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Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

Open AccessPublished:April 24, 2015DOI:https://doi.org/10.1074/mcp.M114.045179
      Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.
      Lipid rafts are cholesterol-enriched membrane microdomains, thought to be present in all cells, that concentrate and organize cell-surface signal transduction events in several signaling cascades, including those of the Toll-like receptors (TLRs) (
      • Fessler M.B.
      • Parks J.S.
      Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.
      ). The selectivity of rafts for particular proteins, and, consequently, the signal strength of pathways initiating from ligated raft-resident receptors, are thought to derive in large part from the high cholesterol content of raft microdomains (
      • Foster L.J.
      • De Hoog C.L.
      • Mann M.
      Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.
      ,
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ,
      • Fessler M.B.
      • Arndt P.G.
      • Frasch S.C.
      • Lieber J.G.
      • Johnson C.A.
      • Murphy R.C.
      • Nick J.A.
      • Bratton D.L.
      • Malcolm K.C.
      • Worthen G.S.
      Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
      ). In vitro, altering raft cholesterol of living cells downward or upward with chemical tools (e.g. cyclodextrins) leads to parallel changes in raft protein abundance (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ,
      • Fessler M.B.
      • Arndt P.G.
      • Frasch S.C.
      • Lieber J.G.
      • Johnson C.A.
      • Murphy R.C.
      • Nick J.A.
      • Bratton D.L.
      • Malcolm K.C.
      • Worthen G.S.
      Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
      ). The relevance of cholesterol-driven alterations in the raft proteome to disease is suggested by reports that hypercholesterolemia cholesterol-loads macrophage rafts and amplifies their responsiveness to lipopolysaccharide (LPS) (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ,
      • Fessler M.B.
      • Arndt P.G.
      • Frasch S.C.
      • Lieber J.G.
      • Johnson C.A.
      • Murphy R.C.
      • Nick J.A.
      • Bratton D.L.
      • Malcolm K.C.
      • Worthen G.S.
      Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
      ). Proteomic strategies have recently been applied to raft isolates from a variety of cell types, aiming to better understand the identity of proteins tonically present in rafts, as well as proteins dynamically recruited to rafts upon cell stimulation (
      • Foster L.J.
      • De Hoog C.L.
      • Mann M.
      Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.
      ,
      • Bae T.J.
      • Kim M.S.
      • Kim J.W.
      • Kim B.W.
      • Choo H.J.
      • Lee J.W.
      • Kim K.B.
      • Lee C.S.
      • Kim J.H.
      • Chang S.Y.
      • Kang C.Y.
      • Lee S.W.
      • Ko Y.G.
      Lipid raft proteome reveals ATP synthase complex in the cell surface.
      ,
      • Bini L.
      • Pacini S.
      • Liberatori S.
      • Valensin S.
      • Pellegrini M.
      • Raggiaschi R.
      • Pallini V.
      • Baldari C.T.
      Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering.
      ,
      • Blonder J.
      • Hale M.L.
      • Lucas D.A.
      • Schaefer C.F.
      • Yu L.R.
      • Conrads T.P.
      • Issaq H.J.
      • Stiles B.G.
      • Veenstra T.D.
      Proteomic analysis of detergent-resistant membrane rafts.
      ,
      • Li N.
      • Mak A.
      • Richards D.P.
      • Naber C.
      • Keller B.O.
      • Li L.
      • Shaw A.R.
      Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation.
      ). To date, however, most reports have used cell lines of uncertain physiological relevance. In addition, although raft cholesterol levels are regulated in vivo by intracellular cholesterol trafficking (
      • Fessler M.B.
      • Parks J.S.
      Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.
      ), no reports to date have sought to define how the raft proteome is physiologically regulated by cholesterol trafficking proteins.
      ATP binding cassette (ABC) A1, a member of the ABC transporter superfamily, plays a key role in regulating levels of cholesterol in macrophages and other cells via promoting efflux of cellular cholesterol to extracellular acceptors, in particular lipid-free apolipoprotein (apo) A-I (
      • Smoak K.A.
      • Aloor J.J.
      • Madenspacher J.
      • Merrick B.A.
      • Collins J.B.
      • Zhu X.
      • Cavigiolio G.
      • Oda M.N.
      • Parks J.S.
      • Fessler M.B.
      Myeloid differentiation primary response protein 88 couples reverse cholesterol transport to inflammation.
      ). The importance of ABCA1

      The The abbreviations used are:

      ABCA1
      ATP binding cassette transporter A1
      CtB
      cholera toxin B subunit
      DRM
      detergent-resistant membrane
      LPS
      lipopolysaccharide
      MyD88
      myeloid differentiation primary response 88
      SLP-2
      stomatin-like protein 2
      SPFH
      stomatin-prohibitin-flotillin-HflK/C
      TLR
      Toll-like receptor.
      to human health is clearly illustrated by Tangier disease, a rare ABCA1 mutation syndrome typified by severe HDL deficiency, widespread macrophage foam cells, and premature atherosclerosis (
      • Singaraja R.R.
      • Brunham L.R.
      • Visscher H.
      • Kastelein J.J.
      • Hayden M.R.
      Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene.
      ). In addition, the large number of common ABCA1 polymorphisms that have been associated with human cardiovascular disease (
      • Singaraja R.R.
      • Brunham L.R.
      • Visscher H.
      • Kastelein J.J.
      • Hayden M.R.
      Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene.
      ) suggest a broad-spanning impact of ABCA1 on human health. It remains somewhat controversial whether ABCA1-effluxed cholesterol derives from raft or extra-raft membranes (
      • Jessup W.
      • Gelissen I.C.
      • Gaus K.
      • Kritharides L.
      Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages.
      ). Nonetheless, both human Tangier disease cells and ABCA1-null murine macrophages have been shown to have greatly expanded lipid rafts that contain increased cholesterol and increased TLR4 (
      • Koseki M.
      • Hirano K.
      • Masuda D.
      • Ikegami C.
      • Tanaka M.
      • Ota A.
      • Sandoval J.C.
      • Nakagawa-Toyama Y.
      • Sato S.B.
      • Kobayashi T.
      • Shimada Y.
      • Ohno-Iwashita Y.
      • Matsuura F.
      • Shimomura I.
      • Yamashita S.
      Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-alpha secretion in ABCA1-deficient macrophages.
      ,
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ). These changes are associated with enhanced responsiveness to LPS that can be reversed by cholesterol depletion (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ,
      • Francone O.L.
      • Royer L.
      • Boucher G.
      • Haghpassand M.
      • Freeman A.
      • Brees D.
      • Aiello R.J.
      Increased cholesterol deposition, expression of scavenger receptors, and response to chemotactic factors in ABCA1-deficient macrophages.
      ,
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ). Collectively, these findings indicate that ABCA1 may regulate the raft proteome and innate immune response through control of raft cholesterol. However, no proteomic analysis of rafts from ABCA1-deficient cells has been reported to date.
      Herein, we report a proteomic analysis of raft isolates from naive and LPS-stimulated Abca1+/+ and Abca1−/− primary murine macrophages. Unexpectedly, we found that ABCA1 deletion and LPS stimulation induced many similar changes in the raft proteome. Stomatin-like protein 2 (SLP-2), a lesser known member of the stomatin-prohibitin-flotillin-HflK/C (SPFH) family of membrane scaffolding proteins, was unique among SPFH proteins in being robustly up-regulated in rafts of unstimulated Abca1−/− cells compared with Abca1+/+ counterparts. We found that rafts of SLP-2 knockdown cells were abnormal, displaying increased binding of cholera toxin subunit B–a probe for the raft-specific ganglioside GM1–but markedly decreased protein, including flotillins-1 and -2, and CD14. Whereas SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux, it reduced macrophage responsiveness to LPS and multiple additional TLR ligands. Taken together, we report that ABCA1 regulates the macrophage raft proteome and identify SLP-2 as a novel ABCA1-dependent regulator of raft composition that controls the innate immune response.

      EXPERIMENTAL PROCEDURES

      Reagents

      Escherichia coli 0111:B4 LPS, penicillin, and streptomycin were from Sigma (St. Louis, MO). The Bio-Rad protein assay (Hercules, CA) and BCA protein assay (Pierce) were used. Pam3CSK4, heat-killed Listeria monocytogenes (HKLM), and imiquimod were from Invivogen (San Diego, CA).

      Macrophage Culture and Treatment

      Myeloid-specific (LysM-Cre) Abca1−/− mice and littermate controls were generated and bred as previously described (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ,
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ). All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review by the Animal Care and Use Committee of Wake Forest School of Medicine. Bone-marrow-derived macrophages were prepared and cultured in DMEM containing 20% (v/v) FBS and 30% (v/v) L cell-conditioned medium for 5 days as previously described (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ,
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ). RAW 264.7 macrophages (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% heat-inactivated endotoxin-free fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 2 mm l-glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin under a humidified 5% CO2 atmosphere at 37 °C. Macrophages were stimulated for 2–24 h with buffer (control), LPS (0.1–100 ng/ml), Pam3CSK4 (2 ng/ml), HKLM (1 × 107/ml), or imiquimod (2 μg/ml).

      Macrophage Transfections and Transductions

      Peritoneal macrophages were collected 4 days after injection of 1 ml of 10% brewer's thioglycolate into the peritoneal cavity of 9–15 week-old mice. Following overnight culture in antibiotic-free complete media, 50 nm of control or SLP-2 smart pool siRNA was transfected with DharmaFECT reagent and incubated for 72 h according to the manufacturer's protocol (Dharmacon, Lafayette, CO). siRNA-treated macrophages were then stimulated with 100 ng/ml LPS for 6 h before cell culture supernatant was collected for analysis of cytokine expression by ELISA. Stable macrophage lines were produced using a set of lentiviral shRNAs against murine SLP-2 from Open Biosystems/Thermo-Fisher (Huntsville, AL). Lentiviral packaging was achieved by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) to transiently transfect HEK293T/17 cells (ATCC) with shRNA in a pLKO.1 vector together with vesicular stomatitis virus G glycoprotein and packaging plasmids. Supernatant was collected 48 h posttransfection and concentrated by centrifugation (50,000 × g, 2 h). Pellets were resuspended in PBS and used for infection. Titers were determined by performing quantitative PCR to measure the number of lentiviral particles that integrated into the host genome. In addition, biological titration of viruses that coexpressed fluorescent moieties was determined by flow cytometry. RAW 264.7 macrophages were infected at multiplicity of infection of 100 and selected 48 h postinfection with 10 μg/ml puromycin (Calbiochem) for ∼9 days. The puromycin-selected stable cells (passage 10–16) were used for all further experiments after replating in puromycin-free media. The sequences of the scrambled shRNA and the two shRNAs that were found to effectively silence SLP-2 are shown in Table S2.

      Detergent-Resistant Membrane (DRM) preparation

      DRMs were prepared as previously reported (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ). Raft fractions were detected by immunoblotting for raft (flotillin-1) and non-raft (transferrin receptor) markers and were identified as flotillin-1-positive, transferrin receptor-negative. Fractions were then pooled, sedimented (20,000 × g, 1h, 4 °C centrifugation after fourfold dilution in MES-buffered saline [25 mm MES, pH 6.5, 150 mm NaCl]), and an equal protein mass from each condition resolved by SDS-PAGE.

      Protein Digestion

      In-gel digestion was carried out for the four lipid raft samples. Each SYPRO-Ruby stained gel lane was cut into 24 equal slices and digested in a 96-well tray using a Progest robotic digester (Genomic Solutions, Inc., Ann Arbor, MI). In brief, gel slices were minced and placed in one well of a 96-well plate. Common in-gel digestion protocol was used. Briefly, the gel bands were soaked with 50 μl of 25 mm ammonium bicarbonate for 15 min and dehydrated with 50 μl of acetonitrile. These steps were performed two times before digestion was started with trypsin. Digestion of protein was carried out using 250 ng of trypsin in each well, and the solution was allowed to incubate for 8 h at 37 °C. Proteins were extracted from the gel using 10% formic acid and acetonitrile as well. Supernatants were pooled, lyophilized, and resuspended in 40 μl of 0.1% formic acid. Samples were kept at −80 °C before LC/MS analysis was performed.

      Nano- LC-MS/MS and Bioinformatics Analysis

      Nano LC-MS/MS analysis was performed using an Agilent 1100 nanoLC system online with an Agilent XCT Ultra ion trap mass spectrometer equipped with the Chip Cube interface. Peptides were separated by reverse-phase LC utilizing a trapping column and nanoflow analytical column (Agilent Technologies, Santa Clara, CA) and analyzed both in full MS scan and CID MS/MS modes. Briefly, 20 μl of peptide digest was loaded into an Agilent C18 chip followed by a 15 min wash with 5% (v/v) acetonitrile, 0.1% formic acid (v/v). Peptides were eluted with a linear gradient of increasing concentration of acetonitrile (in 0.1% v/v formic acid) as follows: 0–45 min, 5–50% (v/v) acetonitrile; 45–50 min, acetonitrile increased to 95% (v/v), and 50–60 min acetonitrile maintained at 95%. Mass spectrometer was used in positive ion mode, standard enhanced mode, and included setting of a mass range from 200–2,200 m/z. MS/MS data were acquired using a data-dependent acquisition format, and six more abundant ions were targeted for MS/MS from each MS scan. The automated switching for MS/MS required a threshold of 5,000 counts. Dynamic ion exclusion of two spectra or 1.0 min ion trap resident time was used during MS/MS data acquisition. Tandem mass spectra were extracted by Spectrum Mill version (ReV A.03.03.078). Charge state deconvolution and deisotoping were not performed. All MS/MS samples were also analyzed using Spectrum Mill (Agilent). Spectrum Mill was set up to search the NCBInr.rodent database (selected for RODENT, unknown version, 14,227,560 entries) using the digestion enzyme trypsin. Spectrum Mill was searched with a fragment ion mass tolerance of 0.70 Da and a parent ion tolerance of 2.5 Da. Oxidation of methionine was specified in Spectrum Mill as a variable modification. Scaffold (version Scaffold 3 00 08, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability as specified by the Peptide Prophet algorithm (Keller, A et al. Anal. Chem. 2002;74(20):5383–92). Protein identifications were accepted if they could be established at greater than 80.0% probability and contained at least two identified peptides per protein. Annotated spectra and associated information are shown in Fig. S3 for two proteins that were identified on the basis of high-quality data from single peptides; for both proteins, additional distinct peptides were identified in independent sample runs. Protein probabilities were assigned by the Protein Prophet algorithm (
      • Nesvizhskii A.I.
      • Keller A.
      • Kolker E.
      • Aebersold R.
      A statistical model for identifying proteins by tandem mass spectrometry.
      ). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. For high and low confidence identification, these criteria were relaxed or tightened (∼90% protein and peptide probability) and specified in the respective data analysis in the manuscript and the respective figures. For protein identifications, Table S1 shows spectral counts, Table S3 shows peptide counts, and Table S4 shows percentage amino acid coverage.

      NF-κB Activity Assays

      Activation of the p65 component of NF-κB in the nuclear fraction from cells (NE-PER kit; Pierce) was quantified by use of a sandwich ELISA (p65 TransAM™ kit, Active Motif, Carlsbad, CA) upon equalized nuclear protein input (BCA protein assay).

      Cholera Toxin B, Filipin, and Rhodamine-Phalloidin Staining and Cell Imaging

      GM1 was stained using Alexa Fluor® 488-Cholera Toxin B (CtB)(Invitrogen/Molecular Probes), a raft probe with high affinity for the raft ganglioside GM1. Cells in growth medium were incubated in AF488-CtB (1 μg/ml, 10 min, room temperature), washed, and cross-linked with anti-CtB antibody provided by the manufacturer (200-fold dilution, 15 min, room temperature). Cells were fixed in chilled 1x PBS containing 4% paraformaldehyde (15 min, room temperature) and then washed several times with 1x PBS. Cholesterol was imaged by staining with filipin III (Sigma). Briefly, cells were fixed (4% paraformaldehyde, 20 min, room temperature), quenched (50 mm NH4Cl, 10 min, room temperature), washed, and then stained with 100 μg/ml Filipin III (20 min, room temperature), either with or without prior detergent permeabilization, with similar results. Actin was imaged by staining with rhodamine-phalloidin. Cells were fixed as above, washed, permeabilized with PBS containing 0.1% Triton X-100 (15 min, room temperature), washed, and then stained with 5 U/ml rhodamine-phalloidin (Molecular Probes, Eugene, OR)(20 min, room temperature). Cells were washed and mounted with ProLong Antifade Kit (Molecular Probes). Imaging was performed with a Zeiss 710 LSM microscope with a 40x objective/1.30 oil and ZEN software. For AF488-CtB, optical filters provided excitation/emission at 501 nm/566 nm; for filipin, a UV 375 nm filter set with a 385 nm long pass filter and a 364 nm laser was used; for rhodamine-phalloidin, a rhodamine filter set with a 458/561 dichroic beam splitter and a 561 nm laser was used. For analysis of fluorescence intensity, at least 10 region of interest (which included a minimum of 6–10 cells) from 2–3 individual experiments were analyzed using MetaMorph software.

      Cytokine Analysis

      Cytokines were quantified by ELISA (Biolegend, San Diego, CA).

      Cholesterol Efflux Assay

      Cholesterol efflux to BSA or apoA-I was quantified as previously reported (
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ).

      Western Blotting

      An equal protein mass (BCA assay) of whole cell lysate or DRMs was resolved on 4–20% Tris-HCl gels (BioRad), transferred to nitrocellulose (Amersham Biosciences), and probed with primary antibodies. Rabbit anti-MyD88 (1:100) was from EMD/Millipore, and mouse anti-flotillin-1, mouse anti-flotillin-2, and rabbit anti-CD14 (all 1:1,000) were from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were washed in Tween Tris buffered saline (TTBS), and exposed to species-specific HRP-conjugated secondary antibody (GE Healthcare; 1:5,000; 60 min) in 5% milk/TTBS. After further TTBS washes, signal was detected with HyGlo (Quick Spray) chemiluminescent HRP antibody detection reagent, followed by film exposure (HyBlot CL, Denville Scientific).

      Statistical Analysis

      Analysis was performed using GraphPad Prism statistical software (San Diego, CA). Data are represented as mean ± S.E. Two-tailed student's t test was applied for comparisons of two groups, and analysis of variance (ANOVA) for comparisons of >2 groups. For all tests, p < .05 was considered significant.

      RESULTS

      Proteins Detected in Macrophage Raft Isolates

      To define the effect of ABCA1 on the raft proteome of resting and LPS-exposed macrophages, we cultured bone-marrow-derived macrophages from myeloid-specific Abca1−/− mice (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ) and littermate Abca1+/+ controls. In four independent experiments, Abca1+/+ and Abca1−/− macrophages were left unstimulated or exposed to E. coli 0111:B4 LPS (100 ng/ml, 30 min). Following this, detergent-resistant membranes (DRMs), a widely accepted surrogate for rafts (
      • Fessler M.B.
      • Parks J.S.
      Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.
      ,
      • Fessler M.B.
      • Arndt P.G.
      • Frasch S.C.
      • Lieber J.G.
      • Johnson C.A.
      • Murphy R.C.
      • Nick J.A.
      • Bratton D.L.
      • Malcolm K.C.
      • Worthen G.S.
      Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
      ), were isolated using standard sucrose density gradient centrifugation methods (
      • Fessler M.B.
      • Arndt P.G.
      • Frasch S.C.
      • Lieber J.G.
      • Johnson C.A.
      • Murphy R.C.
      • Nick J.A.
      • Bratton D.L.
      • Malcolm K.C.
      • Worthen G.S.
      Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
      ). Gradient fractions containing DRMs were identified by immunoblotting to detect presence of the raft-specific protein flotillin-1, and absence of the non-raft marker transferrin receptor (Fig. S1). DRM fractions were pooled and sedimented by ultracentrifugation, resolved by 10% SDS-PAGE, and imaged with SYPRO Ruby protein stain (Fig. S2). In-gel tryptic digest was then performed on the gel lanes, followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with spectral counting.
      As shown in Table S1, 383 unique proteins were collectively identified (0.1% protein false discovery rate, 0.7% peptide false discovery rate, ≥2 peptides) in raft isolates from at least one of the conditions across four experiments. Many raft proteins previously reported by our group and others (
      • Foster L.J.
      • De Hoog C.L.
      • Mann M.
      Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.
      ,
      • Bae T.J.
      • Kim M.S.
      • Kim J.W.
      • Kim B.W.
      • Choo H.J.
      • Lee J.W.
      • Kim K.B.
      • Lee C.S.
      • Kim J.H.
      • Chang S.Y.
      • Kang C.Y.
      • Lee S.W.
      • Ko Y.G.
      Lipid raft proteome reveals ATP synthase complex in the cell surface.
      ,
      • Bini L.
      • Pacini S.
      • Liberatori S.
      • Valensin S.
      • Pellegrini M.
      • Raggiaschi R.
      • Pallini V.
      • Baldari C.T.
      Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering.
      ,
      • Li N.
      • Mak A.
      • Richards D.P.
      • Naber C.
      • Keller B.O.
      • Li L.
      • Shaw A.R.
      Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation.
      ,
      • Dhungana S.
      • Merrick B.A.
      • Tomer K.B.
      • Fessler M.B.
      Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide.
      ) were detected, including flotillins-1 and -2, stomatin, CD18, CD44, CD36, aminopeptidase N, syntaxins, annexins, SNAREs, various heterotrimeric G-protein subunits, and Rab, Ras, and Rap family members. Of interest, we detected three proteins that regulate intracellular trafficking of TLRs but that have not previously been reported in rafts to our knowledge. Niemann-Pick C1, a protein that regulates ABCA1-dependent cholesterol efflux (
      • Choi H.Y.
      • Karten B.
      • Chan T.
      • Vance J.E.
      • Greer W.L.
      • Heidenreich R.A.
      • Garver W.S.
      • Francis G.A.
      Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease.
      ) and TLR4 trafficking to the plasma membrane (
      • Suzuki M.
      • Sugimoto Y.
      • Ohsaki Y.
      • Ueno M.
      • Kato S.
      • Kitamura Y.
      • Hosokawa H.
      • Davies J.P.
      • Ioannou Y.A.
      • Vanier M.T.
      • Ohno K.
      • Ninomiya H.
      Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann–Pick disease type C (NPC) fibroblasts: a potential basis for glial cell activation in the NPC brain.
      ), was noted in all ABCA1 genotype and LPS conditions, without evident alteration in abundance as judged by spectral count. Rab10, a protein that promotes TLR4 signaling by regulating TLR4 recycling to the plasma membrane (
      • Wang D.
      • Lou J.
      • Ouyang C.
      • Chen W.
      • Liu Y.
      • Liu X.
      • Cao X.
      • Wang J.
      • Lu L.
      Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of.
      ), was detected in rafts from all experimental conditions but without notable change across them. Last, Unc93b1, a protein that regulates intracellular trafficking of several TLRs other than TLR4 (
      • Akashi-Takamura S.
      • Miyake K.
      TLR accessory molecules.
      ), was found in rafts from all four experimental conditions, with a notable increase in unstimulated Abca1−/− rafts (detected in four of four experiments) as compared with unstimulated Abca1+/+ rafts (detected in one of four experiments).
      Several previously reported components of the LPS receptor complex were detected, including CD14, HSP70, HSP90, and moesin (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ,
      • Tohme Z.N.
      • Amar S.
      • Van Dyke T.E.
      Moesin functions as a lipopolysaccharide receptor on human monocytes.
      ), albeit without any consistent change in spectral abundance across conditions. RP105, a TLR4 homologue that inhibits LPS signaling by directly inhibiting TLR4 binding of LPS (
      • Divanovic S.
      • Trompette A.
      • Atabani S.F.
      • Madan R.
      • Golenbock D.T.
      • Visintin A.
      • Finberg R.W.
      • Tarakhovsky A.
      • Vogel S.N.
      • Belkaid Y.
      • Kurt-Jones E.A.
      • Karp C.L.
      Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor homolog RP105.
      ), was detected in both unstimulated Abca1+/+ and unstimulated Abca1−/− rafts in two of four experiments but was not detected in LPS-stimulated Abca1+/+ rafts in any of the four experiments, perhaps suggesting that LPS promotes its exclusion from macrophage rafts. By contrast, although TLR7 and TLR13 were detected in individual experiments, we did not detect spectra for the LPS receptor itself, TLR4. As we have previously confirmed TLR4 in Abca1+/+ and Abca1−/− bone-marrow-derived macrophage rafts by immunoblotting (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ) but have not detected it in RAW 264.7 rafts by LC-MS/MS (
      • Dhungana S.
      • Merrick B.A.
      • Tomer K.B.
      • Fessler M.B.
      Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide.
      ), we speculate that this nondetection by MS reflects low protein abundance and/or poor peptide ionizability.

      Impact of ABCA1 Deletion and LPS Exposure on the Macrophage Raft Proteome

      Depiction of the abundance of individual raft proteins across experimental conditions and replicate experiments in the format of a heat map (Fig. 1A) demonstrated a significant degree of biological variation, a challenge that has been previously noted in proteomic analysis of primary cells (
      • Fessler M.B.
      • Malcolm K.C.
      • Duncan M.W.
      • Worthen G.S.
      A genomic and proteomic analysis of activation of the human neutrophil by lipopolysaccharide and its mediation by p38 mitogen-activated protein kinase.
      ). Initial overlay of proteins detected in the various ABCA1 and LPS conditions in the form of Venn diagrams nevertheless revealed a significant number of proteins both common and unique to the various conditions, indicating evident effects of both ABCA1 deletion and LPS exposure on the macrophage raft proteome (Fig. 1B). An initial spectral count-based evaluation of quantitative changes induced in raft proteins by ABCA1 deletion and LPS (i.e. ratio of spectral count in unstimulated Abca1−/− or LPS-stimulated Abca1+/+ rafts over that in unstimulated Abca1+/+ rafts) yielded modest (i.e. less than twofold) changes in a short list of proteins as well as considerable interexperiment variability (data not shown). Given this, we focused our efforts on using qualitative protein changes (i.e. proteins that were not detected in unstimulated Abca1+/+ rafts but that were detected in unstimulated Abca1−/− and/or in LPS-stimulated Abca1+/+ rafts) as an initial screen for identifying raft proteins that were more robustly impacted by ABCA1 and LPS.
      Figure thumbnail gr1
      Fig. 1LPS- and ABCA1-dependent changes in the macrophage raft proteome. (A) Abca1+/+ and Abca1−/− murine bone-marrow-derived macrophages were treated with buffer (control) or LPS in four independent experiments, after which detergent-resistant membranes (DRMs) were isolated and profiled for their proteome by LC-MS/MS. The heat map depicts expression in the form of normalized spectral counts (SC) (quantitative value, calculated by Scaffold [Proteome Software, Portland, OR]) for unique proteins (rows) identified in all conditions (0.1% protein false discovery rate, 0.7% peptide false discovery rate, minimum two peptides per protein for identification). (B) Venn diagrams indicating the number of common and unique proteins identified in DRM preparations from the indicated experimental conditions.
      We examined proteins that were not detected in rafts from unstimulated Abca1+/+ cells but that were detected in rafts from unstimulated Abca1−/− cells (13 proteins; Fig. 2A) or in rafts from LPS-stimulated Abca1+/+ cells (11 proteins; Fig. 2B). Of interest, comparison of the two protein lists shows them to be nearly identical. Nine of the 11 proteins not detected in unstimulated Abca1+/+ rafts but detected in LPS-stimulated Abca1+/+ rafts were also detected in rafts of unstimulated Abca1−/− cells. Nine of the 13 proteins not detected in unstimulated Abca1+/+ rafts but detected in unstimulated Abca1−/− rafts were also detected in LPS-stimulated Abca1+/+ rafts. Taken together, these results indicate a striking degree of convergence in the changes induced in the raft proteome by LPS and ABCA1 deletion. While the full significance of this convergence of protein expression changes is uncertain, given that LPS down-regulates ABCA1 in macrophages (
      • Majdalawieh A.
      • Ro H.S.
      LPS-induced suppression of macrophage cholesterol efflux is mediated by adipocyte enhancer-binding protein 1.
      ,
      • Baranova I.
      • Vishnyakova T.
      • Bocharov A.
      • Chen Z.
      • Remaley A.T.
      • Stonik J.
      • Eggerman T.L.
      • Patterson A.P.
      Lipopolysaccharide down regulates both scavenger receptor B1 and ATP binding cassette transporter A1 in RAW cells.
      ), it may interestingly suggest that a significant proportion of alterations induced in the raft proteome by LPS occur secondary to its repression of ABCA1.
      Figure thumbnail gr2
      Fig. 2LPS treatment and ABCA1 deletion induce specific changes in raft-localization of proteins in the macrophage. (A) The datasets were queried to identify proteins absent in untreated Abca1+/+ rafts but detected in unstimulated Abca1−/− rafts, including both high-confidence (≥2 peptides/dataset; 95% protein probability) and lower confidence (≥1 peptide; 80% confidence) proteins. Among these proteins, only those that had ≥8 spectral counts (SCs) across all datasets were further considered. The heat map displays SC values for these proteins across all conditions in four independent experiments. Green = 0 SCs (nondetected); black = 1 SC; red = 2 or more SCs. (B) The datasets were queried to identify proteins absent in untreated Abca1+/+ rafts but detected in LPS-stimulated Abca1+/+ rafts, using the same criteria as applied in panel A. Color scheme is as described for A. (C) Detergent-resistant membranes (DRMs) were isolated from cell/treatments as indicated, and equal protein loads immunoblotted for the indicated protein targets. In parallel, whole cell lysates from Abca1+/+ and Abca1−/− macrophages were immunoblotted for select targets displaying abundance changes in the DRM preparations. Representative of two to three independent experiments.
      In order to validate proteins not detected by LC-MS/MS in unstimulated Abca1+/+ rafts but detected in unstimulated Abca1−/− rafts and/or LPS-stimulated Abca1+/+ rafts, we next immunoblotted raft isolates. We confirmed up-regulation of Cdc42 by both LPS exposure and ABCA1 deletion, up-regulation of l-plastin by ABCA1 deletion, and up-regulation of SLP-2 by ABCA1 deletion, as well as, more modestly, by LPS stimulation (Fig. 2C). Generally consistent with the spectral count data (Table S1), immunoblotting revealed prohibitin-2 to be modestly reduced with respect to its levels in unstimulated Abca1+/+ rafts, in both LPS-stimulated Abca1+/+ rafts and unstimulated Abca1−/− rafts, whereas flotillin-1, another member of the SPFH family of membrane scaffolding proteins, was unchanged. Similarly, CD14 was unchanged across ABCA1 and LPS conditions. Notably, immunoblotting of whole cell lysates for all of these targets revealed unchanged whole-cell expression, indicating that the differences reflect distributive changes to lipid rafts. Although the qualitative raft protein changes as identified by LC-MS/MS and quantitative raft protein changes as confirmed by immunoblotting are intriguing, these results must be considered as at most interesting leads that will require future quantitative and functional validation.

      SLP-2 Silencing Reduces Macrophage Response to TLR Ligands

      SPFH proteins are characterized by a conserved SPFH (also called “prohibitin homology”) domain, and, while poorly understood, are thought to serve structural/organizational roles in rafts through targeting proteins to rafts, linking rafts to the actin cytoskeleton, and/or promoting nucleation of raft lipid species (
      • Browman D.T.
      • Hoegg M.B.
      • Robbins S.M.
      The SPFH domain-containing proteins: More than lipid raft markers.
      ). Among family members, SLP-2 is unique in not possessing an N-terminal hydrophobic domain, instead being more loosely associated with rafts via myristoylation and palmitoylation (
      • Wang Y.
      • Morrow J.S.
      Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues.
      ,
      • Owczarek C.M.
      • Treutlein H.R.
      • Portbury K.J.
      • Gulluyan L.M.
      • Kola I.
      • Hertzog P.J.
      A novel member of the STOMATIN/EPB72/mec-2 family, stomatin-like 2 (STOML2), is ubiquitously expressed and localizes to HSA chromosome 9p13.1.
      ). Our attention was drawn to SLP-2, as, unlike SPFH members flotillins-1 and -2, prohibitins-1 and -2, and erlins-1 and -2, all of which were decreased or unchanged in Abca1−/− rafts (Table S1, Fig. 2), we found SLP-2 to be dramatically increased (Fig. 2C). Moreover, recent work indicates that SLP-2 plays a role in membrane microdomain organization in the mitochondrion and also in the plasma membrane, where it associates with the T cell receptor and promotes T cell receptor signaling (
      • Christie D.A.
      • Mitsopoulos P.
      • Blagih J.
      • Dunn S.D.
      • St-Pierre J.
      • Jones R.G.
      • Hatch G.M.
      • Madrenas J.
      Stomatin-like protein 2 deficiency in T cells is associated with altered mitochondrial respiration and defective CD4+ T cell responses.
      ,
      • Christie D.A.
      • Kirchhof M.G.
      • Vardhana S.
      • Dustin M.L.
      • Madrenas J.
      Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation.
      ,
      • Kirchhof M.G.
      • Chau L.A.
      • Lemke C.D.
      • Vardhana S.
      • Darlington P.J.
      • Márquez M.E.
      • Taylor R.
      • Rizkalla K.
      • Blanca I.
      • Dustin M.L.
      • Madrenas J.
      Modulation of T cell activation by stomatin-like protein 2.
      ,
      • Christie D.A.
      • Lemke C.D.
      • Elias I.M.
      • Chau L.A.
      • Kirchhof M.G.
      • Li B.
      • Ball E.H.
      • Dunn S.D.
      • Hatch G.M.
      • Madrenas J.
      Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function.
      ). Given this, we hypothesized that SLP-2 might serve an important role in macrophage raft function, perhaps either promoting the enhanced TLR responses of Abca1−/− macrophages, or regulating ABCA1-dependent cholesterol efflux.
      In order to define SLP-2 function in macrophages, we used siRNA to silence it in Abca1+/+ and Abca1−/− murine macrophages (Fig. 3A). As previously reported by us and others (
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ,
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ), Abca1−/− macrophages (transfected with control, nontargeting siRNA) were hyperresponsive to LPS, producing elevated IL-6 (Fig. 3B). SLP-2 silencing equivalently reduced LPS-induced IL-6 in both Abca1+/+ and Abca1−/− macrophages, indicating that it is required for intact TLR4 signaling but suggesting that it does not account for the enhanced TLR4 response of the latter genotype. We next tested the effect of SLP-2 silencing on apoA-I-induced cholesterol efflux, the hallmark function of ABCA1 (
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ), in wild-type murine macrophages. As shown in Fig. 3C, apoA-I-induced cholesterol efflux was unchanged by SLP-2 silencing, suggesting that SLP-2 does not appreciably impact ABCA1 efflux function. SLP-2 silencing also did not impact diffusional cholesterol efflux to bovine serum albumin.
      Figure thumbnail gr3
      Fig. 3SLP-2 regulates the LPS response of macrophages. (A) Abca1+/+ and Abca1−/− murine bone marrow-derived macrophages were transfected with control non-targeting siRNA or SLP-2 siRNA and then immunoblotted as shown. (B) Cells under the same conditions as in panel A were stimulated with LPS, and then cell media IL-6 was quantified by ELISA. Representative of two independent experiments performed in triplicate. ****, p < .0001; ***, p < .001. (C) Abca1+/+ macrophages were transfected with control nontargeting siRNA or SLP-2 siRNA. Cholesterol efflux to bovine serum albumin or apoA-I was then quantified. Representative of three independent experiments. BSA = bovine serum albumin; apoA-I = apolipoprotein A-I.
      Aiming to further validate and define the role of SLP-2 in TLR4 signaling, we generated two stable SLP-2 knockdown cell lines via lentiviral shRNA transduction of RAW 264.7 macrophages with two alternate constructs (Fig. 4A). We then exposed these SLP-2-silenced lines, along with a scrambled (nontargeting) shRNA control line, to LPS. As shown in Fig. 4B, the two SLP-2 knockdown macrophage lines produced lower TNFα than the control line at two concentrations of LPS, in general agreement with our siRNA results in primary murine macrophages. Lipid rafts are thought to regulate signaling by virtually all TLRs. Given this, we next evaluated the effect of SLP-2 silencing upon macrophage responses to a broader panel of ligands to both plasma membrane-localized (TLR1, -2) and endosomal (TLR7) TLRs. Both, Pam3CSK4 (TLR2/TLR1) and heat-killed Listeria monocytogenes (TLR2) induced less TNFα in SLP-2 knockdown macrophages than in controls (Fig. 4C). In addition, imiquimod, a TLR7 agonist, induced reduced TNFα in SLP-2 knockdown macrophages. Similar to our findings in SLP-2-silenced primary murine macrophages (Fig. 3), SLP-2 knockdown also reduced IL-6 output from macrophages stimulated with LPS, Pam3CSK4, or imiquimod (Fig. 4D). LPS induces TNFα and other proinflammatory cytokines via rapid activation of the transcription factor, NF-κB. Consistent with its attenuation of LPS-induced cytokines, SLP-2 silencing significantly reduced LPS-induced activation of NF-κB (Fig. 4E). Taken together, these findings indicate that SLP-2 is required for intact signaling from a broad array of both plasmalemmal and endosomal TLRs.
      Figure thumbnail gr4
      Fig. 4SLP-2 regulates the innate immune response triggered by multiple TLRs. (A) Stable SLP-2 lentiviral shRNA knockdown RAW 264.7 macrophage lines were generated and then probed for SLP-2 and β-actin (loading control) as shown. Significant SLP-2 silencing was achieved with lentiviral constructs #1 and #2, whereas #3 was not effective. (B) Stable RAW 264.7 macrophage lines transduced with a scrambled (scr; i.e. nontargeting) vector or with SLP-2 #1 or #2 constructs as in panel A were exposed to buffer or to two concentrations of LPS for 2 h, after which TNFα was quantified by ELISA in the cell media and normalized to cell protein. Representative of three independent experiments. *, p < .05; **, p < .01. (C) Scrambled or SLP-2 #2 knockdown RAW 264.7 macrophages as in panel B were exposed for 2 h to buffer, Pam3CSK4 (Pam), heat-killed L. monocytogenes (HKLM), or imiquimod (Imiq), after which cell media TNFα was quantified. Representative of three independent experiments. **, p < .01; ***, p < .001. (D) Macrophages as in panel c were exposed for 24 h to buffer, LPS, Pam3CSK4, or imiquimod, after which cell media IL-6 was quantified. Representative of three independent experiments. †, p < .0001. (E) Macrophages as in panels C and D were exposed to buffer or LPS for 30 min, after which DNA binding of p65 NF-κB was quantified in nuclear isolates. Fold-change p65 activation in LPS as compared with buffer is shown. Representative of six biological replicates. **, p < .01. All statistical comparisons are to the corresponding “scr” exposure control.

      SLP-2 Regulates Raft Composition in Macrophages

      Given that rafts regulate TLR signaling, (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ,
      • Fessler M.B.
      • Arndt P.G.
      • Frasch S.C.
      • Lieber J.G.
      • Johnson C.A.
      • Murphy R.C.
      • Nick J.A.
      • Bratton D.L.
      • Malcolm K.C.
      • Worthen G.S.
      Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
      ,
      • Zhu X.
      • Owen J.S.
      • Wilson M.D.
      • Li H.
      • Griffiths G.L.
      • Thomas M.J.
      • Hiltbold E.M.
      • Fessler M.B.
      • Parks J.S.
      Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
      ) and SLP-2 is reported to play a role in membrane microdomain organization in T cells (
      • Christie D.A.
      • Mitsopoulos P.
      • Blagih J.
      • Dunn S.D.
      • St-Pierre J.
      • Jones R.G.
      • Hatch G.M.
      • Madrenas J.
      Stomatin-like protein 2 deficiency in T cells is associated with altered mitochondrial respiration and defective CD4+ T cell responses.
      ,
      • Christie D.A.
      • Lemke C.D.
      • Elias I.M.
      • Chau L.A.
      • Kirchhof M.G.
      • Li B.
      • Ball E.H.
      • Dunn S.D.
      • Hatch G.M.
      • Madrenas J.
      Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function.
      ), we next sought to determine whether SLP-2 is required for normal raft composition in macrophages. GM1 is a raft-specific ganglioside that is commonly used (via imaging with the high-affinity ligand, cholera toxin B subunit [CtB]) as a marker of rafts (
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ) but is itself reported to regulate raft function. Cell surface GM1, as assessed by CtB, is increased in Abca1−/− macrophages (
      • Zhu X.
      • Lee J.Y.
      • Timmins J.M.
      • Brown J.M.
      • Boudyguina E.
      • Mulya A.
      • Gebre A.K.
      • Willingham M.C.
      • Hiltbold E.M.
      • Mishra N.
      • Maeda N.
      • Parks J.S.
      Increased cellular free cholesterol in macrophage-specific ABCA1 knock-out mice enhances pro-inflammatory response of macrophages.
      ). GM1 content in rafts is also up-regulated during cell stimulation (
      • Slaughter N.
      • Laux I.
      • Tu X.
      • Whitelegge J.
      • Zhu X.
      • Effros R.
      • Bickel P.
      • Nel A.
      The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation.
      ) and in disorders characterized by abnormal raft signaling (e.g. lupus (
      • Jury E.C.
      • Kabouridis P.S.
      • Flores-Borja F.
      • Mageed R.A.
      • Isenberg D.A.
      Altered lipid raft-associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus.
      )). Of interest, we found that, compared with scrambled controls, SLP-2 knockdown macrophages bound dramatically increased levels of CtB (Figs. 5A and 5B), consistent with increased cell surface GM1. By contrast, staining with filipin, a fluorescent probe that binds cholesterol in lipid rafts and also in internal membranes, revealed no significant distributive or quantitative differences between scrambled control and SLP-2 knockdown cells in cholesterol, either in the plasma membrane, or internally (Fig. 5C). This is generally consistent with our finding that SLP2 silencing did not modify apoA-I-induced cholesterol efflux (Fig. 3C), and together suggests that SLP-2 does not modify intracellular cholesterol trafficking. As a screen for possible effects of SLP-2 silencing on the actin cytoskeleton, we also stained with the F-actin probe rhodamine-phalloidin. Phalloidin staining revealed no distributive or quantitative differences between scrambled control and SLP-2 knockdown cells either in the unstimulated state (Fig. 5D) or following stimulation with LPS or the chemotactic formylated tripeptide fMLP (data not shown), arguing against significant effects of SLP-2 on cytoskeletal remodeling.
      Figure thumbnail gr5
      Fig. 5SLP-2 silencing dysregulates raft composition. (A) RAW 264.7 macrophage lines stably transduced with either scrambled (scr; i.e. non-targeting) or SLP-2 lentiviral shRNA (SLP-2 #2 shRNA was used throughout this figure) were left untreated or treated with LPS (10 ng/ml), and then stained with Alexa488-cholera toxin B (CtB), and imaged using a Zeiss 710 LSM microscope (40x objective). A representative experiment of three is depicted. (B) Alexa488-CtB signal was quantified across three independent experiments as described in panel A. †, p < .0001. (C) Cells were stained with the cholesterol probe filipin and imaged using a 63X oil objective with a further 1.5X zoom. Overall intensity was quantified using Metamorph software, as shown at right (p = NS). (D) Cells were stained with the actin probe rhodamine-phalloidin and imaged using a 63X oil objective. Overall intensity was quantified using Metamorph software, as shown at right (p = NS). (E) Rafts were isolated from unstimulated SLP-2-silenced and scrambled shRNA control macrophages, and raft protein mass quantified as shown. Normalized data across three independent experiments is shown. **, p < .01. (F) SLP-2 lentiviral shRNA macrophages and scrambled shRNA controls were left unstimulated, or exposed to LPS. Raft fractions were then isolated, pooled, and sedimented for each of the four conditions, and an equal protein mass (35 μg) immunoblotted for the targets shown. Data are representative of 2–3 independent experiments.
      Pursuing the effect of SLP-2 silencing upon raft protein composition, we next exposed SLP-2 knockdown and scrambled macrophage lines to LPS (or left them unexposed) and then isolated, pooled, and sedimented rafts. Notably, total protein mass in rafts from unstimulated SLP-2-silenced macrophages was significantly lower than that in unstimulated control cells (Fig. 5E), suggesting a global effect of SLP-2 on raft protein integrity. An equal protein mass from all four experimental conditions was next processed for immunoblotting. Although rafts from SLP-2 knockdown macrophages excluded the non-raft marker transferrin receptor as expected (not depicted), they were dramatically depleted of the hallmark raft proteins flotillin-1 and flotillin-2 and also had a significant reduction in CD14, a raft-localized protein that serves as a co-receptor for signaling by TLR4 and other TLRs (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ) (Fig. 5F). Myeloid differentiation primary response 88 (MyD88), an adaptor protein required for signaling from all TLRs other than TLR3, is rapidly recruited to rafts in response to LPS, where it associates with TLR4 (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ). An LPS-induced increase in MyD88 in rafts was observed in control macrophages but not in SLP-2-silenced macrophages (Fig. 5F). Taken together, these findings indicate that SLP-2 is required for lipid/protein integrity of macrophage rafts and for the raft-compartmental signaling that underlies the innate immune response.

      DISCUSSION

      The protein repertoire of rafts has been thought to be regulated by raft cholesterol (
      • Fessler M.B.
      • Parks J.S.
      Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.
      ,
      • Foster L.J.
      • De Hoog C.L.
      • Mann M.
      Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.
      ). However, to date, there has been no proteome-scale investigation of whether/how cells physiologically regulate the composition of their rafts. In the present report, we identify for the first time a suite of proteins whose abundance in macrophage rafts is modified by deletion of the cholesterol efflux transporter ABCA1. We find, remarkably, that LPS exposure induces many of the same changes in the raft proteome that are induced by ABCA1 deletion. This suggests the interesting possibility that LPS, a known repressor of ABCA1 (
      • Baranova I.
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      Lipopolysaccharide down regulates both scavenger receptor B1 and ATP binding cassette transporter A1 in RAW cells.
      ), may modify the raft proteome, at least in part, via ABCA1 down-regulation.
      Several proteins we identified in DRMs in this study have been previously localized to mitochondria. Multiple prior reports across a variety of cell types, including one by our group in RAW 264.7 macrophages (
      • Dhungana S.
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      Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide.
      ) have identified several “mitochondrial” proteins in raft preparations (
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      ,
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      Proteomic analysis of detergent-resistant membrane rafts.
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      ,
      • Kim B.W.
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      • Cho B.R.
      • Min D.S.
      • Yoon G.
      • Ko Y.G.
      Mitochondrial oxidative phosphorylation system is recruited to detergent-resistant lipid rafts during myogenesis.
      ,
      • Kim K.B.
      • Lee J.W.
      • Lee C.S.
      • Kim B.W.
      • Choo H.J.
      • Jung S.Y.
      • Chi S.G.
      • Yoon Y.S.
      • Yoon G.
      • Ko Y.G.
      Oxidation-reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts.
      ). While some have suggested that raft-like domains exist in mitochondria (
      • Sorice M.
      • Mattei V.
      • Matarrese P.
      • Garofalo T.
      • Tinari A.
      • Gambardella L.
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      • Misasi R.
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      Dynamics of mitochondrial raft-like microdomains in cell life and death.
      ) or that these proteins represent technical artifact (
      • Foster L.J.
      Lessons learned from lipid raft proteomics.
      ), stringent in situ approaches such as microscopy and cell-surface labeling have convincingly demonstrated “ectopic” plasma membrane localization of several of these proteins, including prohibitin, porin, NADH dehydrogenase, ubiquinol-cytochrome C reductase, and ATP synthase (
      • Kim B.W.
      • Lee C.S.
      • Yi J.S.
      • Lee J.H.
      • Lee J.W.
      • Choo H.J.
      • Jung S.Y.
      • Kim M.S.
      • Lee S.W.
      • Lee M.S.
      • Yoon G.
      • Ko Y.G.
      Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.
      ,
      • Kim B.W.
      • Lee J.W.
      • Choo H.J.
      • Lee C.S.
      • Jung S.Y.
      • Yi J.S.
      • Ham Y.M.
      • Lee J.H.
      • Hong J.
      • Kang M.J.
      • Chi S.G.
      • Hyung S.W.
      • Lee S.W.
      • Kim H.M.
      • Cho B.R.
      • Min D.S.
      • Yoon G.
      • Ko Y.G.
      Mitochondrial oxidative phosphorylation system is recruited to detergent-resistant lipid rafts during myogenesis.
      ).
      Several putative mitochondrial proteins were detected by LC-MS/MS in Abca1−/− but not Abca1+/+ DRMs, suggesting they are increased in the former. These include SLP-2, NADH dehydrogenase Fe-S protein 8, NADH dehydrogenase 1 alpha subcomplex subunit 13, NADH dehydrogenase subunit 1, cytochrome C oxidase subunit 7C, mitochondrial trifunctional protein β-subunit (Hadhb), and electron transferring flavoprotein alpha polypeptide (supplemental Table S1). By contrast, prohibitins-1 and -2, which play key roles in mitochondrial function, were relatively reduced in Abca1−/− rafts. ABCA1 deficiency was recently reported to reduce mitochondrial-mediated death in cancer cells by increasing mitochondrial cholesterol (
      • Smith B.
      • Land H.
      Anticancer activity of the cholesterol exporter ABCA1 gene.
      ), and it has been proposed that this finding could underlie other recent reports linking ABCA1 deficiency to cancer (
      • Lee B.H.
      • Taylor M.G.
      • Robinet P.
      • Smith J.D.
      • Schweitzer J.
      • Sehayek E.
      • Falzarano S.M.
      • Magi-Galluzzi C.
      • Klein E.A.
      • Ting A.H.
      Dysregulation of cholesterol homeostasis in human prostate cancer through loss of ABCA1.
      ,
      • Schimanski S.
      • Wild P.J.
      • Treeck O.
      • Horn F.
      • Sigruener A.
      • Rudolph C.
      • Blaszyk H.
      • Klinkhammer-Schalke M.
      • Ortmann O.
      • Hartmann A.
      • Schmitz G.
      Expression of the lipid transporters ABCA3 and ABCA1 is diminished in human breast cancer tissue.
      ). Given this, some of the targets we found increased in Abca1−/− DRMs may possibly underlie the cancer cell survival advantage recently associated with ABCA1 deficiency. Of interest in this regard, SLP-2 up-regulation has recently been associated with worse outcomes in human cancers (
      • Cao W.
      • Zhang B.
      • Ding F.
      • Zhang W.
      • Sun B.
      • Liu Z.
      Expression of SLP-2 was associated with invasion of esophageal squamous cell carcinoma.
      ,
      • Cui Z.
      • Zhang L.
      • Hua Z.
      • Cao W.
      • Feng W.
      • Liu Z.
      Stomatin-like protein 2 is overexpressed and related to cell growth in human endometrial adenocarcinoma.
      ,
      • Zhang L.
      • Ding F.
      • Cao W.
      • Liu Z.
      • Liu W.
      • Yu Z.
      • Wu Y.
      • Li W.
      • Li Y.
      • Liu Z.
      Stomatin-like protein 2 is overexpressed in cancer and involved in regulating cell growth and cell adhesion in human esophageal squamous cell carcinoma.
      ). Mitochondrial reactive oxygen species and other intermediates have also recently been shown to play a key role in the LPS response of cells (
      • Bulua A.C.
      • Simon A.
      • Maddipati R.
      • Pelletier M.
      • Park H.
      • Kim K.Y.
      • Sack M.N.
      • Kastner D.L.
      • Siegel R.M.
      Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS).
      ). Thus, whether the enhanced LPS response of Abca1−/− macrophages reported by us and others is in part driven by altered mitochondrial function is also an important question worthy of further investigation.
      SLP-2, previously found by us and others in raft preparations from a variety of cell types (
      • Bini L.
      • Pacini S.
      • Liberatori S.
      • Valensin S.
      • Pellegrini M.
      • Raggiaschi R.
      • Pallini V.
      • Baldari C.T.
      Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering.
      ,
      • Blonder J.
      • Hale M.L.
      • Lucas D.A.
      • Schaefer C.F.
      • Yu L.R.
      • Conrads T.P.
      • Issaq H.J.
      • Stiles B.G.
      • Veenstra T.D.
      Proteomic analysis of detergent-resistant membrane rafts.
      ,
      • Dhungana S.
      • Merrick B.A.
      • Tomer K.B.
      • Fessler M.B.
      Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide.
      ), is thought to reside in both mitochondrial and plasma membrane pools (
      • Christie D.A.
      • Kirchhof M.G.
      • Vardhana S.
      • Dustin M.L.
      • Madrenas J.
      Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation.
      ). Rigid topographic distinctions between mitochondria and plasma membrane may actually be unnecessary in this context, as it was recently reported that net exchange of membrane material between the two may occur during T cell activation, when mitochondria, plasma membrane, and increased SLP-2 coalesce at the immunological synapse (
      • Christie D.A.
      • Kirchhof M.G.
      • Vardhana S.
      • Dustin M.L.
      • Madrenas J.
      Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation.
      ). This is consistent with additional past reports documenting translocation of mitochondria to the plasma membrane (
      • Quintana A.
      • Schwindling C.
      • Wenning A.S.
      • Becherer U.
      • Rettig J.
      • Schwarz E.C.
      • Hoth M.
      T cell activation requires mitochondrial translocation to the immunological synapse.
      ,
      • Campello S.
      • Lacalle R.A.
      • Bettella M.
      • Mañes S.
      • Scorrano L.
      • Viola A.
      Orchestration of lymphocyte chemotaxis by mitochondrial dynamics.
      ).
      Roles for SLP-2 as an organizer of membrane microdomains have been proposed in both the plasma membrane and mitochondrial inner membrane. In the former site, SLP-2 is recruited to lipid rafts of T cells upon cell activation, where it associates with T cell receptor signalosome components and the actin cytoskeleton and is required for T cell receptor signaling, consistent with a scaffolding function (
      • Christie D.A.
      • Kirchhof M.G.
      • Vardhana S.
      • Dustin M.L.
      • Madrenas J.
      Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation.
      ,
      • Kirchhof M.G.
      • Chau L.A.
      • Lemke C.D.
      • Vardhana S.
      • Darlington P.J.
      • Márquez M.E.
      • Taylor R.
      • Rizkalla K.
      • Blanca I.
      • Dustin M.L.
      • Madrenas J.
      Modulation of T cell activation by stomatin-like protein 2.
      ). In the mitochondrion, SLP-2 promotes formation of cardiolipin-enriched membrane domains in which respiratory chain components and prohibitins are optimally assembled and stabilized and enhances mitochondrial biogenesis and ATP production (
      • Wang Y.
      • Morrow J.S.
      Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues.
      ,
      • Christie D.A.
      • Mitsopoulos P.
      • Blagih J.
      • Dunn S.D.
      • St-Pierre J.
      • Jones R.G.
      • Hatch G.M.
      • Madrenas J.
      Stomatin-like protein 2 deficiency in T cells is associated with altered mitochondrial respiration and defective CD4+ T cell responses.
      ,
      • Christie D.A.
      • Lemke C.D.
      • Elias I.M.
      • Chau L.A.
      • Kirchhof M.G.
      • Li B.
      • Ball E.H.
      • Dunn S.D.
      • Hatch G.M.
      • Madrenas J.
      Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function.
      ,
      • Da Cruz S.
      • Parone P.A.
      • Gonzalo P.
      • Bienvenut W.V.
      • Tondera D.
      • Jourdain A.
      • Quadroni M.
      • Martinou J.C.
      SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability.
      ,
      • Hájek P.
      • Chomyn A.
      • Attardi G.
      Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2.
      ). We are unaware of prior reports of SLP-2 in relation to macrophages, ABCA1, or TLR signaling.
      In this report, we show that, unlike other SPFH proteins, SLP-2 was robustly and specifically increased in DRMs of Abca1−/− macrophages. Given this, we speculate that SLP-2 may subserve a key compensatory role in raft organization of ABCA1-deficient cells and, moreover, that this may be relevant to both Tangier disease and more common functional ABCA1 polymorphisms found in humans. Consistent with a fundamental role for SLP-2 in raft organization, we found SLP-2-silenced cells to have markedly abnormal rafts, with dramatically increased cell-surface ganglioside as assessed by CtB, reduced raft protein mass, including reductions in key raft proteins, including the flotillins and CD14, but unchanged raft cholesterol as indicated by filipin.
      Moreover, we found that SLP-2 impacts LPS signaling in macrophages, albeit somewhat modestly. The LPS response has previously been shown by us and others to require raft integrity, as TLR4 and its proximal adaptors, including MyD88, translocate to the vicinity of CD14 in raft microdomains (
      • Triantafilou M.
      • Miyake K.
      • Golenbock D.T.
      • Triantafilou K.
      Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
      ). We speculate that the effects of SLP-2 silencing on TLR signaling are most likely indirect (i.e. a secondary consequence of changes to lipid rafts). Thus, although our findings suggest that SLP-2 “regulates” the innate immune response in a general sense (i.e. TLR signaling is attenuated in SLP-2-depleted cells), this should not be construed to indicate that SLP-2 necessarily plays a direct regulatory role in the underlying signaling cascade. Of some interest, we found that SLP-2 silencing led to a relatively larger reduction in IL-6 than TNFα in response to TLR2, TLR4, and TLR7 stimulation (Figs. 4B4D). While the specific underlying mechanism is not clear, this may suggest a somewhat selective effect of SLP-2 on downstream proinflammatory pathway output by TLRs.
      Although GM1 has widely been treated as a simple surrogate measure for rafts, several papers have shown that GM1 levels can be dissociated from levels of raft proteins and raft cholesterol (
      • Nagafuku M.
      • Kabayama K.
      • Oka D.
      • Kato A.
      • Tani-ichi S.
      • Shimada Y.
      • Ohno-Iwashita Y.
      • Yamasaki S.
      • Saito T.
      • Iwabuchi K.
      • Hamaoka T.
      • Inokuchi J.
      • Kosugi A.
      Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor.
      ,
      • Marwali M.R.
      • Rey-Ladino J.
      • Dreolini L.
      • Shaw D.
      • Takei F.
      Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity.
      ,
      • Molander-Melin M.
      • Blennow K.
      • Bogdanovic N.
      • Dellheden B.
      • Månsson J.E.
      • Fredman P.
      Structural membrane alterations in Alzheimer brains found to be associated with regional disease development; increased density of gangliosides GM1 and GM2 and loss of cholesterol in detergent-resistant membrane domains.
      ). Indeed, GM1 up-regulation has itself been linked to mislocalization of raft proteins, reduced membrane fluidity, and abnormal raft-dependent signaling (
      • Jury E.C.
      • Kabouridis P.S.
      • Flores-Borja F.
      • Mageed R.A.
      • Isenberg D.A.
      Altered lipid raft-associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus.
      ,
      • Hamamura K.
      • Tanahashi K.
      • Furukawa K.
      • Hattori T.
      • Hattori H.
      • Mizutani H.
      • Ueda M.
      • Urano T.
      • Furukawa K.
      GM1 expression in H-ras-transformed NIH3T3 results in the suppression of cell proliferation inducing the partial transfer of activated H-ras from non-raft to raft fraction.
      ,
      • Nishio M.
      • Fukumoto S.
      • Furukawa K.
      • Ichimura A.
      • Miyazaki H.
      • Kusunoki S.
      • Urano T.
      • Furukawa K.
      Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells.
      ). Exogenous GM1 inhibits LPS signaling (
      • Mond J.J.
      • Witherspoon K.
      • Yu R.K.
      • Perera P.Y.
      • Vogel S.N.
      Inhibition of LPS-mediated cell activation in vitro and in vivo by gangliosides.
      ), and a naturally occurring GM1hi human monocyte subset has recently been shown to display a reduced LPS response compared with GM1lo counterparts (
      • Moreno-Altamirano M.M.
      • Aguilar-Carmona I.
      • Sánchez-García F.J.
      Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness.
      ). It is thus possible that SLP-2 deficiency may attenuate LPS signaling in rafts via leading to increased GM1 in raft microdomains.
      In summary, we report that ABCA1 regulates the macrophage raft proteome as assessed through DRM preparations and identify a suite of proteins whose abundance in rafts is up- or down-regulated in the absence of ABCA1. Among these, we submit that SLP-2, robustly and specifically up-regulated in Abca1−/− rafts, represents a promising new target in studies of the molecular pathogenesis of ABCA1 deficiency, lipid raft structural organization, and the innate immune response. Future studies employing advanced quantitative proteomic techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), AQUA, and/or isobaric tags for relative and absolute quantification (ITRAQ) may foreseeably identify additional important effects of ABCA1 deletion upon the raft proteome with enhanced precision and sensitivity.

      Acknowledgments

      We acknowledge the NIEHS Viral Vector Core Facility and NIEHS Fluorescence Microscopy & Imaging Center.

      Supplementary Material

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