Maud Arnal-Levron

Yinan Chen

Peter Greimel

Federica Calevro

Karen Gaget

Fabien Riols

Aurélie Batut

Justine Bertrand-Michel

Françoise Hullin-Matsuda

Vesa M Olkkonen

Isabelle Delton

Céline Luquain-Costaz

Céline Luquain

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Keywords: Bis(monoacylglycero)phosphate, macrophages, cholesterol, oxysterols, late endosome, oxysterol binding protein

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Oxysterols are found enriched in pathologic structures such as macrophage foam cells and atherosclerotic lesions [START_REF] Shibata | Macrophages, oxysterols and atherosclerosis[END_REF]. They originate from a massive uptake of oxLDL-containing oxysterols in subendothelial macrophages. Moreover, macrophages are able to produce oxysterols intracellularly in late endosomes (LE), from both LDL-associated and cellular cholesterol [START_REF] Chen | THP1 macrophages oxidized cholesterol, generating 7-derivative oxysterols specifically released by HDL[END_REF]. The large family of oxysterols includes sterols oxidized at the ring, mainly at carbon 7 (e.g., [START_REF] Olkkonen | Macrophage oxysterols and their binding proteins: roles in atherosclerosis[END_REF]-ketocholesterol and 7α/β-hydroxycholesterol) and those oxidized at the sidechain, such as 24S-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. Generally, ring-oxidized sterols tend to be formed non-enzymatically, whereas side-chain oxidation is usually catalyzed by specific enzymes belonging to the cytochrome P450 family. The bulk of oxysterols in oxLDL are oxidized at carbon 7, such as 7β-hydroxycholesterol (7β-HC) and 7-ketocholesterol (7-keto). These oxysterols are responsible for the ability of oxLDL to induce cellular oxidative stress and cytotoxicity, mainly via apoptosis [START_REF] Larsson | Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects[END_REF][START_REF] Vejux | Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis[END_REF][START_REF] Arnal-Levron | Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL[END_REF][START_REF] Nègre-Salvayre | Dual signaling evoked by oxidized LDLs in vascular cells[END_REF].

Oxysterols are associated with a large variety of cellular effectors linked to atherosclerosis. Among them, oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of lipid binding/transfer proteins conserved in eukaryotes [START_REF] Olkkonen | Macrophage oxysterols and their binding proteins: roles in atherosclerosis[END_REF][START_REF] Olkkonen | Oxysterols and their cellular effectors[END_REF][START_REF] Dye | The Fundamental And Pathological Importance Of Oxysterol Binding Protein And Its Related Proteins[END_REF]. Several reports have addressed the role of ORPs in intracellular cholesterol trafficking and homeostasis, for example : control of endoplasmic reticulum-late endosomes (ER -LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cholesterol egress from LE (ORP5), macrophage lipid and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11) [START_REF] Pietrangelo | Bridging the molecular and biological functions of the oxysterolbinding protein family[END_REF][START_REF] Kentala | OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites[END_REF][START_REF] Olkkonen | Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism[END_REF]. The silencing of ORP8 or ORP1L altered the lipidome in RAW macrophages emphasizing their important role in cellular lipid homeostasis [START_REF] Vihervaara | Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins[END_REF].

Another important effector of oxysterols is bis(monoacylglycero)phosphate (BMP), a phospholipid preferentially found in LE membranes. BMP participates in cholesterol metabolism/trafficking in macrophages and regulates cholesterol efflux to HDL [START_REF] Arnal-Levron | Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL[END_REF][START_REF] Delton-Vandenbroucke | Anti-bis(monoacylglycero)phosphate antibody accumulates acetylated LDL-derived cholesterol in cultured macrophages[END_REF][START_REF] Chevallier | Lysobisphosphatidic acid controls endosomal cholesterol levels[END_REF][START_REF] Kobayashi | Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport[END_REF][START_REF] Luquain-Costaz | Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol redistribution, attenuates liver-X receptor/ATP-Binding cassette transporter A1/ATP-binding cassette transporter G1 pathway, and impairs cholesterol efflux[END_REF]. We recently showed that BMP exerts a protective action against the pro-apoptotic effect of oxLDL via a reduced production of intracellular oxysterols [START_REF] Arnal-Levron | Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL[END_REF], but the underlying molecular mechanism remains to be characterized. However, the emerging roles of OSBP/ORPs in sterol trafficking between organelles suggest their putative involvement. BMP has already been linked to two key proteins implicated in intracellular cholesterol traffic, Niemann-Pick C1 (NPC1) and C2 (NPC2) that are mutated in the Niemann-Pick disease type C [START_REF] Vanier | Complex lipid trafficking in Niemann-Pick disease type C[END_REF]. NPC1 and NPC2 are required to deliver free cholesterol from the endosomal compartment to the plasma membrane and ER [START_REF] Zhang | Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking[END_REF][START_REF] Vazquez | Oxidative stress: a pathogenic mechanism for Niemann-Pick type C disease[END_REF]. In vivo NPC cells are characterized by aberrant BMP accumulation and oxidative stress [START_REF] Chevallier | Lysobisphosphatidic acid controls endosomal cholesterol levels[END_REF]. Additionally, BMP and NPC2 cooperatively stimulate the efflux of cholesterol from the endosomal system [START_REF] Abdul-Hammed | Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion[END_REF][START_REF] Xu | Regulation of sterol transport between membranes and NPC2[END_REF][START_REF] Gallala | Biological function of the cellular lipid BMP-BMP as a key activator for cholesterol sorting and membrane digestion[END_REF].

In the present study, we focused on the concerted effect of BMP and members of the ORP family to protect against oxLDL cytotoxicity. ORP transcription level analysis identified ORP11 as the most promising candidate. Indeed, ORP11 not only localizes to the Golgi-LE interface [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF] but also a strong association of Osbpl11 gene polymorphisms with cardiovascular risk factors and diabetes has been reported [START_REF] Bouchard | Association of OSBPL11 gene polymorphisms with cardiovascular disease risk factors in obesity[END_REF]. We stably silenced ORP11 in RAW264.7 macrophages and demonstrated that ORP11 knockdown abolished the protective effect of BMP by impairing BMP-induced egress of cholesterol/oxysterols from endosomal compartments. Our results suggest a functional interplay of BMP and ORP11 to protect macrophages from oxLDL-induced cytotoxicity, with implications for atherogenesis.

BMP/ORP11 cooperation in sterol traffic 3

Materials and methods

Cell culture and treatments

RAW 264.7 cells were cultured in MEM supplemented with non-essential amino acids, 10% foetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin. They were routinely grown in T-75 flasks at 37°C in an atmosphere of 5% CO 2 and subcultured by trypsination at a 1:5 ratio. Experiments were started 24 h after seeding by pre-incubation without (control) or with 30 µM dioleoylphosphatidylglycerol (DOPG) liposomes (BMP-enriched) [START_REF] Bouvier | Selective decrease of BMP/ORP11 cooperation in sterol traffic 13 bis(monoacylglycero)phosphate content in macrophages by high supplementation with docosahexaenoic acid[END_REF] for 24 h. The addition of PG was maintained throughout the experiments. Cells were then incubated in basal conditions (unloaded) or in presence of native (nLDL) or oxLDL (loaded) for 24h at physiological concentrations (100-200 µg/ml). Incubations with LDL were done in medium-containing 5% lipoprotein deficient serum (LPDS). Other details of incubation conditions are given below and/or in the figure legends. 293FT cells used for packaging of lentiviruses were cultured in Opti-MEM supplemented with 10% FBS and 1% GlutaMAX. They were grown in T-75 flasks at 37°C in an atmosphere of 5% CO2. Transfections were carried out at 24 h after seeding the cells in serum-free Opti-MEM GlutaMAX.

Preparation of lentiviruses and infection of RAW264.7 cells

Short hairpin RNA (shRNA) encoding pLKO.1 lentiviral transfer vectors were purchased from Thermo Scientific TRC (The RNAi Consortium) or from Sigma MISSION ® TRC-Mm 1.0 (Mouse) shRNA library (Sigma-Aldrich, St. Louis, MO, USA). After plating and selecting bacteria, a plasmid purification (Plasmid Midi Kit, Qiagen) and DNA quantification (NanoDrop®) were performed. A transfection complex was prepared using 4 µg shRNA transfer vector, 3 µg packaging plasmid p8.91 and 2 µg VSV-G expressing plasmid pMD2.G. This complex was transfected into 293FT packaging cells using FuGENE HD transfection reagent (Roche) in a 5:2 reagent:DNA proportion. After two days, viral supernatants were collected, filtered and used to infect RAW 264.7 cells. Lentiviral transduction was performed using 8 µg/ml hexadimethrine bromide and selection was carried out using puromycin 10 µg/ml. The silencing efficiency of five ORP11 targeting shRNA (shORP11) constructs was determined by quantitative real-time RT-PCR and the shRNA with the best silencing efficiency was selected for the following experiments. ORP3-specific shRNA lentivirus [START_REF] Vihervaara | Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins[END_REF] was included for a comparison, and a non-target shRNA (shNT) SHC002V was used as a control plasmid. Early passages of the cell pools (until P8) were used for experiments, at which time no significant reduction in the silencing efficiency was observed.

Lipoprotein preparation and oxidation

Human LDLs were isolated from plasma by sequential ultracentrifugation [START_REF] Havel | The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum[END_REF]. LDL oxidation was achieved during 5h dialysis at 37°C against 10 mM Tris, 150 mM NaCl, pH 7.4 supplemented with 10 µM of CuSO 4 . Subsequent overnight dialysis was performed at 4°C against 10 mM Tris, 150 mM NaCl, pH 7.4, containing 2 mM EDTA to eliminate CuSO 4 and stop oxidation. LDL oxidation was evaluated by GC-MS/MS quantification of their associated oxysterols compared to native LDL.

Evaluation of cytotoxicity

After treatment, cells were washed with PBS and their viability was assessed using a colorimetric MTT assay (Cell proliferation Kit I, Roche) according to the manufacturer's instructions. MTT cleavage was determined by reading the absorbance at 560 nm. Cell viability in control and BMP enriched-cells was expressed as the percentage of maximum cell viability relative to unloaded-control cells.

Extraction of RNA and proteins

After treatment, RNA and proteins for ORPs analysis were extracted from cell lysates using NucleoSpin RNA/Protein kit (Macherey Nagel), according to manufacturer's instructions. For ABCG1 and LXRα mRNA analysis, total RNA was isolated using Trizol reagent (Invitrogen) according to manufacturer's instructions. RNA concentrations were determined with a NanoDrop® spectrophotometer and protein concentrations estimated with a Qubit® fluorometer.

ORPs mRNA analysis

For each RNA sample, the respective cDNA first-stand was generated with SuperScript® VILO cDNA Synthetis kit (Invitrogen) according to manufacturer's instructions. Each cDNA sample was amplified in triplicate for the genes of interest, using mouse 36B4 as a normalization gene in each run. Quantitative real-time RT-PCR reactions were performed using the Light Cycler® 480 SYBR Green I Master (Roche). The threshold was set in the linear range of fluorescence, and a threshold cycle (Ct) was measured for each well. The data was analyzed according to the comparative Fit Points method. The silencing efficiencies were evaluated by comparing the expression levels between the control (shNT) cells and the cell pool of interest. Primers are available on request.

ABCG1 and ABCA1 mRNAs analysis

Total RNAs were treated with RQ1 RNase-Free DNase I (Promega Corporation, Madison, WI, USA), and reverse-transcribed with the SuperScript TM III First-Strand Synthesis System (Life Technologies, Carlsbad, CA, USA), using Oligo(dT) primers (Invitrogen by Life Technologies,) in order to obtain first-strand cDNA. The quality of the cDNA synthesized was examined by PCR. As a negative control, the remainder of the DNase treated RNA was examined by PCR using the same conditions. Quantitative real-time RT-PCR reactions were performed on a LightCycler® 480 Real-Time PCR System (Roche, Penzberg, Germany) using 1:2.5 diluted cDNAs and a LightCycler® 480 SYBR Green I Master Mix (Roche, Penzberg, Germany) according to manufacturer's instructions. For each gene, triplicate assays were performed. Two normalization gene candidates (GAPDH and HPRT) were tested for gene expression variation in all the experimental conditions used in this work. The HPRT gene was employed as a normalization gene as it met the criteria imposed by the BestKeeper software analysis (data not shown). The primer sequences are available on request.

Western Blot analysis

ORP11 protein was electrophoresed on 6% polyacrylamide gels. After blocking unspecific binding, antibody incubations were carried out in blocking buffer (5% fat-free powdered milk in TBS containing 0.05% Tween 20). The bound primary antibodies were visualized with peroxidase-conjugated anti-rabbit IgG (Bio-Rad). Rabbit polyclonal antibody against ORP11 [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF] was used at 1:800 dilution and mouse polyclonal antibody against tubulin (Sigma) at 1:1000 dilution. All data was quantified with ImageJ software.

Immunofluorescence

Cells were plated on coverslips and, after treatments, fixed at room temperature for 30 min with 4% paraformaldehyde. Subsequently, cells were incubated for 30 min at room temperature with 0.1% BSA to block unspecific antibody binding and for 60 min with primary antibodies (anti-BMP, anti-ORP11 or anti-GM130) in PBS containing 0.05% saponin, followed by incubation with Alexa 488-or Alexa 546-conjugated secondary antibodies. The specimens were mounted with Mowiol and examined with a Zeiss LSM 510 confocal microscope equipped with the C-Apochromat 63XW Korr (1.2 n.a.) objective. The data was quantified with ImageJ software using JACoP (Just Another Colocalization Plugin). Colocalization scores using Mander's overlap coefficient (M1 and M2) were calculated for individual cells representative of a cell population. Thresholds were set to remove the background signal while remaining representative of the confocal image.

Incorporation of [ 3 H]oleate into CE

Macrophages were exposed to 1 µCi/ml [ 3 H]oleate during 24 h of nLDL or oxLDL loading. Unloaded macrophages were labeled by suspending [ 3 H]oleate into the growth medium utilizing an ethanolic stock solution, while maintaining final ethanol concentration <0.1%. After treatments, total lipids were extracted from cell lysates according to the method of Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF], separated by TLC (hexane/diethyl ether/acetic acid, 80:20:1, v/v) and quantified with a radioactivity analyzer (Raytest, France). Degree of cholesterol esterification was expressed as percentage of cholesteryl [ 3 H]oleate relative to total radioactivity recovered.

Sterol efflux to HDL

Cells were pre-incubated with 2 µCi/ml [ 3 H]cholesterol for 24 h. Sterol efflux under 6 h was measured in basal conditions (5% LPDS) or in response to HDL (100 µg/ml) in 5% LPDS-containing medium. The total radioactivity in cells and in media was determined by liquid scintillation counting. Total lipids from both cell and media were extracted according to the method of Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] and sterols were separated by TLC (hexane/diethyl ether/acetic acid/methanol, 50:50:1:5, v/v). The radioactivity associated with each sterol fraction (cholesterol and oxysterols, free or esterified) was measured with a radioactivity analyzer (Rayster, France) and converted to DPM according to liquid scintillation counting. The efflux of each sterol was expressed as the percentage of radioactivity released into the medium relative to total radioactivity in cells plus media.

Quantification of BMP and PG species by LC-MS/MS

After treatments, samples were spiked with C14:0/C14:0-BMP (DMBMP) and C14:0/C14:0-PG (DMPG) as internal standards, and total lipids were extracted from cell lysates and media according to the method of Bligh and Dyer [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF]. The resulting samples were dried under nitrogen flow and resuspended in methanol prior to measurement. LC-MS/MS was carried out on an Agilent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA) integrated with an 4000 QTrap mass spectrometer (AB SCIEX, Foster City, CA) using a Shiseido CAPCELL PAK C18 MGIII Type column (5µm, 2.0 x 50mm) (Shiseido CO., LTD., Tokyo, Japan). LC separation (2 µl sample injection) was performed utilizing methanol:acetonitrile (95:5) with 25 mM ammonium formate and 0.1% formic acid as monocratic mobile phase at a flow rate of 100µl/min. The mass spectrometer was operated in positive ion mode utilizing the following optimized parameters for BMP and PG for multi reaction monitoring (MRM) mode: declustering potential 55V (for BMP) and 70V (for PG), collision energy 30V, collision cell exit potential 15V, entrance potential 10V, Q1 resolution set to unit and Q3 resolution set to low. Internal standards for quantification were monitored at 684.6/285.1 Da (for DMBMP) and at 684.6/495.5 Da (for DMPG).

Quantification of total oxysterols by GC-MS

Oxysterol were quantified by a modified protocol based on Iuliano [START_REF] Iuliano | Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status[END_REF]. After treatments, each cell pellet was crushed with a FastPrep ®-24 Instrument (MP Biomedical) in Methanol/EGTA (2:1, v/v) solution. Total lipids were extracted by a modified method of Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] from cell lysates and media in dichloromethane/methanol/water (2.5:2.5:2, v/v), spiked with internal standards 19-hydroxycholesterol and vitamin E. Esters were hydrolyzed with KOH-CH 2 Cl 2 and neutralized with phosphoric acid addition prior to a second Bligh and Dyer extraction. Next, a Solid Phase Extraction (SPE) using SiOH 96-well plate (100 mg/well, Macherey Nagel) was performed. After sample application, the extraction plate was washed with 2-propranol/heptane (0.5:99.5, v/v) to eliminate neutral lipids like cholesterol. After drying under aspiration, oxysterols were eluted with 2-propanol/heptane (0.3:0.7, v/v). Oxysterols were converted to trimethysilyl ether by addition of BSTFA/ACN (bis(trimethylsilyl)trifluoroacetamide/acetonitrile, 1:1; v:v) at 55°C for 60 min. Gas chromatography-mass spectrometric analysis was performed on a ThermoScientific Trace GC coupled to a Trace ISQ Mass selective detector (ThermoScientific). The silylated oxysterols were separated on an Agilent J&W HP-5MS capillary column (30 m, 0.25 mm, 0.25 µm phase thickness). The oven temperature program was as follows: 180°C for 1 min, 20°C/min to 250°C, 5°C/min to 300°C where the temperature was kept for 8 min, and then 35°C/min to 325°C. High purity helium was used as carrier gas at a flow rate of 0.8 mL/min in constant flow mode. The samples were injected in a splitless mode with an injection volume of 1 µL. The injector, transfer line and source temperatures were 270°C, 280°C and 250°C respectively. The mass spectrometer was operated in the selected ion monitoring (SIM) mode, and the following ions were monitored for analysis: 7α-hydroxycholesterol 456-367 m/z, 7βhydroxycholesterol 456-367 m/z, 25-hydroxycholesterol 546-456 m/z, 7-ketocholesterol 472-382 m/z. Peak detection, integration and quantitative analysis were executed using Xcalibur Quantitative browser (ThermoScientific) based on calibration lines built with commercially available oxysterols standards (Avanti Polar Lipids or Interchim).

GC-MS quantification of free cholesterol

After treatments, total lipids were extracted from cell lysates according to the method of Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] in chloroform/methanol/water (2:2:1.8; v/v), spiked with stigmasterol as internal standard, followed by sterols separation on TLC (hexane, diethyl ether, methanol, acetic acid, 50:50:5:1, v/v). Cholesterol was extracted from silica (hexane/diethyl ether, 1:1; v/v), dried under nitrogen and converted to its respective trimethylsilyl ether by treatment with BSTFA at room temperature, overnight. Gas chromatographic analysis was performed with a Hewlett Packard (HP-6890) and a J & W 122-4762 (60 m x 0.25 mm) capillary column. The eluted compounds were detected at the column outlet by a mass spectrometer (Hewlett Packard MS-5973) and quantified using the internal standard stigmasterol.

Results

Analysis of OSBP and ORPs mRNA expression in RAW264.7 macrophages

First, we established the cellular response in RAW264.7 macrophages to native (nLDL) or oxidized LDL (oxLDL) loading, by determining the respective mRNA expression levels of OSBP/ORP. After exposure to nLDL the mRNA levels of OSBP, ORP1L, ORP8, ORP9 and ORP11 increased significantly compared to unloaded conditions (Fig. 1). ORP2, ORP9 and most strikingly ORP11 mRNA levels were higher in oxLDL loaded macrophages. ORP11 has recently been described to be associated with both Golgi elements and endosomal compartments [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF], indicating a putative role in the non-vesicular lipid transport between Golgi and endosomes. Furthermore, OSBPL11 gene has been reported to be involved in cholesterol metabolism in obese patients [START_REF] Bouchard | Association of OSBPL11 gene polymorphisms with cardiovascular disease risk factors in obesity[END_REF] and facilitate cholesterol trafficking in skin fibroblasts [START_REF] Kara | Severe neurodegenerative disease in brothers with homozygous mutation in POLR1A[END_REF]. Consequently, we focused our further efforts on ORP11, investigating its possible involvement in BMP mediated oxLDL-derived sterol regulation.

BMP/ORP11 cooperation in sterol traffic 7

3.2.ORP11, a candidate implicated in BMP-mediated oxysterol traffic

Next, we examined whether BMP accumulation would impact ORP11 expression in RAW264.7 macrophages. Consistent with the change in mRNA expression, ORP11 protein level was increased upon nLDL incubation and by almost two-fold upon oxLDL incubation (Fig. 2A). In oxLDL BMP-enriched cells, ORP11 was further up-regulated, suggesting a functional relationship between BMP and ORP11. This prompted us to determine the endogenous subcellular distribution of ORP11 in RAW264.7 macrophages compared to BMP. Consistent with earlier reports in HEK293 and HuH7 cells [START_REF] Vazquez | Oxidative stress: a pathogenic mechanism for Niemann-Pick type C disease[END_REF], ORP11 strongly, but not exclusively, colocalized (M2=0.89±0.03 vs. M1=0.67±0.04) with GM130, a Golgi marker displaying a typical compact juxtanuclear distribution (Fig. 2B). Additionally, ORP11 labelling also colocalized with BMP (M2=0.91±0.05), known to be an endosomal resident in RAW264.7 macrophages and representing a typical punctated perinuclear pattern [START_REF] Luquain-Costaz | Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol redistribution, attenuates liver-X receptor/ATP-Binding cassette transporter A1/ATP-binding cassette transporter G1 pathway, and impairs cholesterol efflux[END_REF], consistent with a possible cooperation between these two factors involved in sterol trafficking.

Impact of ORP11 silencing on protective action of BMP towards oxLDL

To further investigate the function of endogenous ORP11 protein in macrophages, we generated stably silenced RAW264.7 cells using shRNA lentiviruses targeting ORP11 mRNA, and ORP3 mRNA used as a non-relevant ORP protein. The gene silencing efficiency was determined by quantitative real-time RT-PCR after lentiviral transduction and selection. Cell pools transduced with each virus were independently selected two times and the silencing efficiencies were between 0 and 69%. shORP11.2 and shORP3.2 cell pools with the highest knock-down efficiency (69% and 70%, respectively) was chosen for further experiments and silencing efficiencies were determined at the protein level (Fig. 3A). ORP11 and ORP3 knockdown efficiencies in these cell pools corresponded well to the level of mRNA reduction (Data not shown). ORP3 was chosen as a control for BMP enrichment, as well as cell viability, as it mediates cell adhesion, independently of cholesterol homeostasis [START_REF] Weber-Boyvat | OSBP-related protein 3 (ORP3) coupling with VAMPassociated protein A regulates R-Ras activity[END_REF][START_REF] Lehto | The R-Ras interaction partner ORP3 regulates cell adhesion[END_REF]. Furthermore, ORP3 is localized in plasma membrane/ER Membrane Contact Sites (MCS) [START_REF] Olkkonen | Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism[END_REF][START_REF] Lehto | Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7[END_REF], but excluded from LE.

To fully validate our cell models, we ensured that BMP enrichment after PG supplementation was maintained after ORP11 and ORP3 silencing. As expected [START_REF] Bouvier | Selective decrease of BMP/ORP11 cooperation in sterol traffic 13 bis(monoacylglycero)phosphate content in macrophages by high supplementation with docosahexaenoic acid[END_REF], incubation of control (shNT) RAW264.7 macrophages with PG lead to a 3-4 fold increase of cellular BMP in all tested conditions (Fig. 3B). Importantly, BMP accumulation was maintained at similar levels in both ORP11 and ORP3 silenced cells (shORP11 and shORP3) compared to their respective controls. Furthermore, as shown in Table 1, BMP molecular species composition was similar between shNT11 and shOPR11 cells, as well as shNT3 and shORP3 cells.

In a previous report [START_REF] Arnal-Levron | Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL[END_REF], we showed that oxLDL exposure induced cell death that was partially restored by BMP enrichment. These effects were not observed in both unloaded and nLDL loading conditions. Consistently, cell viability in both shNT11 and shNT3 cells was drastically affected after oxLDL exposure compared to nLDL-loaded cells (Fig. 4A), and BMP enrichment reduced oxLDL negative effect as cell viability leapt from 2 % up to 20 %. By contrast, cell viability remained low at only 9% in BMP-enriched shORP11 cells, indicating that functional ORP11 is required for the protective effect of BMP. Importantly, this is specific to ORP11, as silencing of ORP3 had a different effect on the protective action of BMP against oxLDL-induced cytotoxicity. Contrariwise, the increase in cell viability upon BMP enrichment was higher in shORP3 cells compared to shNT3 cells.

BMP/ORP11 cooperation in sterol traffic

Cytotoxicity of oxLDL has been shown to be at least partly mediated by C-7 proapoptotic oxysterols [START_REF] Nègre-Salvayre | Dual signaling evoked by oxidized LDLs in vascular cells[END_REF]. Consistently, oxLDL exposure was correlated to increased amounts of C-7 oxysterols (7α-HC, 7β-HC and 7-keto) in both shNT11 and shORP11 cells (Fig 4B ), as well as in shNT3 and shORP3 cells (Fig 4C). As expected, the production of oxysterol was very low in both control and nLDL conditions (Fig 4B). BMP accumulation significantly reduced C-7 oxysterols amounts in shNT11 and shNT3 cells. Importantly, silencing of ORP11 but not ORP3 suppressed this BMP effect. The content of enzymatically and non-enzymatically accessible 25-hydroxycholesterol (25-OH), which has greater or lesser cytotoxicity depending on the cell considered [START_REF] Vejux | Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis[END_REF], was also increased upon oxLDL exposure in both shNT11 and ORP11 cells. No 25-OH was detected in shNT3 or shORP3 cells. In contrast to C-7 oxysterols, the production of 25-OH was unaffected by BMP enrichment (Fig. 4C). Together these results demonstrate the dependency of functional ORP11, but not ORP3, in BMP protective effect against both oxLDL induced cell death and proapoptotic C-7 oxysterols formation. This suggests a functional interplay of ORP11 and BMP selectively affecting the proapoptotic production of oxysterols and a concerted action of ORP11 and BMP for sterol trafficking and metabolism in macrophages.

3.4.Impact of ORP11 silencing on the fate of oxLDL-derived cholesterol

We further aimed to investigate the possible cooperation between BMP and ORP11 in regulating cholesterol homeostasis in RAW264.7 macrophages. Free cholesterol (FC) levels, were compared in shNT11 and shORP11 cells. As shown in (Fig. 5A), in shNT11 cells, FC levels were similar in all tested conditions regardless nLDL/oxLDL exposure or BMP enrichment. ORP11 silencing did not change FC content in unloaded or nLDL-loaded cells, independently of BMP enrichment. In contrast, it induced FC accumulation upon oxLDL loading, which was further enhanced in BMP-enriched cells. These increases in cellular FC may be related to differences in cholesterol esterification or cholesterol efflux. Cholesterol esterification was measured as a proportion of radioactivity recovered in cholesterol ester (CE) after incubation with [ 3 H]oleate for 24 h. Neither BMP enrichment nor ORP11 silencing modified the rate of cholesterol esterification markedly in oxLDL loaded cells (Fig. 5B).

As macrophage cholesterol efflux is essentially dependent on the expression of ATPbinding cassette transporters ABCG1 and ABCA1 [START_REF] Yvan-Charvet | Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses[END_REF], we measured their mRNA expression in shORP11 cells by quantitative real-time RT-PCR. ABCG1 mRNA levels were significantly reduced in ORP11 silenced cells compared to control (Fig. 5C) and further decreased under BMP-enrichment conditions. In contrast, no differences were observed in ABCA1 mRNA levels (Fig. 5D). We then measured cholesterol efflux to HDL which is dependent of ABCG1 in macrophages whereas efflux to ApoA1 is only dependent of ABCA1 [START_REF] Phillips | Molecular mechanisms of cellular cholesterol efflux[END_REF]. As expected from our previous report [START_REF] Arnal-Levron | Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL[END_REF], BMP enrichment slightly but significantly reduced cholesterol efflux to HDL in oxLDL-loaded shNT11 cells (Fig. 6E). Cholesterol efflux was reduced in shORP11 cells compared to shNT11 cells, and completely abolished in BMP-enriched shORP11 cells. This is in good agreement with the above observed reduction of ABCG1 expression upon ORP11 silencing. Even if it is difficult to compare changes in mRNA levels and % of efflux, these two parameters lead in the same direction. Together, these observations suggest that ORP11 is intrinsically and specifically involved in oxLDL-derived cholesterol retention/efflux and support a facilitating role of BMP in that process.

Discussion

We previously demonstrated that BMP exerts a protective action against the proapoptotic effect of oxLDL on macrophages via a reduced production of intracellular proapoptotic oxysterols [START_REF] Arnal-Levron | Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL[END_REF]. In the present study, we report a new role for ORP11 in modulating the protective action of BMP against oxLDL cytotoxic effects, putatively via the control of the intracellular trafficking of cholesterol/oxysterols. Our data suggest that in cultured macrophages BMP and ORP11 together control the sterol metabolism to dampen the generation of cytotoxic 7-oxysterols.

Human and mouse ORPs genes are expressed in a large variety of tissues [START_REF] Lehto | Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7[END_REF][START_REF] Laitinen | Family of human oxysterol binding protein (OSBP) homologues. A novel member implicated in brain sterol metabolism[END_REF][START_REF] Lehto | The OSBP-related protein family in humans[END_REF][START_REF] Jaworski | A family of 12 human genes containing oxysterol-binding domains[END_REF][START_REF] Anniss | An oxysterol-binding protein family identified in the mouse[END_REF], but there are clear differences in tissue-specific expression patterns between the members of the ORPs family [START_REF] Vihervaara | Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins[END_REF][START_REF] Yan | Expression of human OSBP-related protein 1L in macrophages enhances atherosclerotic lesion development in LDL receptor-deficient mice[END_REF][START_REF] Yan | OSBP-related protein 8 (ORP8) suppresses ABCA1 expression and cholesterol efflux from macrophages[END_REF]. In the present study, we confirmed the expression of eight OSBP/ORPs genes in mouse RAW264.7 macrophages and found that most ORP mRNAs are induced after exposure of the cells to nLDL, a major sterol carrier. More interestingly, three ORP genes were up-regulated upon incubation of the cells with oxLDL: ORP2, ORP9 and ORP11. ORP2 regulates the membrane contact between ER and lipid droplets, providing a plausible explanation for its up-regulation under oxLDL exposure [START_REF] Orso | Oxidized LDL-induced endolysosomal phospholipidosis and enzymatically modified LDL-induced foam cell formation determine specific lipid species modulation in human macrophages[END_REF][START_REF] Kentala | Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure[END_REF][START_REF] Weber-Boyvat | Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites[END_REF][START_REF] Kentala | OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism[END_REF]. ORP9 localizes at the Golgi/ER interface and regulates phosphatidylinositol-4-phosphate (PI4P) metabolism in Golgi membranes, competitively with ORP4S [START_REF] Liu | Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9)[END_REF]. Recently, ORP11 was demonstrated to dimerize with ORP9 and to localize at the interface of Golgi complex and LE [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF]. This is consistent with their concomitant upregulation upon oxLDL treatment, as oxLDL promotes endolysosomal lipid storage in macrophage foam cells [START_REF] Schmitz | Endolysosomal phospholipidosis and cytosolic lipid droplet storage and release in macrophages[END_REF].

Little is known about the functions of ORP11, but given its intracellular localization at the Golgi-LE interface [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF], and that of BMP almost exclusively in LE [START_REF] Kobayashi | Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport[END_REF][START_REF] Hullin-Matsuda | Bis(monoacylglycero)phosphate, a peculiar phospholipid to control the fate of cholesterol: Implications in pathology[END_REF], we envision that these two molecules could work together to egress oxLDL-derived cholesterol and/or oxysterols out of the endosomal compartment. Of interest, upregulation of ORP11 protein expression upon oxLDL exposure was enhanced in BMP enriched cells. Furthermore, a strong colocalization of BMP and ORP11 in the endosomal compartment was observed. To further analyze the function of ORP11, we generated stably silenced ORP11 depleted RAW264.7 macrophages. ORP3 silenced cells were chosen as non-relevant controls as ORP3 expression was not modulated by nLDL/oxLDL exposure in our cell model. Moreover, so far known functions of ORP3 are not related to sterol metabolism [START_REF] Weber-Boyvat | OSBP-related protein 3 (ORP3) coupling with VAMPassociated protein A regulates R-Ras activity[END_REF]. We observed as a key result that the protection of BMP against the cytotoxic effect of oxLDL was specifically impaired by ORP11 knock-down. Importantly, this could not be attributed to the lack of BMP or different BMP molecular species in shORP11/shORP3 cells since both amount and composition of BMP were similar to respective shNT cells. Impacts on cell death/survival have been reported for ORP8 [START_REF] Zhong | Oxysterol-binding proteinrelated protein 8 (ORP8) increases sensitivity of hepatocellular carcinoma cells to Fas-mediated apoptosis[END_REF][START_REF] Li | Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol[END_REF], OSBP [START_REF] Burgett | Natural products reveal cancer cell dependence on oxysterol-binding proteins[END_REF], ORP4 [START_REF] Charman | Oxysterol-binding protein (OSBP)-related protein 4 (ORP4) is essential for cell proliferation and survival[END_REF], and ORP9 [START_REF] Lessmann | Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation[END_REF], via protein-protein interactions or impacts on membrane lipid domains [START_REF] Olkkonen | Macrophage oxysterols and their binding proteins: roles in atherosclerosis[END_REF]. ORP11 affected cell viability significantly only after BMP accumulation, suggesting that this protein-phospholipid interaction is an essential link of the cell survival-promoting machinery, and that BMP needs ORP11 to protect cell viability. This hypothesis was supported by the oxysterol profile in ORP11 knock-down as the negative impact of ORP11 silencing on cell viability was correlated with an elevated level of proapoptotic 7-oxysterols. Cells overexpressing ORP11 displayed multilamellar bodies associated with the Golgi complex related to ORP11 intracellular localization at the LE-Golgi interface [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF]. Moreover, ORP11 interacts with ORP9, a family member implicated in cholesterol transport between ER and Golgi membranes [START_REF] Ngo | Oxysterol binding protein-related Protein 9 (ORP9) is a cholesterol transfer protein that regulates Golgi structure and function[END_REF].

Vihervaara et al. [START_REF] Vihervaara | Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins[END_REF] tested the effect of knocking down ORP1L, ORP3 or ORP8 in RAW264.7 macrophages, on the cellular lipidome. In basal conditions, levels of FC and CEs were elevated in ORP1L and ORP8 knock-down cells. Levels of PIs, PEs, or SMs were differently modified, revealing distinct roles of the different ORPs as regulators of macrophage lipid composition. In the present work we showed that both ORP11 and ORP3 BMP/ORP11 cooperation in sterol traffic 10 silencing did not interfere with BMP metabolism, as similar amounts of BMP were observed compared to shNT controls. The fact that BMP molecular species were not changed may suggest that ORP11 and ORP3 do not impact either on fatty acid metabolism. By contrast, ORP11 silencing increased the cellular cholesterol content in the presence of oxLDL, the effect being amplified after BMP accumulation, indicating a cumulative effect of ORP11 and BMP to regulate LE cholesterol levels. The increase of free cholesterol in ORP11 knockdown cells was not associated with a reduction of cholesterol esterification, indicating that ORP11 is not required for cholesterol transport from LE to the ER. However, cholesterol efflux to HDL was markedly reduced in ORP11 silenced cells after BMP accumulation, concomitant with reduced ABCG1 expression. Related functions have been assigned for several ORP family members: Knock-down of ORP8 up-regulated cholesterol biosynthesis in hepatic cells through sterol regulatory element binding protein (SREBP) activity [START_REF] Zhou | OSBP-related protein 8 (ORP8) regulates plasma and liver tissue lipid levels and interacts with the nucleoporin Nup62[END_REF] and induced FC accumulation in macrophages [START_REF] Vihervaara | Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins[END_REF] apparently via the inhibition of its efflux through ABCA1 [START_REF] Yan | OSBP-related protein 8 (ORP8) suppresses ABCA1 expression and cholesterol efflux from macrophages[END_REF]. ORP1L deficient macrophage displayed an increase of FC and CE, due to the inhibition of cholesterol efflux [START_REF] Vihervaara | Sterol binding by OSBP-related protein 1L regulates late endosome motility and function[END_REF], and it was proposed that ORP1L impacts the endosomal cholesterol transport via the formation of MCSs between LE and ER [START_REF] Rocha | Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning[END_REF][START_REF] Rowland | ER contact sites define the position and timing of endosome fission[END_REF][START_REF] Zhao | Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System[END_REF]. ORP5 was suggested to mediate cholesterol egress from the LE limiting membrane in concert with Niemann-Pick C1 [START_REF] Du | A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking[END_REF]; however, ORP5 and ORP8 were also identified as dual PS/PI4P transporters [START_REF] Chung | INTRACELLULAR TRANSPORT. PI4P/phosphatidylserine countertransport at ORP5-and ORP8-mediated ER-plasma membrane contacts[END_REF][START_REF] Ghai | ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P 2) and regulate its level at the plasma membrane[END_REF]. The closely related ORP2 has the capacity to enhance the transport of newly synthesized cholesterol to the cell surface [START_REF] Hynynen | Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis[END_REF] and during steroid hormone biosynthesis via LXR [START_REF] Escajadillo | Oxysterol-related-binding-protein related Protein-2 (ORP2) regulates cortisol biosynthesis and cholesterol homeostasis[END_REF]. Moreover, OSBP negatively regulates ABCA1 protein stability and consequently the cellular cholesterol efflux capacity [START_REF] Bowden | OSBP negatively regulates ABCA1 protein stability[END_REF].

OSBP/ORPs proteins have been originally characterized as sterol sensors and/or transporters at membrane contact sites (MCS) between ER and other organelles [START_REF] Kentala | OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites[END_REF][START_REF] Olkkonen | Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism[END_REF]. More recently, this role was expanded to the binding /transfer of glycerophospholipids [START_REF] Olkkonen | OSBP-related proteins: liganding by glycerophospholipids opens new insight into their function[END_REF], such as PI4P [START_REF] Pietrangelo | Bridging the molecular and biological functions of the oxysterolbinding protein family[END_REF][START_REF] Chung | INTRACELLULAR TRANSPORT. PI4P/phosphatidylserine countertransport at ORP5-and ORP8-mediated ER-plasma membrane contacts[END_REF][START_REF] De Saint-Jean | Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers[END_REF][START_REF] Mesmin | A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP[END_REF][START_REF] Moser Von Filseck | Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate[END_REF][START_REF] Antonny | The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol[END_REF], phosphatidylinositol bisphosphate (PIP 2 ) [START_REF] Ghai | ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P 2) and regulate its level at the plasma membrane[END_REF][START_REF] Raychaudhuri | Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides[END_REF] or Phosphatidylserine (PS) [START_REF] Moser Von Filseck | Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate[END_REF][START_REF] Maeda | Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins[END_REF][START_REF] Iwama | Osh6p, a homologue of the oxysterolbinding protein, is involved in production of functional cytochrome P450 belonging to CYP52 family in n-alkane-assimilating yeast Yarrowia lipolytica[END_REF]. Similarly, ORP11 localizes via its N-terminal domain in Golgi and LE membranes, and is implicated in non-vesicular communication between these two organelles [START_REF] Zhou | OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface[END_REF]. We propose that, in cooperation with BMP, ORP11 could act as a key component of a new pathway of non-vesicular lipid transport between LE and Golgi. BMP could be considered as a potential ligand of the ORD on ORP11, which could act as lipid signal to stimulate the egress of cholesterol from LE. This non-vesicular transport could be facilitated by a MCS between these two organelles [START_REF] Kentala | OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites[END_REF][START_REF] Prinz | Bridging the gap: membrane contact sites in signaling, metabolism, and organelle dynamics[END_REF]. ORP11 could act directly as a cholesterol carrier stimulated by BMP or via a yet known partner like NPC2 regulation as described for NPC1 [START_REF] Storch | Niemann-Pick C2 (NPC2) and intracellular cholesterol trafficking[END_REF][START_REF] Mccauliff | Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport[END_REF][START_REF] Luo | Routes and mechanisms of post-endosomal cholesterol trafficking: A story that never ends[END_REF]. However, Maeda et al. [START_REF] Maeda | Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins[END_REF] classified ORP11 within a clade that may not bind/transport cholesterol. Therefore, we find it possible that ORP11 may promote sterol transport indirectly via regulation of the assembly or function of a MCS. Further study of a putative molecular interaction between BMP and ORP11 by employing biomimetic membranes and liposomes will be crucial for elucidating the mechanism of ORP11 action and its modulation by BMP.

To conclude, the present work identifies ORP11 as a regulator of macrophage cholesterol homeostasis. ORP11 regulates the cytotoxicity of oxLDL and oxysterols via the endosomal phospholipid, BMP, as well as cholesterol efflux from macrophages. Consistent with the genetic findings of Bouchard et al. [START_REF] Bouchard | Association of OSBPL11 gene polymorphisms with cardiovascular disease risk factors in obesity[END_REF], ORP11 may thus act as a modulator of atherosclerosis.

Juselius Foundation, and the Magnus Ehrnrooth Foundation (V.M.O). GC-MS analyses were performed on the Toulouse INSERM Metatoul-Lipidomique Core Facility-MetaboHub ANR-11-INBS-010

Figures legends

Fig. 1 .Fig. 2 .

 12 Fig.1. Expression level of major OSBP/ORP mRNA in RAW264.7 macrophages. Cells were incubated in the absence or presence of 100µg/ml nLDL or 200µg/ml oxLDL for 24h. Cells were analyzed for different mRNA content by qPCR. Data are means±SD of 3 wells and are representative of 2 independent experiments. a, p<0,05 compared to unloaded; b, p<0,05 compared to nLDL

Fig. 3 .

 3 Fig.3. Characterization of RAW264.7 macrophages subjected to ORP11 knock-down. (A) A representative Western Blotting of RAW macrophages showing efficiency of silencing for ORP11 (90%) and ORP3 (85%) at the protein level ORP11 (shORP11.2) and ORP3 (shORP3.2) (B) Effect of ORP11 silencing on BMP level. Unloaded and BMP-enriched shNT, shORP11, shNT3 or shORP3 cells where incubated with 100µg/ml nLDL or 200µg/ml oxLDL for 24h. BMP was quantified by liquid chromatography-mass spectrometry as described in M&M. Data are the means±SD of 3 wells and representative of 3 independent experiments. a, p<0,05 compared to control; b, p<0,05 compared to nLDL; c, p<0,05 compared to oxLDL; d, p<0,05 compared to oxLDL BMP enriched in shNT cells.

Fig. 4 .

 4 Fig.4. Effect of ORP11 silencing on protective action of BMP towards oxLDL. Control and BMPenriched shNT, shORP11 or shOPR3 cells where incubated with 100µg/ml nLDL or 200µg/ml oxLDL for 24h. (A) Cell viability was evaluated. Data are the means±SD of 3 wells and representative of 3 independent experiments. a, p<0,05 compared to nLDL; b, p<0,05 compared to oxLDL; c, p<0,05 compared to shNT oxLDL BMP enriched cells; d, p<0,05 compared to shNT3 oxLDL BMP enriched. Control and BMP-enriched shNT or shORP11 cells where incubated in the absence or presence of 100µg/ml nLDL or 200µg/ml oxLDL for 24h. (B and C) 7-oxycholesterols (7α-, 7βhydroxycholesterol , and 7-ketocholesterol) and 25-hydoxycholesterols (D) were quantified by GC-MS/MS as described in M&M. Data are the means±SD of 3 wells and representative of 3 independent experiments. a, p<0,05 compared oxLDL, b, p<0,05 compared to shNT oxLDL BMP enriched.

Fig. 5 .

 5 Fig.5. Effect of ORP11 silencing on cellular cholesterol fate. Unloaded and BMP-enriched shNT or shORP11 cells where incubated in the absence or presence of 100µg/ml nLDL or 200µg/ml oxLDL for 24h. (A) Free cholesterol (FC) was quantified by GC/MS as described in M&M. Data are the means±SD of 3 wells and representative of 2 independent experiments. a, p<0,05 compared to shNT oxLDL cells; b, p<0,05 compared to shNT oxLDL BMP enriched; c, p<0,05 compared to shORP11 oxLDL . (B) Cells where incubated with [ 3 H] oleate and cholesterol esterification was measured as the proportion of radioactivity recovered in CE. ABCG1 (C) and ABCA1 (D) mRNA content was measured by RT-PCR as described in M&M. a, p<0,05 compared to shNT oxLDL; b, p<0,05 compared to shNT oxLDL BMP enriched; c, p<0,05 compared to shORP11 oxLDL. (E) Cells where incubated with [ 3 H] cholesterol for 24h. Cholesterol efflux was stimulated by 100µg/ml HDL for 6 hours. Data are the means±SD of 3 wells and representative of 3 independent experiments. a, p<0,05 compared to shNT oxLDL; b, p<0,05 compared to shNT oxLDL; c, p<0,05 compared to shNT oxLDL BMP enriched; d, p<0,05 compared to shORP11 oxLDL.

  Figure 1

Table 1

 1 /18:1 18:1/18:1 18:1/18:1 18:1/18:1 18:1/18:1 18:1/18:1 18:1/18:1 18:1/18:1

		shNT11 cells	shORP11 cells	shNT3 cells	shORP3 cells
	BMP 18:1molecular PG	PG	PG	PG	PG	PG	PG	PG
	species	mol%	oxLDL	mol%	oxLDL	mol%	oxLDL	mol%	oxLDL
			mol %		mol %		mol %		mol %
	16:0/18:1	4.21	6.63	2.56	5.68	2.99	5.83	2.36	7.25
	18:1/18:2	4.38	14.05	3.23	13.74	4.32	12.59	5.39	12.74
	18:1/18:1	72.73	66.88	80.47	69.77	73.55	65.97	65.74	58.83
	18:2/18;2	0.00	0.39	0.00	0.41	0.01	0.30	0.00	0.40
	18:1/20:4	1.75	1.99	1.82	2.17	2.27	3.07	2.12	2.64
	18:2/20:4	0.01	0.09	0.01	0.07	0.03	0.10	0.01	0.21
	18:1/22:6	16.00	9.24	10.87	7.44	13.75	10.20	20.33	14.83
	18:1/22:5	0.92	0.74	1.04	0.73	3.08	1.96	4.06	3.10

Acknowledgments

We gratefully acknowledge Riikka Kosonen for skilled technical assistance.

Funding

This study was supported by grants from INSA-Lyon, INSERM, from the Avenir Lyon Saint-Etienne program and Rhône-Alpes region, from the Academy of Finland (285223), the Sigrid

Conflict of interest:

The authors declare that they have no conflicts of interest with the contents of this article.