Xiong et al. J Transl Med (2017) 15:234 DOI 10.1186/s12967-017-1336-z Journal of Translational Medicine

RESEARCH Open Access The C‑type lectin receptor CLEC5A (MDL‑1) expression is associated with early plaque progression and promotes macrophage survival Weixin Xiong1, Haibo Wang2, Lin Lu1,2, Rui Xi1, Fang Wang1, Gang Gu1* and Rong Tao1*

Abstract Background: Biomarkers of early plaque progression are still elusive. Myeloid DAP12-associating lectin-1 (MDL-1), also called CLEC5A, is a C-type lectin receptor implicated in the progression of multiple acute and chronic infamma- tory diseases. However, the relationship between its level and atherosclerosis is unknown. In this study, we aimed to investigate the correlation between macrophage MDL-1 expression and early atherosclerosis progression. Methods: Immunofuorescence staining, real-time PCR and western blot were performed to analyze MDL-1 expres- sion in aorta or mice . The role of MDL-1 in macrophage survival was further investigated by adenovirus infection and TUNEL assay. Results: Signifcant MDL-1 expression was found in advanced human and apoE / mice atherosclerotic plaques, especially in lesional macrophages. In the model of atherosclerosis regression, we− found− MDL-1 expression was highly downregulated in lesional macrophages from ldlr / mouse regressive plaques, coincident with a reduction in lesional macrophage content and marker of M1 proinfammatory− − macrophages. Furthermore, we found MDL-1 was signifcantly expressed in infammatory M1 subtype polarized bone marrow-derived macrophages. In vitro experi- ments, the level of MDL-1 was remarkably elevated in macrophages treated with pathophysiological drivers of plaque progression, such as oxidized low-density lipoprotein (ox-LDL) and hypoxia. Mechanistically, we demonstrated that MDL-1 overexpression notably promoted macrophage survival and decreased cleaved caspase-3 expression under ox-LDL stimulation, which suggested that it could maintain lesional macrophage survival and cause its accumulation. Conclusions: This study frstly demonstrated that MDL-1 is mainly expressed in atherosclerotic lesional macrophages and increased macrophage MDL-1 expression is associated with early plaque progression and promotes macrophage survival. Keywords: Atherosclerosis, Plaque progression, Macrophage, Apoptosis, Myeloid DAP12-associating lectin-1

Background /macrophage, is central in the disease and Atherosclerosis is both a metabolic and an immunoin- their accumulation is relevant to plaque development fammatory disease [1]. Either disturbance of lipid level and also a major contributor to the persistence of dis- or immune system balance might contribute to athero- ease state [4, 5]. In the early phase of atherosclerosis, genesis [2, 3]. Te role of infammatory cells, especially recruit and transmigrate to the subendothe- lial layer of the aortic wall and diferentiate into mac- *Correspondence: [email protected]; [email protected] rophages. Tese infammatory macrophages could then 1 Department of Cardiology, Ruijin Hospital, Jiao Tong University School either emigrate from the site of localized infammation or of Medicine, No. 197, Ruijin Er Road, Huangpu District, Shanghai 200025, undergo apoptosis process, which reduces plaque burden China Full list of author information is available at the end of the article and causes atherosclerosis regression [6, 7]. However,

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xiong et al. J Transl Med (2017) 15:234 Page 2 of 13

atherosclerotic infammation is not readily resolved and to macrophage accumulation during early atherosclerotic these macrophages become foam cells persisting in the plaque progression. arterial wall, which leads to plaque instability and disease progression. Terefore, fnding the target and biomarker Methods of lesional macrophage retention and accumulation is Clinical samples promising for prevention of later adverse cardiovascular Human femoral arteries with atherosclerotic plaques outcome occurrence. were obtained from three patients who underwent leg Myeloid DAP12-associated lectin-1 (MDL-1) is amputation surgery, and the arteries were then embed- also called C-type lectin domain family 5-member A ded in parafn. Te internal thoracic arteries (n = 3) (CLEC5A), which has a short cytoplasmic region lacking were obtained as normal artery. Baseline demograph- particular signaling motifs and associates noncovalently ics, risk factors for atherosclerosis, clinical biochemical with DAP-12 to transmit downstream signaling via DAP- results and medications were recorded. Te study pro- 12 immuno-receptor tyrosine-based activation motif tocol was approved by the Ethics Committee of Ruijin (ITAM) [8]. Te endogenous ligand for MDL-1 has yet to Hospital, Shanghai Jiaotong University School of Medi- be found and dengue virus particle is the only ligand iden- cine, and written informed consent was obtained from all tifed in immune cells [9]. Recent researches showed that patients. Since the study did not involve any intervention, MDL-1 is expressed on mouse , monocytes/ it was not prospectively registered. macrophages and human peripheral blood monocytes as well as myeloid cell precursors [9, 10]. Studies also dem- Isolation of human peripheral blood monocytes (PBMCs) onstrated a strong expression of MDL-1 and DAP-12 in We obtained samples of peripheral blood from signif- mature and diferentiated monocytes/macrophages in cant CAD patients with stable angina (n = 3) who had contrast to undiferentiated bone marrow ­CD34+ stem at least a major epicardial coronary artery luminal diam- cells or monocytes [11]. Functionally, MDL-1 acts as a eter narrowing ≥ 50% observed in coronary angiography signaling receptor for proinfammatory cytokine release examination and healthy volunteers (n = 3). We then iso- [12, 13] and is implicated in mediating multiple acute lated PBMCs using dextran sedimentation and a Ficoll– and chronic infammatory diseases, including hemor- Hypaque density-gradient separation and enriched by a rhagic fever, lethal shock, virus-induced brain damage, magnetically negative depletion protocol (Miltenyi Bio- chronic obstructive pulmonary disease and development tec) as previously described [20]. Purifed PBMCs were of autoimmune arthritis [14–18]. However, the correla- lysed in a lysis bufer and a protease inhibitor cocktail tion of MDL-1 with atherosclerosis and its behavior are and stored at − 80 °C until use. still elusive and remain unknown. Moreover, investiga- tions on 9P21.3 coronary artery disease (CAD) risk locus Experimental animals and diet of human macrophages exhibited remarkable diferences All animal experiments were conducted according to in expression including CCL-8, CCL-2 and MDL-1 the experimental protocols approved by the Committee between myocardial infarction patients and controls [19], on Animal Resources of Shanghai Jiaotong University. which suggested macrophage MDL-1 might be associ- 6-week old apoE−/− and ldlr−/− mice were purchased ated with CAD progression. Terefore, in this study, from Model Animal Research Center of Nanjing Uni- we aim to study the relationship between macrophage versity and maintained in a pathogen-free facility in the MDL-1 expression and early atherosclerosis progression Animal Experiment Center of Ruijin Hospital, Shanghai and its role in macrophage apoptosis. Jiaotong University School of Medicine. All apoE−/− Here, we showed that MDL-1 is abundantly expressed mice were fed a chow diet for 2 weeks and half of them in macrophages of human and apoE−/− as well as were then changed into a high-fat diet (HFD, 21% fat, ldlr−/− mice advanced atherosclerotic lesions and 0.2% cholesterol, 23% protein, and 40.5% carbohy- downregulated during early atherosclerotic plaque drate; Research Diets, Inc.) for 8 weeks before sacrifce. regressive phase. Moreover, we further found MDL-1 was In the model of regression, 8-week old ldlr−/− mice highly expressed in M1 subtype macrophage and elevated were placed on a HFD for 8 weeks and were either sac- when treated with pathophysiological drivers of plaque rifced (baseline) or switched to chow diet for 4 weeks progression (e.g. ox-LDL or hypoxia). Mechanistically, we as described [21]. Aortic and heart tissues were then found MDL-1 overexpression notably inhibited ox-LDL- exposed and obtained. Blood samples and serum were induced macrophage apoptosis and amplifed monocyte collected both in baseline and plaque regression ldlr−/− chemotactic protein-1 (MCP-1) production, thereby mice group. Cholesterol levels were measured by HDL implicating that it might maintain lesional macrophage and LDL/VLDL cholesterol assay kit (Abcam) according survival and link abnormal hyperlipidemic environment to the manufacturer’s protocol. Xiong et al. J Transl Med (2017) 15:234 Page 3 of 13

Histology and immunostaining sulphate in phosphate bufered saline (PBS) at 37 °C Serial frozen sections of gelatin permeabilized aortic for 24 h before oxidation was terminated with ethylene sinus lesions were prepared from OCT embedded heart diamine tetraacetic acid (EDTA). Te level of oxidation tissue using a microtome as described [22]. For lesion was measured using thiobarbituric acid reactive (TBAR) immunostaining, 10 μm sections were blocked with 10% determination according to a malondialdehyde standard. normal serum for 1 h and then incubated with primary Te migration factor of ox-LDL was 2.2. To assess MDL-1 antibodies recognizing MDL-1 (1:50 dilution, abcam), cellular efects, we infected the macrophages with adeno- CD68 (1:100 dilution, AbD Serotec), MOMA-2 (Rat anti- viral vector overexpressing MDL-1 before treatment with mouse MOMA-2, 1:50 dilution, AbD Serotec), DAP-12 ox-LDL. (1:50 dilution, Santa Cruz), Ly-6G (1:100 dilution, AbD Serotec), iNOS (1:100 dilution, BD biosciences) or Arg-1 Western blot (1:100 dilution, BD biosciences) overnight at 4 °C. All Te concentrations of all tissue and cell lysate protein sections were washed and incubated with fuorescent samples were examined according to the method of secondary antibodies (1:1000 dilution, invitrogen) for Bradford as described [25]. After normalizing for equal 60 min. Sections without primary antibody served as protein concentration, the protein samples were resus- negative controls. Finally, all the sections were washed in pended in SDS protein bufer before separation by SDS- TBS and mounted in glycerol-based mounting medium PAGE. We then transferred the samples to polyvinylidene containing antifade reagent with DAPI (Invitrogen) and difuoride membranes. Te membranes were probed with then photomicrographed and digitized with an Olympus primary antibodies overnight at 4 °C and horseradish DP50-CU digital camera mounted on an Olympus BX51 peroxidase-conjugated secondary antibodies for 60 min microscope. Lesional MDL-1 positive macrophages were at room temperature. We then detected the protein determined as the percentage of MDL-1 and MOMA-2 bands using an electrochemiluminescence (ECL) system double positive macrophages per lesional area. All immu- (GE Healthcare Biosciences, Pittsburgh, PA, USA). Den- nostaining images were analyzed with the NIH Image- sitometry analysis of the protein bands was carried out Pro Plus software (Media Cybernetics, Bethesda, MD). using ImageJ software from the National Institutes of Health (NIH). Cell culture Bone marrow-derived macrophages (BMDMs) were Quantitative real‑time polymerase chain reaction (RT‑PCR) prepared from mice bone marrow and resuspended Total BMDM RNA was isolated using a Trizol reagent and cultured in RPMI 1640 medium (Gibco) supple- (Invitrogen, Carlsbad, CA, USA). For reverse transcrip- mented with 10 ng/mL monocyte-colony stimulating tion, we converted 1 ug of total RNA into frst strand factor (M-CSF, R&D systems, Minneapolis, MN, USA), complementary DNA (cDNA) in a 20 μL reaction vol- 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL ume using a reverse transcription kit (Promega, Madison, penicillin and 50 U/mL streptomycin as described [23]. WI, USA) according to the manufacturer’s instruction. For macrophage subtype polarization, we changed to Quantitative real-time PCR analysis was performed (Ste- fresh stimulation medium: for M1 activation, use RPMI pOne, Applied Biosystems) using SYBR Green (Takara, 1640 containing 10% FBS and 100 ng/mL LPS (Sigma Otsu, Japan). Gene expression levels were normalized Aldrich); for M2 activation, use RPMI 1640 contain- with GAPDH, and data were analyzed with StepOne soft- ing 10% FBS with 10 ng/mL IL-4 (Peprotech). Peritoneal ware v2.1 (Applied BioSystems). Following primers were primary macrophages were obtained from 6-week old used: 5′-TACCCCCAATGTGTCCGTC-3′ for mouse C57BL/6 mice by peritoneal lavage 4 days after intra- GAPDH-F and 5′- GGTCCTCAGTGTAGCCCAAG-3′ peritoneal injection of 3% thioglycollate (Sigma Aldrich). for mouse GAPDH-R. 5′-TCGGGGCTTATCGTAG- Te cells were washed and resuspended and then cul- TAGTG-3′ for mouse MDL-1-F and 5′-TGTAGGCATG- tured in RPMI 1640 medium supplemented with 10% GTACTTTCGTCAT-3′ for mouse MDL-1-R. heat-inactivated FBS, 50 U/mL penicillin and 50 U/mL streptomycin as previously described [24]. To study cel- Terminal deoxynucleotidyl transferase dUTP nick end lular MDL-1 expression, we maintained peritoneal mac- labeling (TUNEL) staining rophages to subconfuence and stimulated them with Macrophage apoptosis assessment was detected by 50 μg/mL ox-LDL/LDL (AbD Serotec) or ­CoCl2 (Sigma TUNEL staining using the in situ cell death detection Aldrich) with or without pretreatment of 10 μg/mL HDL kit (Roche) according to the instruction. Briefy, adher- (Sigma Aldrich) or 100 μmol/L HIF-1α inhibitor (Calbio- ent cell chamber slides were washed several times and chem, EMD Millipore). LDL was purifed to homogeneity fxed in freshly prepared 4% paraformaldehyde solu- by ultracentrifugation, oxidized using 20 μmol/L cupric tion. We then incubated the slides in 0.1% Triton X-100 Xiong et al. J Transl Med (2017) 15:234 Page 4 of 13

solution for 2 min on ice. After washed with PBS, the Table 1 Baseline characteristics and medical treatment slides were incubated with TUNEL reaction mixture for of patients 60 min at 37 °C in a humidifed atmosphere in the dark. Variables Patients (n 3) = Te slides were then washed and mounted in glycerol- Age (years) 60.7 7.4 based mounting medium containing antifade reagent ± Male, n (%) 3 (100) with DAPI (invitrogen) and then photomicrographed BMI (kg/m2) 27.3 1.4 and digitized with an Olympus DP50-CU digital camera ± Smoking, n (%) 1 (33.3) mounted on an Olympus BX51 microscope. Te average Hypertension, n (%) 3 (100) percent of TUNEL­ + cells was determined as the percent- Hyperlipidemia, n (%) 2 (66.7) age of ­TUNEL+ nuclei per feld. Leukocyte ­(109/L) 6.0 0.6 ± Platelet ­(109/L) 197.3 10.7 Adenovirus infection ± FBG (mmol/L) 5.5 1.0 Freshly prepared peritoneal macrophages were seeded ± 2h-PG (mmol/L) 10.5 3.4 in 6-well dish and maintained in RPMI 1640 medium ± HbA1c (%) 7.0 1.1 for 24 h. Te recombinant adenovirus with or with- ± Blood urea nitrogen (mmol/L) 4.5 0.3 out MDL-1 overexpressed adenoviral vector (pAdeno- ± Creatinine (μmol/L) 75.0 6.7 MCMV-EGFP-P2A-MDL1-3FLAG) was added to cell ± Uric acid (mmol/L) 290.0 37.6 with MOI (multiplicity of infection) of 1000 plaque-form- ± hs-CRP (mg/L) 1.0 0.5 ing units per cell in RPMI 1640 medium without FBS for ± Triglyceride (mmol/L) 1.2 0.2 2 h. Infection was carried out with the indicated MOI ± Total cholesterol (mmol/L) 4.7 0.3 for 48 h, after which the infection medium was aspirated ± HDL-cholesterol (mmol/L) 1.1 0.2 and replaced with fresh complete medium. Te ability of ± LDL-cholesterol (mmol/L) 3.2 0.3 infected macrophages to express MDL-1 was assessed by ± Lipoprotein (a) (g/L) 0.1 0.03 Western blot analysis. ± Medications, n (%) Statistical analysis Anti-platelet 3 (100) Statistical analyses were performed using Student’s t test Beta-blocker 2 (66.7) or ANOVA when data followed a normal distribution or ACE-I/ARB 2 (66.7) Mann–Whitney U test when data did not follow a nor- Statin 3 (100) mal distribution, using the software SPSS 16.0 (SPSS Inc., BMI body mass index, FBG fasting blood glucose, 2h-PG 2h-postprandial glucose, Chicago, IL, USA). A p value below 0.05 was considered hs-CRP high sensitivity C-reactive protein, HDL high-density lipoprotein, LDL low-density lipoprotein, ACE-I angiotensin converting enzyme inhibitor, ARB statistically signifcant. angiotensin receptor blocker

Results MDL‑1 is mainly expressed in macrophages of advanced Figure S1), which suggests MDL-1 associates with DAP- atherosclerotic plaques and its level decreases 12 and plays its role on advanced atherosclerotic lesion. in regressive plaques Since these MDL-1 expressed lesional macrophages To demonstrate the macrophage expression of MDL-1 derived from peripheral blood circulating monocytes and at protein level in atherosclerotic lesions, human femo- MDL-1 was also found to be expressed in monocytes, we ral arteries with atherosclerotic plaques and internal tho- detected MDL-1 expression in PBMCs from both CAD racic arteries (as controls) were obtained and prepared patients and healthy volunteers. As we expected, MDL-1 (donor baseline characteristics in Table 1). In these arte- was demonstrated to be expressed in circulating mono- rial sections, we uncovered that MDL-1 was mainly colo- cytes from CAD patients, much higher than healthy con- calized with CD68 in advanced human atherosclerotic trols (p < 0.01; Additional fle 2: Figure S2), which showed plaques, which indicates MDL-1 is abundantly expressed increased MDL-1 expression in circulating monocytes in lesional macrophages (Fig. 1a). Tis fnding was correlated to atherosclerotic progression. also demonstrated in early advanced atheromata from To understand the dynamic changes of macrophage apoE−/− mice fed a 8-week HFD, which exhibited sig- MDL-1 expression in atherosclerosis, we used an estab- nifcant colocalization with MOMA-2 (Fig. 1b). However, lished model of plaque regression where ldlr−/− mice we did not fnd MDL-1 expression in normal control fed a 8-week HFD were either sacrifced for baseline arterial sections (Fig. 1a, b). Furthermore, staining for lesion measurements or switched to a chow diet for MDL-1 adaptor DAP-12 also colocalized with MDL-1 in another 4 weeks. Tis switch to chow is associated with early advanced apoE−/− mice plaques (Additional fle 1: signifcant reductions in total serum cholesterol and LDL Xiong et al. J Transl Med (2017) 15:234 Page 5 of 13

Fig. 1 MDL-1 is mainly expressed by lesional macrophages in human and apoE / mice atherosclerotic plaques. a MDL-1 (green) or CD68 (red) − − were stained in human femoral arterial plaques (top panel) and normal internal thoracic arteries (bottom panel). Areas of colocalization are shown in yellow in the merged image. Images are representative of 3 subjects. b MDL-1 (green) or MOMA-2 (red) were stained in apoE / mice aortic − − root sections from western diet group (top panel) and chow diet group (bottom panel). Areas of colocalization are exhibited in yellow in the merged image. Images are representative of 3 mice

cholesterol (all p < 0.05; Fig. 2). We observed that mac- investigate whether MDL-1 expression is related to rophage MDL-1 expression was remarkably decreased infammatory lesional macrophage phenotype, we accompanied with reduced MOMA-2 positive mac- both measured MDL-1 expression in BMDM polarized rophage content and iNOS expression in regressive in vitro and costained iNOS and MDL-1 in advanced plaques, which suggested decreased MDL-1 expression and regressive atherosclerotic plaques in vivo. As what might be related to reduced number of lesional mac- we hypothesized, the mRNA level of MDL-1 was signif- rophages in regressive plaques and this is possibly caused cantly elevated in M1 subtype macrophages (p < 0.05; by cell apoptosis process in early atherosclerotic lesion Fig. 3d), much higher than M2 subtype macrophages, (p < 0.05; Fig. 3a–c). Besides, we also found an increased and MDL-1 protein expression notably colocalized Arg-1 expression in regressive plaques, a marker of with iNOS in early advanced atheromata from baseline M2 subtype macrophages (Fig. 3b). Furthermore, to group (Additional fle 3: Figure S3), which refected its Xiong et al. J Transl Med (2017) 15:234 Page 6 of 13

after 48 h (all p < 0.05; Fig. 4a). However, native LDL did not cause this alteration of MDL-1 expression (data not shown). Because ox-LDL induces plaque oxidative stress and its downstream transcription factor HIF-1α, we pretreated macrophages with HIF-1α inhibitor which could block its downstream transcription factor activity. Notably, we found ox-LDL-caused elevation of MDL-1 expression was blocked after pretreatment with HIF-1α inhibitor (all p < 0.05; Fig. 4c). Since hypoxia and HIF-1α have also been demonstrated in promoting atheroscle- rotic progression [28, 29], we then treated peritoneal macrophages with a chemical mimic of hypoxia, ­CoCl2, which can stabilize the downstream transcriptional factor HIF-1α. Similarly, we found ­CoCl stimulation Fig. 2 The serum total and LDL cholesterol levels were signifcantly 2 lowered in LDLr / mice when switching to a chow diet. LDLr / remarkably induced MDL-1 expression and the increase − − − − mice fed a 8-week HFD were either sacrifced for baseline or switched of MDL-1 expression was also inhibited after pretreat- to a chow diet for another 4 weeks. Total cholesterol and LDL/VLDL ment with HIF-1α inhibitor (all p < 0.05; Fig. 4b, d). cholesterol concentration were decreased and HDL cholesterol was Moreover, we observed that the increase of MDL-1 elevated in switching group compared with baseline group mice expression was partly reversed after treatment of HDL, a (n 4–5 mice/group). *p < 0.05 compared with baseline group mice. = LDLr low-density lipoprotein receptor, HFD high-fat diet, LDL/VLDL lipoprotein implicated in promotion of cholesterol efux low-density lipoprotein/very low-density lipoprotein from the cells and blocks infammatory efects of ox-LDL in atherosclerotic regression (all p < 0.05; Fig. 4e) [30]. Whereas, treatment with HDL alone did not cause any change of MDL-1 level. Tese results refect the dynamic proinfammatory role in disease progression. Consider- changes of macrophage MDL-1 expression regulated by ing MDL-1 also expressed in and neutrophil both pro- and anti-atherosclerotic stimuli. being an important contributor to plaque progression, we costained MDL-1 and neutrophil both in baseline MDL‑1 overexpression protects macrophages from Ox‑LDL and switch diet groups. Although MDL-1 expression was induced apoptosis in vitro observed in a few lesional neutrophils, however, no sig- Previous research on mouse myeloid lineage cells found nifcant diference was found between these two groups MDL-1 stimulation helps to maintain 32Dcl3 cell survival (Additional fle 4: Figure S4). [10]. Terefore, we detected whether increased MDL-1 In another established atherosclerotic model, apoE−/− expression could afect macrophage apoptosis under ath- mice were either fed a HFD or chow diet for 8 weeks and erogenic stimuli (e.g. ox-LDL). Here, we used a MDL-1 the aortic tissues were obtained after sacrifce. Notably, overexpressed adenovirus, to infect peritoneal mac- MDL-1 was abundantly expressed in aortic tissues of rophages before exposed to ox-LDL (Fig. 5a). In TUNEL HFD-fed group mice compared with chow diet group, assay, primary macrophages underwent apoptotic pro- which suggested MDL-1 level increases in an early pro- cess after high-dose ox-LDL stimulation. Surprisingly, gressive environment such as hypercholesterolemic con- these macrophages were largely protected from apoptosis text (Fig. 3e). when pretreated with adenovirus overexpressing MDL-1 (Ad-MDL-1) compared with control virus (all p < 0.05; MDL‑1 expression is regulated by pathophysiological Fig. 5b, c). However, treatment with Ad-MDL-1 alone did agents implicated in atherosclerotic progression in vitro not exhibit any diference compared with control group To further investigate molecular mechanisms which (Fig. 5b, c). afected MDL-1 expression in the progression and Furthermore, we next detected active cleaved cas- regression of atherosclerosis and demonstrate what we pase-3 expression in peritoneal macrophages, molecular observed in mouse models and polarized BMDMs, we mechanism associated with cell apoptosis, under ox-LDL used several pathophysiological agents implicated in stimulation. As what we expected, the level of cleaved plaque progression (i.e. ox-LDL and hypoxia) or regres- caspase-3 was signifcantly elevated after ox-LDL stimu- sion (i.e. HDL) to stimulate macrophages and detected lation and this increased expression was notably inhibited MDL-1 expression in vitro [26, 27]. Remarkably, we in Ad-MDL-1 infected macrophages (all p < 0.05; Fig. 6a, found MDL-1 expression was upregulated after ox-LDL b), which was coincident with the results of TUNEL stimulation for 24 h and more signifcant than baseline assay. Moreover, these Ad-MDL-1 infected macrophages Xiong et al. J Transl Med (2017) 15:234 Page 7 of 13

Fig. 3 MDL-1 expression is downregulated in regressive plaques and has a proinfammatory phenotype in the lesion. a Immunofuorescent stain- ing of MDL-1 (green) and MOMA-2 (red) in aortic sinus plaques in a progressive (ldlr / mice fed a 8-week HFD) or regressive (ldlr / mice fed − − − − a 8-week HFD and then switched to 4-week chow diet) environment. b Immunofuorescent staining of iNOS (M1 subtype macrophage marker, red) or Arg-1 (M2 subtype macrophage marker, red) in aortic sinus plaques both in progressive and regressive context. c Quantifcation of MDL-1 positive macrophages in a. d Real-time PCR analysis of MDL-1 mRNA in bone marrow-derived macrophages (BMDMs) polarized toward M1, M2, or unpolarized (M0). e Western-blot analysis of aortic lysates from apoE / mice fed a 8-week HFD or chow diet. All images are representative of at − − least three mice per group. Values are expressed as mean SEM. *p < 0.05 compared with baseline group mice or unpolarized BMDMs ±

induced more MCP-1 production under ox-LDL stimula- can cause more macrophage migration and accumu- tion compared with control virus group (Additional fle 5: lation. Tese results indicate that increased expres- Figure S5), which manifested the survival macrophages sion of MDL-1 promotes macrophages survival under Xiong et al. J Transl Med (2017) 15:234 Page 8 of 13

Fig. 4 MDL-1 expression is regulated by pathophysiological agents implicated in atherosclerotic progression and regression in vitro. Western blot

analyses of MDL-1 protein level in C57BL/6 mice peritoneal macrophages treated with a 50 μg/mL ox-LDL for the indicated times or b ­CoCl2 at the concentrations indicated for 24 h. Band densitometry are quantifed. c Macrophages were stimulated with 50 μg/mL ox-LDL with or without pre-

treatment with 100 μmol/L HIF-1α inhibitor. d Macrophages were stimulated with 200 μmol/L CoCl­ 2 with or without pretreatment with 100 μmol/L HIF-1α inhibitor. Band densitometry quantifcation are shown. e Macrophages were treated with 50 μg/mL ox-LDL with or without pretreatment with 10 μg/mL HDL. Images are representative of three independent experiments. Values are expressed as mean SEM. *p < 0.05; #p < 0.01 ± Xiong et al. J Transl Med (2017) 15:234 Page 9 of 13

Fig. 5 MDL-1 overexpression promotes macrophage survival under ox-LDL stimulation. a Western-blot analysis of MDL-1 expression in peritoneal macrophages after infected with adenovirus overexpressing MDL-1 (Ad-MDL-1) or control virus (Ad-null). b Macrophage apoptosis (green) meas- ured by TUNEL assay of peritoneal macrophages stimulated with 50 μg/mL ox-LDL with or without pretreatment with Ad-MDL-1 or Ad-null in RPMI 1640 media. Nuclei were costained with DAPI (blue). Quantifcation of tunel assay from each group (n 3/group) was performed in (c). Values are ≥ expressed as mean SEM. *p < 0.05 compared with ox-LDL with Ad-null treatment group; #p < 0.01 compared with PBS or Ad-null group ± atherogenic stimuli, which could be predicted to foster promotes atherosclerotic plaque regression and sta- lesional macrophage accumulation and increase foam cell bility [3, 32, 33]. However, the mechanisms in lesional burden during early atherosclerosis progression. macrophage retention and accumulation are, to a large extent, still elusive and unknown. Our study frstly dem- Discussion onstrated that MDL-1, a C-type lectin receptor, is mainly Atherosclerosis progression is characterized by persis- expressed in lesional macrophages and its expression is tent accumulation of lipids and immune cells in plaques, associated with early atherosclerotic plaque progres- which is the leading cause of atherosclerotic plaque sion both in two well-established models (apoE−/− and rupture and contributes to myocardial infarction or ldlr−/− mice). Furthermore, we observed macrophage other adverse cardiovascular outcomes. Rupture-prone MDL-1 expression is notably decreased accompanied plaques feature with a thin fbrous cap and a large lipid with reduced lesional macrophage content and M1 sub- necrotic core [31]. Lesional macrophages are central type macrophage marker iNOS expression in plaque in these pathophysiological processes and recent stud- regression group mice and it is signifcantly expressed in ies suggest macrophage appears in a dynamic balance polarized M1 macrophage, refecting its proinfamma- in the lesion and strategies aiming at reducing mac- tory role in the disease. Moreover, our in vitro experi- rophage-derived foam cell burden could signifcantly ments found MDL-1 expression is driven by several Xiong et al. J Transl Med (2017) 15:234 Page 10 of 13

Fig. 6 MDL-1 overexpression reduces apoptosis-associated molecule expression in macrophages under ox-LDL stimulation. a Representative West- ern blot analyses of cleaved caspase-3 and caspase-3 expression in cell lysates from peritoneal macrophages treated with 50 μg/mL ox-LDL with or without pretreatment of adenovirus overexpressing MDL-1 (Ad-MDL-1) or control virus (Ad-null) in RPMI 1640 media. Images are representative of three independent experiments. Quantifcation of band densitometry is shown in b. Values are expressed as mean SEM. *p < 0.05 compared with ± Ad-null or ox-LDL with Ad-null treatment group

pathophysiological agents implicated in plaque progres- being an important contributor to plaque progression sion (e.g. ox-LDL and hypoxia) and the increased MDL-1 [32], these cells might come from neutrophils. Tis con- expression can be blocked by regressive agents like HDL clusion was also demonstrated in our immunofuores- and HIF-1α inhibitor. In addition, we found MDL-1 over- cent costaining of ldlr−/− mice lesional neutrophil and expression could maintain macrophage survival, down- MDL-1, which MDL-1 was found to be expressed in a regulate cleaved caspase-3 expression and induce MCP-1 few of neutrophils. However, we did not fnd signifcant production under atherosclerotic agents stimulation (e.g. diference on neutrophil MDL-1 expression between pro- ox-LDL). Terefore, MDL-1 is predicted to cause infam- gressive and regressive plaques. Tis may attribute to dif- matory lesional macrophage accumulation in the context ferent MDL-1 expression pattern in neutrophil and the of early atherosclerosis and these discoveries provide a extent of neutrophil MDL-1 expression is more relevant novel macrophage receptor biomarker regarding to the to cell diferentiation [10]. Functionally, MDL-1 has been relationship between its level and atherosclerotic plaque increasingly recognized as a key regulator involving in the progression and link abnormal lipid level to macrophage progression of multiple acute and chronic infammatory survival and accumulation, which might further aggra- diseases including hemorrhagic fever, lethal shock, virus- vate plaque foam cell burden and promote atherosclero- induced brain damage, chronic obstructive pulmonary sis progression. disease and the development of autoimmune arthritis [13, MDL-1, also called CLEC5A, associates non-covalently 15–18]. Furthermore, MDL-1 promotes proinfammatory with its adaptor DAP-12 to form receptor complexes and cytokines secretion and macrophage activation in these mediate its signaling pathway [8]. Several previous stud- infammation-associated diseases [12, 34]. In infam- ies showed that MDL-1 is mainly expressed in mouse matory macrophage, activation of Syk-coupled MDL-1 neutrophils, monocytes/macrophages and human periph- induces transcription of pro-interleukin-1β (IL-1β) and eral blood monocytes as well as myeloid cell precursors NLRP-3 infammasome during DV infection [35]. Tese [9–11, 18]. In addition, stronger MDL-1 expression was functions of MDL-1 might partly explain what we have observed in mature diferentiated myelomonocytic cells observed in this study, which signifcant increased MDL-1 [11]. Tese results were coincident with our fnding that expression is found on M1 proinfammatory macrophage MDL-1 is highly expressed in lesional macrophages in subtype, PBMCs from signifcant CAD patients and asso- both human and apoE−/− mice advanced atheromata ciated with infammatory chemokine (e.g. MCP-1) pro- and MDL-1 colocalized with DAP-12 in atherosclerotic duction and atherosclerotic plaque progression. plaques. Besides, we also observed some MDL-1 positive Our in vitro experiments exhibited that MDL-1 expres- cells were not macrophage-derived. Considering numer- sion in protein level is driven by several atherosclerotic ous related studies described above and neutrophil also pathophysiological agents like ox-LDL and hypoxia. We Xiong et al. J Transl Med (2017) 15:234 Page 11 of 13

also found macrophage MDL-1 expression were not macrophage physiological apoptotic process in this stage induced by native LDL. As we know, ox-LDL but not may contribute to lesion progression [6, 41, 42]. Fur- LDL has been described as potential promoter of vas- thermore, we also found MDL-1 overexpression in these cular infammatory responses and oxidative stress dur- macrophages amplifed ox-LDL induced MCP-1 produc- ing atherosclerosis [36]. And MDL-1, a proinfammatory tion. Terefore, the results of this study, which indicated receptor, were found in M1 subtype macrophages and MDL-1 overexpression promotes macrophage survival could be upregulated by hypoxia in our study. Besides, we and subsequently amplifes chemokine production, sug- found that increased MDL-1 expression is blocked after gest MDL-1, a receptor involving in multiple acute and HDL or HIF-1α inhibitor pretreatment. Previous studies chronic infammatory diseases, is associated with early have demonstrated that hypoxia exists in lipid enriched atherosclerotic plaque progression. advanced plaques and it is coexpressed with netrin-1 [28], one of the neuronal guidance cues implicated in Conclusions macrophage retention and leading to foam cell accu- MDL-1 is abundantly expressed in macrophages of mulation [37]. Investigation also showed that HIF-1α, a human and apoE−/− as well as ldlr−/− mice advanced downstream transcription factor of ox-LDL, could afect atherosclerotic lesions and increased macrophage lesional macrophage infammatory profle and promote MDL-1 expression is associated with early plaque pro- development of atherosclerosis [29]. Terefore, increased gression and promotes macrophage survival under MDL-1 expression induced by ox-LDL or hypoxia and proatherosclerotic stimuli (e.g. ox-LDL), thereby impli- inhibited by HDL or HIF-1α inhibitor suggests that cating that it might maintain lesional macrophage sur- MDL-1 might participate in lesional macrophage accu- vival and link abnormal hyperlipidemic environment to mulation and the failure to resolve infammation under macrophage accumulation during early atherosclerotic proatherogenic conditions. Tis conclusion is further plaque progression. Our study indicated MDL-1 may be supported when we found that MDL-1 expression is developed as a potent biomarker for predicting and pre- downregulated in lesional macrophages in a regressive venting early atherosclerotic plaque progression. plaque milieu accompanied with a reduction in lesional Additional fles macrophage content. And as what we observed in regres- sive plaque, this reduced expression of MDL-1 might be Additional fle 1: Figure S1. MDL-1 and its adaptor DAP-12 colocalize due to decreased number of lesional macrophages. in advanced apoE / mice atherosclerotic plaques. Immunostaining of Since our study indicated that changes of macrophage MDL-1 (green) and− DAP-12− (red) in apoE / mice advanced atheroscle- − − MDL-1 expression also accompanied with alterations of rotic plaques. Areas of colocalization are shown in yellow in the merged image (shown with white arrows). Images are representative of 3 mice. lesional macrophage content, we conducted the TUNEL Additional fle 2: Figure S2. Increased MDL-1 level in human peripheral assay and found that overexpression of MDL-1 contrib- blood monocytes is associated with the presence of coronary artery dis- uted to signifcant decrease of macrophage apoptosis ease (CAD). Western-blot analyses of MDL-1 protein expression in periph- under high-dose ox-LDL stimulation. Tis phenomenon eral blood monocytes from signifcant CAD patients (n 3) and healthy volunteers (n 3). Band densitometry are quantifed. Values= are expressed is also in accordance with the fnding that MDL-1 stim- as mean SEM.= #p < 0.01 compared with healthy volunteers. ulation helps to maintain 32Dcl3 cell survival [10]. ± Additional fle 3: Figure S3. MDL-1 and M1 subtype macrophage Although our results showed increased MDL-1 expres- marker iNOS colocalize in early advanced ldlr / mice atherosclerotic sion can be induced by ox-LDL, we here used an high- lesion and both reduce in regressive plaque. Immunofuorescent− − costain- dose extensively oxidized LDL to stimulate the cells for ing of MDL-1 (green) and iNOS (red) in aortic sinus plaques in a progres- sive (ldlr / mice fed a 8-week HFD) or regressive (ldlr / mice fed a 24 h and previous investigations demonstrated low-dose 8-week HFD− − and then switched to 4-week chow diet) environment.− − Areas minimally oxidized LDL could lead to macrophage sur- of colocalization are exhibited in yellow in the merged image. Images are vival [38]. Past research has demonstrated that local representative of at least 3 mice. apoptosis mainly mediates clearance of macrophages Additional fle 4: Figure S4. No signifcant diference is found on neutrophil MDL-1 expression between progressive and regressive from resolving infammation and controls macrophage atherosclerotic plaques. MDL-1 (green) and Ly-6G (red) were costained removal [39]. As we know, in the early stage of athero- in ldlr / mice advanced or regressive atherosclerotic plaques. Areas of − − sclerotic lesions, increased resistance of macrophages to colocalization are exhibited in yellow in the merged image (shown with white arrows). Images are representative of at least 3 mice. apoptosis is associated with macrophage accumulation Additional fle 5: Figure S5. MDL-1 overexpression amplifes ox-LDL and increased plaque burden [40]. Tis conclusion is also induced monocyte chemotactic protein 1 (MCP-1) production in coincident with the results of studies which revealed that peritoneal primary macrophages. Representative Western blot analyses apoptotic cells are only detectable in advanced lesions of MCP-1 expression in cell lysates from peritoneal macrophages treated with 50 μg/mL ox-LDL with or without pretreatment of adenovirus and this refects an efcient apoptosis clearance mecha- overexpressing MDL-1 (Ad-MDL-1) or control virus (Ad-null) in RPMI 1640 nism (e.g. normal eferocytosis function) in the early media. Images are representative of 3 independent experiments. stage of atherosclerotic plaques and any disruption of Xiong et al. J Transl Med (2017) 15:234 Page 12 of 13

Abbreviations 4. Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jafer FA, Libby P, et al. Mono- MDL-1: myeloid DAP12-associating lectin-1; CLEC5A: C-type lectin domain cyte accumulation in mouse atherogenesis is progressive and propor- family 5-member A; ox-LDL: oxidized low-density lipoprotein; ITAM: immuno- tional to extent of disease. Proc Natl Acad Sci USA. 2006;103:10340–5. receptor tyrosine-based activation motif; CAD: coronary artery disease; CCL: 5. Libby P. Infammation in atherosclerosis. Arterioscler Thromb Vasc Biol. chemokine (C–C motif) ligand; HFD: high-fat diet; HDL: high-density lipopro- 2012;32:2045–51. tein; M-CSF: monocyte-colony stimulating factor; BMDMs: bone marrow- 6. Ley K, Miller YI, Hedrick CC. Monocyte and macrophage dynamics during derived macrophages; FBS: fetal bovine serum; PBS: phosphate bufered saline; atherogenesis. Arterioscler Thromb Vasc Biol. 2011;31:1506–16. IL: interleukin; LPS: lipopolysaccharide; EDTA: ethylene diamine tetraacetic 7. Feig JE. Regression of atherosclerosis: insights from animal and clinical acid; TC: total cholesterol; LDL/VLDL: low-density lipoprotein/very low-density studies. Ann Glob Health. 2014;80:13–23. lipoprotein; WD: western diet; MCP-1: monocyte chemotactic protein 1. 8. Bakker AB, Baker E, Sutherland GR, Phillips JH, Lanier LL. Myeloid DAP12- associating lectin (MDL)-1 is a cell surface receptor involved in the activa- Authors’ contributions tion of myeloid cells. Proc Natl Acad Sci USA. 1999;96:9792–6. WXX performed the experiments, analyzed the data, and drafted the manu- 9. Chen ST, Lin YL, Huang MT, Wu MF, Cheng SC, Lei HY, et al. CLEC5A is criti- script. RX carried out q-PCR. FW carried out Western blotting. HBW and LL par- cal for dengue-virus-induced lethal disease. Nature. 2008;453:672–6. ticipated in animal experiments. HBW provided the clinical samples. GG and 10. Aoki N, Kimura Y, Kimura S, Nagato T, Azumi M, Kobayashi H, et al. Expres- RT conceived the study, analyzed the results. GG and RT wrote the manuscript. sion and functional role of MDL-1 (CLEC5A) in mouse myeloid lineage All authors read and approved the fnal manuscript. cells. J Leukoc Biol. 2009;85:508–17. 11. Gingras MC, Lapillonne H, Margolin JF. TREM-1, MDL-1, and DAP12 Author details expression is associated with a mature stage of myeloid development. 1 Department of Cardiology, Ruijin Hospital, Jiao Tong University School Mol Immunol. 2002;38:817–24. of Medicine, No. 197, Ruijin Er Road, Huangpu District, Shanghai 200025, 12. Cheng YL, Lin YS, Chen CL, Tsai TT, Tsai CC, Wu YW, et al. Activation of Nrf2 China. 2 Institute of Cardiovascular Disease, Jiao Tong University School by the dengue virus causes an increase in CLEC5A, which enhances TNF- of Medicine, Shanghai 200025, China. alpha production by mononuclear phagocytes. Sci Rep. 2016;6:32000. 13. Wu MF, Chen ST, Yang AH, Lin WW, Lin YL, Chen NJ, et al. CLEC5A is Acknowledgements critical for dengue virus-induced infammasome activation in human We appreciate the contribution of the colleagues in Animal Experiment macrophages. Blood. 2013;121:95–106. Center of Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine 14. Ward PA. Do MDL-1( ) cells play a broad role in acute infammation? J for maintenance of animal models. Clin Invest. 2011;121:4234–7.+ 15. Cheung R, Shen F, Phillips JH, McGeachy MJ, Cua DJ, Heyworth PG, et al. Competing interests Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal The authors declare that they have no competing interests. shock in mice. J Clin Invest. 2011;121:4446–61. 16. Chen ST, Liu RS, Wu MF, Lin YL, Chen SY, Tan DT, et al. CLEC5A regulates Availability of data and materials virus-induced neuroinfammation and lethality. Available under request. PLoS Pathog. 2012;8:e1002655. 17. 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