD from at least three independent experiments. *, P < 0.05; **, P< 0.01; ***, P< 0.001.

Figure 6: CD137 signaling regulates proangiogenic features of M2 macrophages via STAT6/PPARδ pathway. Macrophages were treated with agonist-CD137 recombinant protein or/and AS1517499, and transfected with PPARδ siRNA. Then, supernatant was collected from macrophages and incubated with MBVECs or HUVECs (A). The proliferation of MBVECs was measured by EdU assay (B). The migration ability of MBVECs was detected by Transwell migration assay (C). The lumen forming ability of HUVECs, including total tube length (μm) and tube numbers, was investigated by Matrigel HUVEC tube formation assay (D). Scale bars=50 μm, n=6. Data are

presented as the mean ± SD from at least three independent experiments. *, P < 0.05;

**, P< 0.01; ***, P< 0.001. HUVECs: human umbilical vein endothelial cells, MBVECs: mouse brain microvascular endothelial cells, EdU: 5-Ethynyl-2’-deoxyuridine.

Highlights

 Macrophage M2 polarization in atherosclerosis is regulated by the STAT6/PPARδ pathway.
 Inhibition of CD137 signaling prevents the activation of the STAT6/PPARδ pathway and rescues the polarity shift of macrophages.
 CD137 signaling may effect the proangiogenic features of M2 macrophages indirectly.

Author contributions
Tianxin Geng and Jinchuan Yan conceived and designed the study. Tianxin Geng, Yang Yan, and Liangjie Xu performed the experiments. Jinchuan Yan and Jun Pu provided reagents and technical support. Mengfei Cao and Yu Xu searched the literature. Tianxin Geng and Yang Yan wrote the paper.

Highlights

 Macrophage M2 polarization in atherosclerosis is regulated by the STAT6/PPARδ pathway.
 Inhibition of CD137 signaling prevents the activation of the STAT6/PPARδ pathway and rescues the polarity shift of macrophages.
 CD137 signaling may effect the proangiogenic features of M2 macrophages indirectly.