Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol [S]

Xuewei Zhu; John S. Owen; Martha D. Wilson; Haitao Li; Gary L. Griffiths; Michael J. Thomas; Elizabeth M. Hiltbold; Michael B. Fessler; John S. Parks

doi:10.1194/jlr.m006486

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol [S]

Zhu, Xuewei;Owen, John S.;Wilson, Martha D.;Li, Haitao;Griffiths, Gary L.;Thomas, Michael J.;Hiltbold, Elizabeth M.;Fessler, Michael B.;Parks, John S. 2010-11-01 00:00:00 Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafﬁ cking to lipid rafts by reduction of lipid raft cholesterol Xuewei Zhu , * John S. Owen , Martha D. Wilson , * Haitao Li , ** Gary L. Grifﬁ ths , ** † § †† 1, , † Michael J. Thomas , Elizabeth M. Hiltbold , Michael B. Fessler , and John S. Parks * † § Departments of Pathology/Lipid Sciences,* Biochemistry, and Microbiology and Immunology, Wake Forest University School of Medicine , Winston-Salem, NC ; Imaging Probe Development Center,** National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, MD ; and Laboratory of †† Respiratory Biology, National Institute of Environmental Health Sciences , Research Triangle Park, NC Abstract We previously showed that macrophages from receptor trafﬁ cking to lipid rafts by reduction of lipid raft macrophage-speciﬁ c ATP-binding cassette transporter A1 cholesterol. J. Lipid Res . 2010. 51: 3196–3206. -M/-M (ABCA1) knockout ( Abca1 ) mice had an enhanced proin- ﬂ ammatory response to the Toll-like receptor (TLR) 4 ago- Supplementary key words free cholesterol � cytokines � immunology � nist, lipopolysaccharide (LPS), compared with wild-type lipid droplets � lymphocytes � retinoids (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft con- tent, and hyper-responsiveness of macrophages to LPS in ATP-binding cassette transporter A1 (ABCA1) is a plasma M/-M WT mice. Abca1 macrophages were also hyper-respon- membrane protein that functions to eliminate excess free sive to speciﬁ c agonists to TLR2, TLR7, and TLR9, but not cholesterol (FC) from tissues by efﬂ uxing cellular FC and TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR phospholipid (PL) to lipid-free apolipoprotein AI, forming agonists by modulation of lipid raft cholesterol and TLR nascent HDL particles ( 1, 2 ). Therefore, ABCA1 plays a -M/-M mobilization to lipid rafts. We demonstrated that Abca1 critical role in the movement of cholesterol from periph- vs. WT macrophages contained 23% more FC in isolated eral tissues to the liver in a process known as reverse choles- lipid rafts. Further, mass spectrometric analysis suggested terol transport. Mutations that inactivate the human ABCA1 raft phospholipid composition was unchanged. Although cell -M/-M gene result in Tangier disease, which is characterized by ex- surface expression of TLR4 was similar between Abca1 tremely low plasma HDL cholesterol concentrations, mildly and WT macrophages, signiﬁ cantly more TLR4 was distrib- -M/-M elevated plasma triglyceride levels, and accumulation of uted in membrane lipid rafts in Abca1 macrophages. -M/-M cholesterol in macrophages ( 3–5 ). ABCA1 protein is ex- Abca1 macrophages also exhibited increased trafﬁ cking of the predominantly intracellular TLR9 into lipid rafts in pressed to a variable extent in most cells in the body, and its response to TLR9-speciﬁ c agonist (CpG). Collectively, expression is regulated by transcriptional activation and our data suggest that macrophage ABCA1 dampens inﬂ am- protein degradation ( 6, 7 ), making it difﬁ cult to determine mation by reducing MyD88-dependent TLRs trafﬁ cking to lipid rafts by selective reduction of FC content in lipid rafts. —Zhu, X., J. S. Owen, M. D. Wilson, H. Li, G. L. Grifﬁ ths, M. J. Thomas, E. M. Hiltbold, M. B. Fessler, and J. S. Parks. +/+ +/ M Abbreviations: Abca1 , wild type mice at the Abca1 locus; Abca1 , Macrophage ABCA1 reduces MyD88-dependent Toll-like -M/-M heterozygous macrophage-speciﬁ c ABCA1 knockout; Abca1 , ho- mozygous macrophage-speciﬁ c knockout; Abca1 , total Abca1 knock- out; ABCG1, ATP-binding cassette transporter G1; BMDM, bone marrow-derived macrophage; CT-B, cholera toxin B; FC, free choles- This work was supported by National Institutes of Health Grants HL-49373, terol; fPEG-chol, ﬂ uorescein ester of polyethylene glycol-derivatized HL-094525, and AT-27820 (J.S.P.), American Heart Association fellowship 09POST2250225 (X.Z.), and the Intramural Research Program of the Na- cholesterol; GLC, gas-liquid chromatography; I B , inhibitory B pro- tional Institutes of Health, National Institute of Environmental Health Sciences tein ; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-acti- Z01 ES102005 (M.B.F.). The fPEG-cholesterol synthesis was supported by the vated protein kinase; M CD, methyl- -cyclodextrin; MyD88, myeloid National Institutes of Health Roadmap for Medical Research Initiative through differentiation primary-response protein 88; NF, nuclear factor; NutSP, its establishment of the Imaging Probe Development Center, administered by the Nutridoma SP; PI, phosphatidylinositol; PL, phospholipid; PM, perito- National Heart, Lung, and Blood Institute. Its contents are solely the respon- neal macrophage; PNS, postnuclear supernatant; TLR, Toll-like recep- sibility of the authors and do not necessarily represent the ofﬁ cial views of the tor; WT, wild type. National Institutes of Health. To whom correspondence should be addressed. Manuscript received 2 March 2010 and in revised form 21 July 2010. e-mail: [email protected] Published, JLR Papers in Press, July 21, 2010 The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M006486 contains supplementary data in the form of Data, Results, and six ﬁ gures. 3196 Journal of Lipid Research Volume 51, 2010 This article is available online at http://www.jlr.org This is an Open Access article under the CC BY license. its physiological role in individual tissues using global Abca1 However, several questions still remain unanswered. First, knockout mice or cells in culture. However, generation of is there a direct relationship among macrophage FC con- cell-speciﬁ c Abca1 knockout mice has helped deﬁ ne the tent, lipid raft content, and the hyper-responsiveness to role of tissue-speciﬁ c ABCA1 expression in whole body HDL TLR4 agonist? Alternatively, is there a change of PL com- biogenesis as well as several unanticipated roles for the position in lipid rafts that leads to increased lipid raft -M/-M transporter. For example, hepatocyte and intestinal epithe- content in Abca1 macrophages? Third, do other lial cell ABCA1 contribute 70–80% and 20–30% of the TLR-speciﬁ c agonists also induce increased inﬂ ammatory -M/-M plasma HDL pool, respectively ( 8, 9 ). Pancreatic cell signaling in Abca1 macrophages? Finally, does the ex- ABCA1 plays a role in insulin secretion ( 10 ) and brain aggerated TLR signaling result from increased trafﬁ cking of TLRs to lipid rafts? In the present study, we demon- ABCA1 regulates neuronal structure and function ( 11 ). Macrophages, a primary cell type involved in innate im- strated: 1 ) a direct association between FC and lipid raft munity, have been implicated in chronic inﬂ ammatory dis- content that leads to hyper-responsiveness of macrophages eases such as atherosclerosis and insulin resistance, where to LPS in WT macrophages; 2 ) ABCA1 deletion in mac- rophages resulted in selective FC accumulation in lipid they accumulate in arteries ( 12, 13 ) and adipose tissue ( 14 ), respectively. Transplantation of bone marrow between wild- rafts without alternation of PL composition and enhanced type (WT) and ABCA1 knockout mice has little effect on activation of other MyD88-dependent TLRs compared plasma HDL concentration ( 15, 16 ). Despite this, lipopoly- with WT macrophages; and 3 ) ABCA1 deletion results in increased TLR4 and TLR9 localization in lipid rafts in saccharide (LPS)-induced sepsis was exacerbated in Abca1 / / / both resting and stimulated states, suggesting a mechanis- Ldlr mice compared with Ldlr mice ( 17 ), suggesting a -M/-M novel antiinﬂ ammatory role for macrophage ABCA1 in in- tic explanation for the hyper-responsiveness of Abca1 nate immunity. However, it was not clear from these studies macrophages to proinﬂ ammatory stimuli. whether the exacerbated proinﬂ ammatory response of / / Abca1 /Ldlr mice to LPS was due to the massive cellular cholesteryl ester accumulation in macrophages, the absence MATERIALS AND METHODS of plasma HDL, or some other alteration of macrophages. In addition, ABCA1 expression decreases cellular plasma mem- Animals brane rigidity by reducing formation of tightly packed lipid +/+ +/ M -M/-M Abca1 (WT), Abca1 (heterozygous), and Abca1 (ho- raft domains in the plasma membrane ( 18 ). Lipid rafts, en- mozygous) mice were generated as described previously ( 24 ). riched in FC and glycosphingolipid, play an important role in Mice were backcrossed to C57BL/6 background for six genera- signal transduction by recruiting and concentrating signaling tions before use in the studies. molecules in the plasma membrane ( 19 ). For instance, LPS Cell culture activation of macrophage results in transient Toll-like recep- Peritoneal macrophages (PMs) were harvested from C57BL/6 tor 4 (TLR4) trafﬁ cking to lipid rafts along with its cognate +/+ -M/-M backcrossed Abca1 and Abca1 littermate mice 4 days after re- adaptor proteins ( 20–23 ) and subsequent secretion of in- ceiving an intraperitoneal injection of 1 ml 10% thioglycolate and ﬂ ammatory cytokines and chemokines. These studies suggest plated in RPMI media containing 1% Nutridoma SP (NutSP) media a link among ABCA1-mediated cellular lipid efﬂ ux, mem- (Roche Applied Science) as previously described ( 24 ). Bone mar- brane lipid raft homeostasis, and activation of macrophages. +/+ +/ M -M/-M row from Abca1 , Abca1 , and Abca1 littermate mice was iso- Nevertheless, the molecular details regarding how ABCA1 lated and cultured in DMEM media containing 20% FBS and 30% expression affects macrophage inﬂ ammatory response are L929 conditioned media for 5–7 days before being used in experi- poorly understood. ments as bone marrow-derived macrophages (BMDMs) ( 24 ). To explore the speciﬁ c role of ABCA1 in macrophages FC, lipid raft content, surface TLR4, and inﬂ ammatory in vivo and in vitro, we generated macrophage-speciﬁ c - cytokine expression in WT macrophages M/-M ABCA1 knockout ( Abca1 ) mice. Using this unique +/+ BMDMs from Abca1 mice were plated in tissue culture dishes mouse model, we demonstrated that macrophages from -M/-M and cultured in RPMI media plus 1% NutSp overnight as de- Abca1 mice have a signiﬁ cant increase in FC and are scribed above. Cholesterol depletion and overloading of mac- more proinﬂ ammatory in vivo and in vitro in response to rophages with methyl- -cyclodextrin (M CD) (Sigma-Aldrich) LPS via TLR4 compared with WT. This response was medi- was performed as described previously ( 24 ). Brieﬂ y, BMDMs were ated through a myeloid differentiation primary-response incubated with or without prewarmed 10 mM M CD at 37°C for protein 88 (MyD88)-dependent pathway and was indepen- 30 min to deplete cholesterol, or macrophages were incubated with M CD-cholesterol (40 or 80 g/ml) (Sigma-Aldrich) at dent of alterations in plasma lipid concentrations ( 24 ). We -M/-M 37°C for 60 min to overload macrophages with cholesterol. further showed that the hypersensitivity of Abca1 mac- After three washes with PBS, macrophages were extracted with rophages to LPS was most likely due to increased lipid raft isopropanol to measure cholesterol content by gas-liquid chro- content, presumably caused by increased intracellular FC matography (GLC) as described before ( 24 ) or incubated ± 100 accumulation. Recently, Yvan-Charvet et al. ( 25 ) observed ng/ml Kdo -lipid A (Avanti Polar Lipids) for 6 h before quantiﬁ - / / a similar inﬂ ammatory phenotype in Abca1 Abcg1 cation of cytokine expression by ELISA kits (BD Bioscience). macrophages compared with WT. Collectively, these stud- ies suggest that ABC transporters suppress macrophage Flow cytometry +/+ -M/-M TLR4 activation, possibly through modulation of mem- Elicited peritoneal cells from Abca1 or Abca1 mice were stained directly or plated in tissue culture dishes and cultured in brane lipid rafts. Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3197 RPMI media plus 1% NutSp overnight as described above. To NaF, 0.1 mM Na VO , 5 g/ml leupeptin, 5 g/ml aprotinin) by 3 4 measure TLR4/MD2 surface expression, elicited peritoneal cells 20 passages through a 22 G syringe needle. The nuclei and cell +/+ -M/-M from Abca1 or Abca1 mice were incubated in the presence debris were pelleted by centrifugation for 10 min at 1000 g . The or absence of 100 ng/ml LPS from Salmonella typhimurium (Sigma) PNS was collected and stored on ice. The pellet was resuspended for 1 h in suspension before staining with PE-F4/80 (BD Biosci- in 1 ml of Buffer B, homogenized, and centrifuged again. The ence) and/or APC-TLR4/MD2 (MTS510) (eBioscience) or iso- two PNSs (2 ml) were mixed with an equal volume (2 ml) of Solu- type control (eBioscience or BD Bioscience). To examine the tion D (50% Optiprep) and then centrifuged through 9 ml of content of lipid rafts or TLR4/MD2 complex in macrophages, 5–20% continuous Optiprep gradient using an SW41 rotor (90 after incubation with 10 mM M CD or 80 g/ml M CD- min, 53,000 g , 4°C). Following centrifugation, thirteen 1 ml frac- cholesterol for 30 min or 1 h, macrophages were gently lifted tions were harvested from top to bottom. using a cell scraper. Live PMs were then stained with 0.5 g/ml Detergent extraction method. Cold detergent resistance mem- cold Alexa ﬂ uor 488-cholera toxin B (CT-B) (Molecular Probes) branes (or lipid rafts) were prepared according to the method de- for 15 min at 4°C or stained with APC-TLR4/MD2 (MTS510) for scribed by Fessler et al. ( 29 ). Brieﬂ y, BMDMs were washed three 30 min at 4°C or PMs were ﬁ xed with 2% paraformaldehyde and times with ice-cold PBS. Following the wash, cells were centrifuged then stained with 10 g/ml fPEG -chol (ﬂ uorescein ester of (1,000 rpm, 10 min, 4°C) and resuspended in 1 ml of ice-cold lysis polyethylene glycol-derivatized cholesterol) ( 26 ) at room tem- buffer (25 mM MES, pH 6.5, 150 mM NaCl, 1% Triton X-100, perature for 10 min. Cell ﬂ uorescence was determined using a 1 mM PMSF, 1 mM NaF, 0.1 mM Na VO , 5 g/ml leupeptin, 5 g/ FACSCalibur ﬂ ow cytometer (BD Bioscience) and data were ana- 3 4 ml aprotinin). Cells were vortexed for 15 s on ice and allowed to sit lyzed using Flowjo software (Tree Star). on ice for 10 min. This step was repeated three times and then the cell lysate was centrifuged (1,000 g , 10 min, 4°C). The supernatant Inﬂ ammatory mediator expression in TLR ( 1 ml) was gently mixed with an equal volume of 80% sucrose in agonist-treated macrophages MBS (25 mM MES, pH 6.5, 150 mM NaCl) and then centrifuged After overnight incubation, PMs were switched to serum- through a 5–30% continuous sucrose gradient using an SW41 ro- free RPMI-1640 media for 2 h before TLR agonist stimulation. tor (20 h, 37,000 rpm, 4°C). Following centrifugation, twelve 1 ml BMDMs were incubated overnight in 1% NutSP media before fractions were harvested from top to bottom. stimulation with various TLR agonists. Macrophages were treated To induce translocation of TLRs to lipid rafts, BMDMs from both with 100 ng/ml Pam SCK (TLR2), 10 g/ml poly (I: C) (TLR3), 3 4 -M/-M WT and Abca1 mice were incubated in 1% NutSP-RPMI-1640 200 M Loxoribine (TLR7), or 1 M CpG (TLR9), or control media overnight and then treated with ±100 ng/ml LPS from Salmo- CpG (agonists from InvivoGen) in serum-free RPMI-1640 media nella typhimurium (Sigma-Aldrich) or ±1 M CpG for 1 h, followed by for 0–12 h, after which the culture supernatant was collected and preparation of lipid rafts and nonraft as described above. Mac- stored at 80°C for cytokine ELISA (BD Bioscience). rophage lipid raft TLR content was calculated as a percentage of total membrane TLR (i.e., lipid rafts + nonraft fractions). Preparation of subcellular membrane fractions BMDMs were used for all lipid raft isolation procedures to in- Immunoblotting and immunoprecipitation crease macrophage yield. The protein concentration in each iso- Western blots were performed using speciﬁ c antibodies against: lated fraction was determined by the Bradford protein assay caveolin-1 (Abcam), CD14 (Abcam) (as lipid raft marker), clathrin (Pierce). (Sigma-Aldrich) (as nonraft marker), TLR4 (cell signaling), or Nondetergent method. Lipid rafts were prepared according to TLR9 (Abcam). TLR9 was immunoprecipitated with a TLR9 poly- the method of Smart et al. ( 27 ). Brieﬂ y, BMDMs were scraped clonal antibody from pooled raft or nonraft fractions, followed by into ice-cold 0.25 M sucrose, 1 mM EDTA, and 20 mM Tris (pH immunoblotting with antibody against TLR9. Blots were devel- 7.8) (Buffer A) with protease inhibitors (1 mM PMSF, 1 mM NaF, oped using HRP-linked secondary antibody. Immunoblots were 0.1 mM Na VO , 5 g/ml leupeptin, 5 g/ml aprotinin) and ho- 3 4 visualized with the Supersignal substrate system (Pierce). Images mogenized in 1 ml cold buffer A by 20 passages through a 22 G were captured and quantiﬁ ed using the LSA-3000 imaging system syringe needle. The nuclei and cell debris were pelleted by cen- and Multi Gauge software (Fujiﬁ lm Life Science). trifugation for 10 min at 1,000 g . The postnuclear supernatant (PNS) was collected and stored on ice. The pellet was resus- Analysis of nuclear factor- B pathway in the pended in Buffer A, homogenized, and centrifuged again. The CpG-treated macrophages two PNSs (2 ml) were combined and layered on top of 23 ml of -M/-M BMDMs from both WT and Abca1 mice were incubated in 1% 30% Percoll (Sigma-Aldrich) in Buffer A and centrifuged for 30 NutSP-RPMI-1640 media overnight before treated ± 1 M CpG for min at 84,000 g . The plasma membrane fraction was collected 0–3 h, after which cells were processed for Western-blot analysis. and sonicated on ice for a total of six bursts (50 J each time). The Antibody to inhibitory B protein (I B ) was purchased from Cell sonicated membrane fraction was made up to 23% Optiprep Signaling Technology, Inc. and antibody to -actin from Sigma- (Sigma-Aldrich) (ﬁ nal volume: 4 ml) by the addition of a 50% Aldrich. Immunoblots were visualized as described above. Optiprep stock in 20 mM Tris, 0.25 M sucrose, and 1 mM EDTA (pH 7.8) (Buffer C) and placed in the bottom of a centrifuge FC measurements tube. An 8 ml gradient from 20% to 10% Optiprep was layered Raft and nonraft fractions isolated using Smart’s nondetergent on top of the sample, which was then centrifuged for 90 min at method were extracted using the Bligh-Dyer method ( 30 ), and cho- 52,000 g in an SW41 swinging bucket rotor. The gradient was lesterol content was determined by GLC as previously described then fractioned into 12 or 13 1 ml fractions from top to bottom. ( 24 ). Simpliﬁ ed nondetergent method. Lipid rafts were prepared ac- Lipidomic assay cording to the method of MacDonald et al. ( 28 ). Brieﬂ y, BMDMs were scraped into ice-cold 0.25 M sucrose, 1 mM CaCl , 1 mM The distribution of PL species in pooled raft or nonraft frac- MgCl , 20 mM Tris-HCl, pH 7.8 (Buffer B), and homogenized in tions or whole cells was measured using ESI/MS/MS as described 1 ml cold buffer B with protease inhibitors (1 mM PMSF, 1 mM in the supplementary data. 3198 Journal of Lipid Research Volume 51, 2010 Statistics ment, macrophages subjected to cholesterol depletion secreted less interleukin (IL)-6 and IL-12p40, whereas Differences were compared with two-tailed Student’s t -test or one-way ANOVA using GraphPad Prism software. P < 0.05 was macrophages overloaded with cholesterol secreted more considered statistically signiﬁ cant. Data are presented as the of these cytokines, suggesting that cytokine secretion in means ± SEM unless indicated otherwise. TLR4 activated macrophages has a signiﬁ cant positive as- sociation with cellular FC content ( Fig. 1B ). To determine whether rapid alteration of FC content changes lipid raft content or TLR4 surface expression, we measured lipid RESULTS raft content of macrophages either depleted of or over- Macrophage FC content is positively associated with lipid loaded with cholesterol by ﬂ ow cytometry. We used two raft content and proinﬂ ammatory response types of raft markers: fPEG-chol ( 26, 31, 32 ), which parti- To directly examine the effect of FC on macrophage tions into membrane raft microdomains in direct relation proinﬂ ammatory activation, we incubated WT mac- to their cholesterol content, and Alexa ﬂ uor 488-CT-B, rophages with 10 mM M CD (cholesterol depletion) or which binds to ganglioside GM1 located in lipid rafts. As M CD loaded with 40 or 80 g/ml of cholesterol (choles- shown in Fig. 1C , cellular ﬂ uorescent intensity of fPEG- terol overloading) before cells were challenged with Kdo2- chol increased with increasing macrophage FC content. lipid A, a speciﬁ c TLR4 agonist. Figure 1A shows that Incubation of cells with M CD also resulted in decreased incubating macrophages with M CD or M CD-cholesterol CT-B binding, presumably due to the disruption of lipid resulted in a 2-fold variation in cellular FC content. Cho- raft structure by M CD, but unlike fPEG-chol, cholesterol lesteryl ester content in macrophages was negligible with overloading did not result in an increase in CT-B binding all treatments (data not shown). Compared with no treat- on the cell surface, suggesting a rapid increase of FC on Fig. 1. Manipulation of cellular FC content alters proinﬂ ammatory status and cell surface lipid raft, but not TLR4, content. A: WT BMDMs were incubated at 37°C with M CD for 0.5 h or with M CD-chol for 1 h before FC measurement by GLC. B: WT BMDMs were incubated with Kdo2-lipid A for 6 h after in- cubation with M CD or M CD-chol, and cytokine expression was measured by ELISA. Cytokine con- centration was plotted against FC content of mac- rophages. The line of best ﬁ t for IL-6 ( r = 0.92) and IL-12p40 ( r = 0.75) is shown. C–E: WT-elicited PMs were treated with M CD or M CD-chol before stained with fPEG-chol (C), Alexa ﬂ uor CT-B (D), or APC-TLR4/MD2 (MTS510) (E). Cells were analyzed using ﬂ ow cytometry. Histogram of individual sam- ples is shown in the left panel and mean ﬂ uorescence intensity is shown in the right panel. Values with unlike letters are signiﬁ cantly different ( P < 0.05). Results represent mean ± SEM of 2 independent experiments; n = 4–5 dishes of cells/group. Chol: M CD-cholesterol. Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3199 -M/-M the cell surface does not alter the distribution of ganglio- Abca1 macrophages are hypersensitive to stimulation by various TLR agonists side GM1 ( Fig. 1D ). Furthermore, disrupting lipid rafts us- ing M CD resulted in a slight decrease of TLR4 surface In addition to TLR4, we also investigated the effect of expression compared with control, but cholesterol over- macrophage ABCA1 deﬁ ciency on the activation of other loading did not signiﬁ cantly change the TLR4 surface ex- TLRs. PMs were treated with speciﬁ c TLR agonists Pam SCK 3 4 pression ( Fig. 1E ). (TLR2), poly (I:C) (TLR3), Loxoribine (TLR7), or CpG -M/-M Fig. 2. Abca1 macrophages are hypersensitive to TLR2, 7, and 9, but not to TLR3 stimulation. A–C: Elicited PMs were treated with SCK (TLR2), Loxoribine (TLR7), or TLR9 for 0–12 h. The secretion of inﬂ ammatory factors was measured by ELISA. D, E: PMs were Pam 3 4 treated with poly (I:C) (TLR3) for 0–12 h. Cytokine or chemokine secretion was measured by ELISA. Results represent mean ± SEM of at +/+ -M/-M : WT; -M/-M: Abca1 . least two independent experiments; n = 3 dishes of cells/group. * P <0.05, compared with WT. 3200 Journal of Lipid Research Volume 51, 2010 (TLR9) for 3–12 h and cytokine secretion from the stim- ing raft (caveolin-1 and CD14) and nonraft (clathrin) ulated cells was measured by ELISA. Compared with markers, we designated the top eight fractions (nos. 1–7) -M/-M WT, Abca1 macrophages secreted more IL-6, IL-12p40, as lipid rafts and the bottom fractions (nos. 9–12) as non- -M/-M and RANTES over the time course after stimulation with rafts ( Fig. 4A ). Interestingly, we found that in Abca1 Pam SCK , Loxoribine, or CpG ( Fig. 2A –C ). A similar pat- cells, each lipid raft fraction contained more FC ( Fig. 4B ), 3 4 tern was observed for TNF- secretion (data not shown). resulting in an average 23.01 ± 2.12% greater FC content However, TLR3 stimulation by poly (I:C) resulted in for the whole lipid raft fraction (i.e., fractions 1–7) com- similar secretion of inﬂ ammatory factors from WT and pared with the same fractions from WT cells ( Fig. 4C ). On -M/-M Abca1 macrophages ( Fig. 2D, E). Similar results were the other hand, FC content was similar in nonraft fractions -M/-M obtained with BMDMs (data not shown). TLR3 is the only of WT and Abca1 cells ( Fig. 4B, C ) . Taken together, member of the TLR family whose signal transduction is our data suggested that ABCA1 deﬁ ciency results in an in- independent of MyD88. The data in Fig. 2 suggest that crease in lipid raft content, likely due to a striking increase ABCA1 deﬁ ciency mainly affects TLR pathways that signal in FC content of this fraction. We also tested an alternative through MyD88. hypothesis that sphingomyelin (SM) content in lipid rafts may be increased in the absence of ABCA1, resulting in -M/-M Lipid rafts from Abca1 macrophages are speciﬁ cally increased lipid raft content (supplementary Figs. II–IV). enriched in FC However, SM content and PL fatty acyl molecular species To determine whether the increase in lipid rafts previ- in raft and nonraft fractions were similar among the three -M/-M ously described in Abca1 macrophages ( 24 ) is the di- genotypes of mice. rect result of increased FC accumulation in lipid rafts, we performed two experiments. First, we examined the inten- Increased lipid raft TLR4 content in -M/-M Abca1 macrophages sity of FC-enriched microdomains by staining macrophages with fPEG-chol. As shown in Fig. 3A , D, compared with TLR4 signaling is initiated by ligand binding and tran- -M/-M WT, Abca1 macrophages had a slight but signiﬁ cantly sient trafﬁ cking of TLR4 to lipid rafts after macrophages brighter staining of fPEG-chol, indicating more FC- are activated ( 20–23 ). Our data suggested that total cellu- enriched microdomains located on the cell surface. Further- lar ( 24 ) and surface expression of TLR4 were indistin- -M/-M more, cholesterol depletion using M CD normalized the guishable between WT and Abca1 macrophages difference in fPEG-chol staining between the two geno- (supplementary Fig. V; see more details in supplementary types ( Fig. 3B , D), whereas the difference of fPEG-chol Results). To investigate whether ABCA1 deﬁ ciency results staining was maintained when cells were overloaded with in enhanced TLR4 recruitment to lipid rafts, we isolated cholesterol ( Fig. 3C, D). To determine whether the in- rafts and nonrafts from BMDMs ± 100 ng/ml LPS and im- -M/-M creased FC in Abca1 macrophages is located exclusively munoblotted for TLR4. In resting macrophages, the ma- in lipid rafts, we isolated lipid rafts and nonrafts from mac- jority of TLR4 was observed in nonraft fractions ( Fig. 5A ). rophages using a nondetergent isolation method ( 27 ). Us- However, a portion of TLR4 was also detected in raft frac- Fig. 3. ABCA1 deﬁ ciency in macrophages resulted in increased membrane lipid raft content. Elicited PMs were incubated with or without M CD for 0.5 h or M CD-chol (80 µg/ml) for 1 h before staining with fPEG-chol. Cell ﬂ uorescence was analyzed using ﬂ ow cytometry. A–C : Histograms show macrophage fPEG-chol staining in the absence (A) or presence (B) of M CD or presence of M CD-chol (C), respec- tively. D: Data from panels A–C are presented as mean ± SEM. Statistical signiﬁ cant differences be- tween groups are indicated (*, P < 0.05; **, P < 0.01). Results are representative of two independent ex- +/+ periments with at least three mice per group. : -M/-M WT; -M/-M: Abca1 . Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3201 Nuclear factor- B pathway and lipid rafts are involved with the enhanced TLR9 signaling in -M/-M Abca1 macrophages TLR7 or TLR9 was shown to translocate from ER to the endosome/lysosome upon stimulation by speciﬁ c agonists, resulting in activation of the nuclear factor (NF)- B and mitogen-activated protein kinase (MAPK) pathways ( 33 ). To examine whether the NF- B pathway is upregulated in -M/-M TLR9 agonist (CpG)-treated Abca1 macrophages, we -M/-M stimulated WT and Abca1 BMDMs with 1 M CpG for 0–3 h before isolating cell lysates for immunoblotting. As shown in Fig. 6A , within 30 min in both genotypes of mac- rophages, CpG induced I B degradation, which is indic- ative of NF B activation. Interestingly, compared with WT, -M/-M Abca1 BMDMs showed a greater and prolonged I B degradation during 3 h of TLR9 stimulation. Because CpG stimulation was shown not only to induce translocation of TLR9 to endosomes but also to induce trafﬁ cking of TLR9 to plasma membrane lipid rafts ( 20, 34, 35 ), we next tested the hypothesis that CpG may induce more TLR9 transloca- -M/-M tion to lipid rafts in Abca1 versus WT macrophages. To test this hypothesis, we isolated lipid rafts and nonrafts -M/-M from WT and Abca1 BMDMs using a simpliﬁ ed nonde- tergent isolation method ( 28 ). The majority of TLR9 was Fig. 4. Macrophage ABCA1 deﬁ ciency results in an increase in located in nonraft fractions in resting BMDMs ( Fig. 6B ). lipid raft FC. A: Lipid raft and nonraft fractions were isolated using Next, we pooled raft (fractions 1–8) and nonraft (fractions a nondetergent method. Each fraction was analyzed by Western 10–13) fractions isolated from BMDMs ± CpG treatment blotting for the indicated proteins. B: FC and protein concentra- and performed Western-blot analysis ( Fig. 6C ) or immu- tions in each fraction in panel A were measured and the results noprecipitation ( Fig. 6D ) to visualize and quantify the traf- were plotted as µg cholesterol/mg protein. Mean ± range (n = 2). Results are representative of two independent experiments. C: ﬁ cking of TLR9 to lipid rafts. As shown in Fig. 6C–E , we Data from panel B are presented as raft (fractions 1–7) and nonraft observed a trend of increased basal lipid raft TLR9 con- -M/-M (fractions 9–12) cholesterol content for each genotype. * P < 0.05, tent in resting Abca1 cells ( P = 0.08). More interest- +/+ -M/-M compared with WT. : WT; -M/-M: Abca1 . ingly, CpG stimulation signiﬁ cantly triggered increased translocation of TLR9 from nonraft to lipid raft fractions -M/-M tions, colocalized with CD14. Interestingly, TLR4 was more in Abca1 vs. WT BMDMs ( Fig. 6C–E ). These combined diffusely distributed in the lipid raft fractions (fractions results suggest that the increased lipid raft content in -M/-M -M/-M Abca1 cells increases MyD88-dependent activation of 1–7) of Abca1 BMDMs ( Fig. 5B ) compared with that in plasma membrane and intracellular TLRs through aug- WT cells ( Fig. 5A ). To visualize the trafﬁ cking of TLR4 induced by LPS stimulation, we pooled raft and nonraft mented translocation or trafﬁ cking to lipid rafts, resulting fractions isolated from BMDMs ± LPS treatment and per- in hyper-activation of downstream signaling transduction formed Western-blot analysis. Lipid raft and nonraft frac- and exaggerated proinﬂ ammatory cytokine expression. tions were identiﬁ ed based on the distribution of raft markers (caveolin-1, CD14, and ﬂ otillin-1) and the non- DISCUSSION raft marker, clathrin ( Fig. 5C ). The majority of TLR4 was The well-known function of ABCA1 is to transport FC distributed in the nonraft membrane fraction in both WT -M/-M and PLs out of cells, protecting cells from excessive choles- and Abca1 BMDMs. However, we observed a signiﬁ - cantly higher TLR4 content in lipid rafts from resting (i.e., terol accumulation. Interestingly, studies from our labora- -M/-M nontreated) Abca1 BMDMs compared with WT cells tory and others suggest an additional antiinﬂ ammatory ( Fig. 5C, D). Upon LPS stimulation, a trend toward in- role of macrophage ABCA1 ( 17, 24, 25, 36 ), but the mech- anism still remains unclear. The present study provides creased translocation of TLR4 into lipid rafts was observed -M/-M in Abca1 relative to WT macrophages ( Fig. 5C, D). As direct evidence showing that ABCA1 deﬁ ciency in mac- a control, another lipid raft protein, caveolin-1, was exam- rophages leads to selective FC accumulation in lipid rafts ined. Unlike TLR4, caveolin-1 content in lipid rafts was without alteration of PL composition. ABCA1 deﬁ ciency unaffected by LPS treatment of macrophages or by the in macrophages also results in more TLR content in lipid genotype of macrophages (supplementary Fig. VI). Col- rafts, consequently leading to enhanced TLR activation. lectively, these data suggest that ABCA1 deﬁ ciency in mac- To our knowledge, this is the ﬁ rst study to directly show rophages results in a signiﬁ cant increase in FC-enriched that ABC transporters downregulate TLR (TLR2, 4, 7, and lipid rafts, which concentrate and recruit more TLR4 in 9) signaling by reducing FC enrichment in lipid rafts and resting and potentially in LPS-activated macrophages. trafﬁ cking of TLRs into rafts. 3202 Journal of Lipid Research Volume 51, 2010 -M/-M Fig. 5. Increased TLR4 in lipid rafts of Abca1 macrophages. A and B: Lipid raft and nonraft mem- brane regions were isolated using a nondetergent method and analyzed for TLR4, clathrin (non-raft marker), and CD14 (raft marker) distribution by Western blotting. C: BMDMs were treated with 100 ng/ml LPS for 1 h and then lipid raft (fractions 1–8) and nonraft (fractions 10–13) fractions were pooled, and the distribution of the indicated proteins was analyzed by Western blotting. Results are representa- tive of two independent experiments. D: TLR4 con- tent of macrophage lipid rafts was calculated as a percentage of total membrane TLR4 (lipid rafts + nonrafts). Data are presented as mean ± range (n = 2). Statistically signiﬁ cant differences between +/+ genotypes are indicated (* P < 0.05). : WT, -M/-M: M/-M Abca1 . Lipid rafts are liquid-ordered membrane microdomains plasma membrane ﬂ uidity but not sufﬁ cient to activate enriched in FC and SM ( 19 ). ABCA1 expression results in TLR signaling without speciﬁ c agonist stimulation. redistribution of SM and FC from lipid raft to nonraft frac- Of the 13 members in the TLR family discovered so far, tions in baby hamster kidney cells ( 18 ). Our previous study TLR2 and TLR4 on the cell surface recognize bacterial suggested that ABCA1 deﬁ ciency in macrophages was as- lipoteichoic acid and LPS, respectively, whereas TLR3, 7, sociated with an increase in lipid rafts, presumably due to and 9, located in intracellular compartments, detect nu- the increase in FC accumulation ( 24 ). To test an alterna- cleic acids derived from pathogens ( 33 ). Beside their criti- -M/-M tive hypothesis that the increase in lipid rafts in Abca1 cal role in innate immunity, TLRs are also involved in the macrophages was the result of increased SM enrichment, development of chronic inﬂ ammatory diseases, such as we analyzed the PL proﬁ le of lipid rafts from macrophages atherosclerosis ( 38, 39 ) and insulin resistance ( 40 ). In ad- using MS. Our data suggested that the relative distribution dition, TLR9 was shown to be involved in macrophage of SM and phosphatidylcholine fatty acyl species in lipid foam cell formation ( 41 ). We previously showed that -M/-M rafts was similar for WT and Abca1 macrophages (sup- ABCA1 deﬁ ciency resulted in an enhanced MyD88-depen- M/-M plementary Fig. IV). Thus, the hypersensitivity of Abca1 dent TLR4 signal ( 24 ). Here, we further demonstrated macrophages to TLR activation was not due to alterations that ABCA1 deﬁ ciency also resulted in enhanced activa- in SM or phosphatidylcholine content of lipid rafts. tion of TLR2, 7, and 9, but not TLR3. Despite having in -M/-M On the other hand, Abca1 macrophages contained common an intracellular localization, TLR7 and TLR9 more FC-enriched lipid rafts, demonstrated by fPEG-chol employ MyD88, whereas TLR3 recruits TRIF to propagate staining and cholesterol quantiﬁ cation in isolated lipid downstream signal transduction. The mechanism by which rafts. ABCA1 deﬁ ciency favors MyD88-dependent TLR pathways Excessive FC in late endosomes or in the plasma mem- remains unknown. / / brane activates the p38 MAPK pathway through TLR3 or WT and Abca1 Abcg1 macrophages had similar TLR4, respectively, without speciﬁ c agonist stimulation cytokine gene expression when cells were treated with ( 37 ), indicating that FC overloading alone is sufﬁ cient to TLR2, TLR7, or TLR9 agonists ( 25 ). In contrast, when / / activate TLRs in certain situations. In our study, when FC cholesterol loaded using M CD-chol, Abca1 Abcg1 content of WT macrophages was varied over a 2-fold range, macrophages were hyper-sensitive to TLR4 stimulation / / we observed a signiﬁ cant positive association among mac- compared with WT cells. In addition, Abca1 Abcg1 rophage FC content, lipid raft content, and the inﬂ amma- macrophages were also hyper-responsive to TLR3 stimula- tory response to TLR4 agonist ( Fig. 1 ). However, in tion with or without cholesterol loading ( 25 ). The differ- M/-M Abca1 macrophages, FC content was only slightly in- ence between our study and the study of Yvan-Charvet / / creased ( 10%) compared with WT cells, and no signiﬁ - et al. ( 25 ) is that Abca1 Abcg1 macrophages (without cant difference in the activation of NF- B and MAPK cholesterol overloading) were hyper-responsive to TLR3 pathways or inﬂ ammatory gene expression was observed and TLR4, but not to TLR2, 7, or 9 stimulation, whereas -M/-M -M/-M between WT and Abca1 macrophages in the absence of our Abca1 macrophages were hyper-responsive to speciﬁ c ligand stimulation ( 24, 25 ), suggesting that a sub- TLR2, 4, 7, or 9 stimulation but not to TLR3 activation. / / tle increase in FC content alone is sufﬁ cient to alter the Notably, Abcg1 Ldlr macrophages had a 60% Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3203 Fig. 6. NF- B pathway and lipid rafts are involved -M/-M with enhanced TLR9 signaling in Abca1 mac- rophages. A: BMDMs were incubated with 1 M CpG for 0–3 h. Cell lysates were harvested and the pro- teins were analyzed by immunoblotting with the indi- cated antibodies. I B was quantiﬁ ed and normalized to -actin. The degradation of I B induced by CpG stimulation is presented as fraction of basal protein level in control WT cells. B: BMDMs were fractioned into raft and nonraft fractions using a simpliﬁ ed non- detergent method. The protein distribution in each fraction was analyzed by Western blotting using the indicated antibodies. C: BMDMs were treated with 1 M CpG for 1 h and then lipid rafts (fractions 1–8) and nonrafts (fractions 10–13) were pooled, and the distribution of the indicated proteins was analyzed by Western blotting. D: Lipid raft and nonraft fractions isolated as described in panel C were pooled and im- munoprecipitated with anti-TLR9 polyclonal anti- body, followed by immunoblotting with anti-TLR9 polyclonal antibody. Results are representative of two independent experiments. E: Lipid raft TLR9 con- tent in macrophages was calculated as a percentage of total TLR9 (lipid rafts + nonrafts). Data are pre- sented as mean ± SEM; n = 3. Statistically signiﬁ cant differences between groups are indicated (* P < 0.05). +/+ -M/-M : WT, -M/-M: Abca1 . increase in FC compared with Ldlr cells ( 25 ). Although and potentially after LPS stimulation compared with WT macrophages ( Fig. 5 ). Evidence suggests that recognition Yvan-Charvet et al. ( 25 ) did not show evidence of FC en- / / of CpG by TLR9 is likely to occur not only in endosomes, richment in endosomes, the excess FC in Abca1 Abcg1 macrophages might accumulate in intracellular compart- but also in plasma membrane lipid rafts ( 20, 34 ). By con- ments, resulting in activation of TLR3 ( 37 ). In our study, trast, TLR3 activation does not occur in the rafts ( 43, 44 ). -M/-M FC content in Abca1 macrophages was only increased With these ﬁ ndings in mind, we hypothesized that the in- -M/-M creased lipid raft content in Abca1 macrophages might 10% compared with WT macrophages, and the FC enrich- ment was selective for lipid rafts. Therefore, the difference also result in enhanced TLR9 trafﬁ cking into rafts and our / / in FC content between Abca1 Abcg1 macrophages results supported this hypothesis ( Fig. 6 ). Because TLR2 -M/-M and Abca1 cells as well as the differential compartmen- and TLR4 (cell surface receptors) or TLR7 and TLR9 (in- tracellular receptors) share many properties and similar tal enrichment may account for the different responsive- ness to TLRs stimulation. activation mechanisms, respectively ( 22, 45, 46 ), we as- TLR2 or TLR4 signaling is thought to be initiated after sume that ABCA1 deﬁ ciency in macrophages results in TLRs rapidly trafﬁ c to lipid rafts in response to speciﬁ c li- increased translocation or recruitment of all MyD88- gands or saturated fatty acids ( 22, 23, 42 ). Based on these dependent TLRs ( 2, 4, 7 and 9 ) into lipid rafts, which leads ﬁ ndings, we hypothesized that the enhanced TLR4 activa- to enhanced downstream signal transduction and augmented -M/-M -M/-M tion in Abca1 macrophages may be the result of an in- proinﬂ ammatory cytokine production in Abca1 creased amount of TLR4 on the plasma membrane or macrophages. increased TLR4 trafﬁ cking into lipid rafts. Our data sug- It is intriguing to speculate that an important physiolog- gested that total cellular and surface expression of TLR4 ical and antiinﬂ ammatory role of macrophage ABCA1 is to -M/-M was indistinguishable between WT and Abca1 mac- suppress activation of TLRs by endogenous (saturated rophages (supplementary Fig. V). Despite similar surface fatty acids) and exogenous (bacterial and viral products) expression of TLR4, there was greater expression of TLR4 stimuli by decreasing trafﬁ cking of TLRs into lipid rafts. 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Publisher: American Society for Biochemistry and Molecular Biology
Copyright: Copyright © 2010 Elsevier Inc.
ISSN: 0022-2275
eISSN: 1539-7262
DOI: 10.1194/jlr.m006486
Publisher site: See Article on Publisher Site

Abstract

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafﬁ cking to lipid rafts by reduction of lipid raft cholesterol Xuewei Zhu , * John S. Owen , Martha D. Wilson , * Haitao Li , ** Gary L. Grifﬁ ths , ** † § †† 1, , † Michael J. Thomas , Elizabeth M. Hiltbold , Michael B. Fessler , and John S. Parks * † § Departments of Pathology/Lipid Sciences,* Biochemistry, and Microbiology and Immunology, Wake Forest University School of Medicine , Winston-Salem, NC ; Imaging Probe Development Center,** National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, MD ; and Laboratory of †† Respiratory Biology, National Institute of Environmental Health Sciences , Research Triangle Park, NC Abstract We previously showed that macrophages from receptor trafﬁ cking to lipid rafts by reduction of lipid raft macrophage-speciﬁ c ATP-binding cassette transporter A1 cholesterol. J. Lipid Res . 2010. 51: 3196–3206. -M/-M (ABCA1) knockout ( Abca1 ) mice had an enhanced proin- ﬂ ammatory response to the Toll-like receptor (TLR) 4 ago- Supplementary key words free cholesterol � cytokines � immunology � nist, lipopolysaccharide (LPS), compared with wild-type lipid droplets � lymphocytes � retinoids (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft con- tent, and hyper-responsiveness of macrophages to LPS in ATP-binding cassette transporter A1 (ABCA1) is a plasma M/-M WT mice. Abca1 macrophages were also hyper-respon- membrane protein that functions to eliminate excess free sive to speciﬁ c agonists to TLR2, TLR7, and TLR9, but not cholesterol (FC) from tissues by efﬂ uxing cellular FC and TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR phospholipid (PL) to lipid-free apolipoprotein AI, forming agonists by modulation of lipid raft cholesterol and TLR nascent HDL particles ( 1, 2 ). Therefore, ABCA1 plays a -M/-M mobilization to lipid rafts. We demonstrated that Abca1 critical role in the movement of cholesterol from periph- vs. WT macrophages contained 23% more FC in isolated eral tissues to the liver in a process known as reverse choles- lipid rafts. Further, mass spectrometric analysis suggested terol transport. Mutations that inactivate the human ABCA1 raft phospholipid composition was unchanged. Although cell -M/-M gene result in Tangier disease, which is characterized by ex- surface expression of TLR4 was similar between Abca1 tremely low plasma HDL cholesterol concentrations, mildly and WT macrophages, signiﬁ cantly more TLR4 was distrib- -M/-M elevated plasma triglyceride levels, and accumulation of uted in membrane lipid rafts in Abca1 macrophages. -M/-M cholesterol in macrophages ( 3–5 ). ABCA1 protein is ex- Abca1 macrophages also exhibited increased trafﬁ cking of the predominantly intracellular TLR9 into lipid rafts in pressed to a variable extent in most cells in the body, and its response to TLR9-speciﬁ c agonist (CpG). Collectively, expression is regulated by transcriptional activation and our data suggest that macrophage ABCA1 dampens inﬂ am- protein degradation ( 6, 7 ), making it difﬁ cult to determine mation by reducing MyD88-dependent TLRs trafﬁ cking to lipid rafts by selective reduction of FC content in lipid rafts. —Zhu, X., J. S. Owen, M. D. Wilson, H. Li, G. L. Grifﬁ ths, M. J. Thomas, E. M. Hiltbold, M. B. Fessler, and J. S. Parks. +/+ +/ M Abbreviations: Abca1 , wild type mice at the Abca1 locus; Abca1 , Macrophage ABCA1 reduces MyD88-dependent Toll-like -M/-M heterozygous macrophage-speciﬁ c ABCA1 knockout; Abca1 , ho- mozygous macrophage-speciﬁ c knockout; Abca1 , total Abca1 knock- out; ABCG1, ATP-binding cassette transporter G1; BMDM, bone marrow-derived macrophage; CT-B, cholera toxin B; FC, free choles- This work was supported by National Institutes of Health Grants HL-49373, terol; fPEG-chol, ﬂ uorescein ester of polyethylene glycol-derivatized HL-094525, and AT-27820 (J.S.P.), American Heart Association fellowship 09POST2250225 (X.Z.), and the Intramural Research Program of the Na- cholesterol; GLC, gas-liquid chromatography; I B , inhibitory B pro- tional Institutes of Health, National Institute of Environmental Health Sciences tein ; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-acti- Z01 ES102005 (M.B.F.). The fPEG-cholesterol synthesis was supported by the vated protein kinase; M CD, methyl- -cyclodextrin; MyD88, myeloid National Institutes of Health Roadmap for Medical Research Initiative through differentiation primary-response protein 88; NF, nuclear factor; NutSP, its establishment of the Imaging Probe Development Center, administered by the Nutridoma SP; PI, phosphatidylinositol; PL, phospholipid; PM, perito- National Heart, Lung, and Blood Institute. Its contents are solely the respon- neal macrophage; PNS, postnuclear supernatant; TLR, Toll-like recep- sibility of the authors and do not necessarily represent the ofﬁ cial views of the tor; WT, wild type. National Institutes of Health. To whom correspondence should be addressed. Manuscript received 2 March 2010 and in revised form 21 July 2010. e-mail: [email protected] Published, JLR Papers in Press, July 21, 2010 The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M006486 contains supplementary data in the form of Data, Results, and six ﬁ gures. 3196 Journal of Lipid Research Volume 51, 2010 This article is available online at http://www.jlr.org This is an Open Access article under the CC BY license. its physiological role in individual tissues using global Abca1 However, several questions still remain unanswered. First, knockout mice or cells in culture. However, generation of is there a direct relationship among macrophage FC con- cell-speciﬁ c Abca1 knockout mice has helped deﬁ ne the tent, lipid raft content, and the hyper-responsiveness to role of tissue-speciﬁ c ABCA1 expression in whole body HDL TLR4 agonist? Alternatively, is there a change of PL com- biogenesis as well as several unanticipated roles for the position in lipid rafts that leads to increased lipid raft -M/-M transporter. For example, hepatocyte and intestinal epithe- content in Abca1 macrophages? Third, do other lial cell ABCA1 contribute 70–80% and 20–30% of the TLR-speciﬁ c agonists also induce increased inﬂ ammatory -M/-M plasma HDL pool, respectively ( 8, 9 ). Pancreatic cell signaling in Abca1 macrophages? Finally, does the ex- ABCA1 plays a role in insulin secretion ( 10 ) and brain aggerated TLR signaling result from increased trafﬁ cking of TLRs to lipid rafts? In the present study, we demon- ABCA1 regulates neuronal structure and function ( 11 ). Macrophages, a primary cell type involved in innate im- strated: 1 ) a direct association between FC and lipid raft munity, have been implicated in chronic inﬂ ammatory dis- content that leads to hyper-responsiveness of macrophages eases such as atherosclerosis and insulin resistance, where to LPS in WT macrophages; 2 ) ABCA1 deletion in mac- rophages resulted in selective FC accumulation in lipid they accumulate in arteries ( 12, 13 ) and adipose tissue ( 14 ), respectively. Transplantation of bone marrow between wild- rafts without alternation of PL composition and enhanced type (WT) and ABCA1 knockout mice has little effect on activation of other MyD88-dependent TLRs compared plasma HDL concentration ( 15, 16 ). Despite this, lipopoly- with WT macrophages; and 3 ) ABCA1 deletion results in increased TLR4 and TLR9 localization in lipid rafts in saccharide (LPS)-induced sepsis was exacerbated in Abca1 / / / both resting and stimulated states, suggesting a mechanis- Ldlr mice compared with Ldlr mice ( 17 ), suggesting a -M/-M novel antiinﬂ ammatory role for macrophage ABCA1 in in- tic explanation for the hyper-responsiveness of Abca1 nate immunity. However, it was not clear from these studies macrophages to proinﬂ ammatory stimuli. whether the exacerbated proinﬂ ammatory response of / / Abca1 /Ldlr mice to LPS was due to the massive cellular cholesteryl ester accumulation in macrophages, the absence MATERIALS AND METHODS of plasma HDL, or some other alteration of macrophages. In addition, ABCA1 expression decreases cellular plasma mem- Animals brane rigidity by reducing formation of tightly packed lipid +/+ +/ M -M/-M Abca1 (WT), Abca1 (heterozygous), and Abca1 (ho- raft domains in the plasma membrane ( 18 ). Lipid rafts, en- mozygous) mice were generated as described previously ( 24 ). riched in FC and glycosphingolipid, play an important role in Mice were backcrossed to C57BL/6 background for six genera- signal transduction by recruiting and concentrating signaling tions before use in the studies. molecules in the plasma membrane ( 19 ). For instance, LPS Cell culture activation of macrophage results in transient Toll-like recep- Peritoneal macrophages (PMs) were harvested from C57BL/6 tor 4 (TLR4) trafﬁ cking to lipid rafts along with its cognate +/+ -M/-M backcrossed Abca1 and Abca1 littermate mice 4 days after re- adaptor proteins ( 20–23 ) and subsequent secretion of in- ceiving an intraperitoneal injection of 1 ml 10% thioglycolate and ﬂ ammatory cytokines and chemokines. These studies suggest plated in RPMI media containing 1% Nutridoma SP (NutSP) media a link among ABCA1-mediated cellular lipid efﬂ ux, mem- (Roche Applied Science) as previously described ( 24 ). Bone mar- brane lipid raft homeostasis, and activation of macrophages. +/+ +/ M -M/-M row from Abca1 , Abca1 , and Abca1 littermate mice was iso- Nevertheless, the molecular details regarding how ABCA1 lated and cultured in DMEM media containing 20% FBS and 30% expression affects macrophage inﬂ ammatory response are L929 conditioned media for 5–7 days before being used in experi- poorly understood. ments as bone marrow-derived macrophages (BMDMs) ( 24 ). To explore the speciﬁ c role of ABCA1 in macrophages FC, lipid raft content, surface TLR4, and inﬂ ammatory in vivo and in vitro, we generated macrophage-speciﬁ c - cytokine expression in WT macrophages M/-M ABCA1 knockout ( Abca1 ) mice. Using this unique +/+ BMDMs from Abca1 mice were plated in tissue culture dishes mouse model, we demonstrated that macrophages from -M/-M and cultured in RPMI media plus 1% NutSp overnight as de- Abca1 mice have a signiﬁ cant increase in FC and are scribed above. Cholesterol depletion and overloading of mac- more proinﬂ ammatory in vivo and in vitro in response to rophages with methyl- -cyclodextrin (M CD) (Sigma-Aldrich) LPS via TLR4 compared with WT. This response was medi- was performed as described previously ( 24 ). Brieﬂ y, BMDMs were ated through a myeloid differentiation primary-response incubated with or without prewarmed 10 mM M CD at 37°C for protein 88 (MyD88)-dependent pathway and was indepen- 30 min to deplete cholesterol, or macrophages were incubated with M CD-cholesterol (40 or 80 g/ml) (Sigma-Aldrich) at dent of alterations in plasma lipid concentrations ( 24 ). We -M/-M 37°C for 60 min to overload macrophages with cholesterol. further showed that the hypersensitivity of Abca1 mac- After three washes with PBS, macrophages were extracted with rophages to LPS was most likely due to increased lipid raft isopropanol to measure cholesterol content by gas-liquid chro- content, presumably caused by increased intracellular FC matography (GLC) as described before ( 24 ) or incubated ± 100 accumulation. Recently, Yvan-Charvet et al. ( 25 ) observed ng/ml Kdo -lipid A (Avanti Polar Lipids) for 6 h before quantiﬁ - / / a similar inﬂ ammatory phenotype in Abca1 Abcg1 cation of cytokine expression by ELISA kits (BD Bioscience). macrophages compared with WT. Collectively, these stud- ies suggest that ABC transporters suppress macrophage Flow cytometry +/+ -M/-M TLR4 activation, possibly through modulation of mem- Elicited peritoneal cells from Abca1 or Abca1 mice were stained directly or plated in tissue culture dishes and cultured in brane lipid rafts. Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3197 RPMI media plus 1% NutSp overnight as described above. To NaF, 0.1 mM Na VO , 5 g/ml leupeptin, 5 g/ml aprotinin) by 3 4 measure TLR4/MD2 surface expression, elicited peritoneal cells 20 passages through a 22 G syringe needle. The nuclei and cell +/+ -M/-M from Abca1 or Abca1 mice were incubated in the presence debris were pelleted by centrifugation for 10 min at 1000 g . The or absence of 100 ng/ml LPS from Salmonella typhimurium (Sigma) PNS was collected and stored on ice. The pellet was resuspended for 1 h in suspension before staining with PE-F4/80 (BD Biosci- in 1 ml of Buffer B, homogenized, and centrifuged again. The ence) and/or APC-TLR4/MD2 (MTS510) (eBioscience) or iso- two PNSs (2 ml) were mixed with an equal volume (2 ml) of Solu- type control (eBioscience or BD Bioscience). To examine the tion D (50% Optiprep) and then centrifuged through 9 ml of content of lipid rafts or TLR4/MD2 complex in macrophages, 5–20% continuous Optiprep gradient using an SW41 rotor (90 after incubation with 10 mM M CD or 80 g/ml M CD- min, 53,000 g , 4°C). Following centrifugation, thirteen 1 ml frac- cholesterol for 30 min or 1 h, macrophages were gently lifted tions were harvested from top to bottom. using a cell scraper. Live PMs were then stained with 0.5 g/ml Detergent extraction method. Cold detergent resistance mem- cold Alexa ﬂ uor 488-cholera toxin B (CT-B) (Molecular Probes) branes (or lipid rafts) were prepared according to the method de- for 15 min at 4°C or stained with APC-TLR4/MD2 (MTS510) for scribed by Fessler et al. ( 29 ). Brieﬂ y, BMDMs were washed three 30 min at 4°C or PMs were ﬁ xed with 2% paraformaldehyde and times with ice-cold PBS. Following the wash, cells were centrifuged then stained with 10 g/ml fPEG -chol (ﬂ uorescein ester of (1,000 rpm, 10 min, 4°C) and resuspended in 1 ml of ice-cold lysis polyethylene glycol-derivatized cholesterol) ( 26 ) at room tem- buffer (25 mM MES, pH 6.5, 150 mM NaCl, 1% Triton X-100, perature for 10 min. Cell ﬂ uorescence was determined using a 1 mM PMSF, 1 mM NaF, 0.1 mM Na VO , 5 g/ml leupeptin, 5 g/ FACSCalibur ﬂ ow cytometer (BD Bioscience) and data were ana- 3 4 ml aprotinin). Cells were vortexed for 15 s on ice and allowed to sit lyzed using Flowjo software (Tree Star). on ice for 10 min. This step was repeated three times and then the cell lysate was centrifuged (1,000 g , 10 min, 4°C). The supernatant Inﬂ ammatory mediator expression in TLR ( 1 ml) was gently mixed with an equal volume of 80% sucrose in agonist-treated macrophages MBS (25 mM MES, pH 6.5, 150 mM NaCl) and then centrifuged After overnight incubation, PMs were switched to serum- through a 5–30% continuous sucrose gradient using an SW41 ro- free RPMI-1640 media for 2 h before TLR agonist stimulation. tor (20 h, 37,000 rpm, 4°C). Following centrifugation, twelve 1 ml BMDMs were incubated overnight in 1% NutSP media before fractions were harvested from top to bottom. stimulation with various TLR agonists. Macrophages were treated To induce translocation of TLRs to lipid rafts, BMDMs from both with 100 ng/ml Pam SCK (TLR2), 10 g/ml poly (I: C) (TLR3), 3 4 -M/-M WT and Abca1 mice were incubated in 1% NutSP-RPMI-1640 200 M Loxoribine (TLR7), or 1 M CpG (TLR9), or control media overnight and then treated with ±100 ng/ml LPS from Salmo- CpG (agonists from InvivoGen) in serum-free RPMI-1640 media nella typhimurium (Sigma-Aldrich) or ±1 M CpG for 1 h, followed by for 0–12 h, after which the culture supernatant was collected and preparation of lipid rafts and nonraft as described above. Mac- stored at 80°C for cytokine ELISA (BD Bioscience). rophage lipid raft TLR content was calculated as a percentage of total membrane TLR (i.e., lipid rafts + nonraft fractions). Preparation of subcellular membrane fractions BMDMs were used for all lipid raft isolation procedures to in- Immunoblotting and immunoprecipitation crease macrophage yield. The protein concentration in each iso- Western blots were performed using speciﬁ c antibodies against: lated fraction was determined by the Bradford protein assay caveolin-1 (Abcam), CD14 (Abcam) (as lipid raft marker), clathrin (Pierce). (Sigma-Aldrich) (as nonraft marker), TLR4 (cell signaling), or Nondetergent method. Lipid rafts were prepared according to TLR9 (Abcam). TLR9 was immunoprecipitated with a TLR9 poly- the method of Smart et al. ( 27 ). Brieﬂ y, BMDMs were scraped clonal antibody from pooled raft or nonraft fractions, followed by into ice-cold 0.25 M sucrose, 1 mM EDTA, and 20 mM Tris (pH immunoblotting with antibody against TLR9. Blots were devel- 7.8) (Buffer A) with protease inhibitors (1 mM PMSF, 1 mM NaF, oped using HRP-linked secondary antibody. Immunoblots were 0.1 mM Na VO , 5 g/ml leupeptin, 5 g/ml aprotinin) and ho- 3 4 visualized with the Supersignal substrate system (Pierce). Images mogenized in 1 ml cold buffer A by 20 passages through a 22 G were captured and quantiﬁ ed using the LSA-3000 imaging system syringe needle. The nuclei and cell debris were pelleted by cen- and Multi Gauge software (Fujiﬁ lm Life Science). trifugation for 10 min at 1,000 g . The postnuclear supernatant (PNS) was collected and stored on ice. The pellet was resus- Analysis of nuclear factor- B pathway in the pended in Buffer A, homogenized, and centrifuged again. The CpG-treated macrophages two PNSs (2 ml) were combined and layered on top of 23 ml of -M/-M BMDMs from both WT and Abca1 mice were incubated in 1% 30% Percoll (Sigma-Aldrich) in Buffer A and centrifuged for 30 NutSP-RPMI-1640 media overnight before treated ± 1 M CpG for min at 84,000 g . The plasma membrane fraction was collected 0–3 h, after which cells were processed for Western-blot analysis. and sonicated on ice for a total of six bursts (50 J each time). The Antibody to inhibitory B protein (I B ) was purchased from Cell sonicated membrane fraction was made up to 23% Optiprep Signaling Technology, Inc. and antibody to -actin from Sigma- (Sigma-Aldrich) (ﬁ nal volume: 4 ml) by the addition of a 50% Aldrich. Immunoblots were visualized as described above. Optiprep stock in 20 mM Tris, 0.25 M sucrose, and 1 mM EDTA (pH 7.8) (Buffer C) and placed in the bottom of a centrifuge FC measurements tube. An 8 ml gradient from 20% to 10% Optiprep was layered Raft and nonraft fractions isolated using Smart’s nondetergent on top of the sample, which was then centrifuged for 90 min at method were extracted using the Bligh-Dyer method ( 30 ), and cho- 52,000 g in an SW41 swinging bucket rotor. The gradient was lesterol content was determined by GLC as previously described then fractioned into 12 or 13 1 ml fractions from top to bottom. ( 24 ). Simpliﬁ ed nondetergent method. Lipid rafts were prepared ac- Lipidomic assay cording to the method of MacDonald et al. ( 28 ). Brieﬂ y, BMDMs were scraped into ice-cold 0.25 M sucrose, 1 mM CaCl , 1 mM The distribution of PL species in pooled raft or nonraft frac- MgCl , 20 mM Tris-HCl, pH 7.8 (Buffer B), and homogenized in tions or whole cells was measured using ESI/MS/MS as described 1 ml cold buffer B with protease inhibitors (1 mM PMSF, 1 mM in the supplementary data. 3198 Journal of Lipid Research Volume 51, 2010 Statistics ment, macrophages subjected to cholesterol depletion secreted less interleukin (IL)-6 and IL-12p40, whereas Differences were compared with two-tailed Student’s t -test or one-way ANOVA using GraphPad Prism software. P < 0.05 was macrophages overloaded with cholesterol secreted more considered statistically signiﬁ cant. Data are presented as the of these cytokines, suggesting that cytokine secretion in means ± SEM unless indicated otherwise. TLR4 activated macrophages has a signiﬁ cant positive as- sociation with cellular FC content ( Fig. 1B ). To determine whether rapid alteration of FC content changes lipid raft content or TLR4 surface expression, we measured lipid RESULTS raft content of macrophages either depleted of or over- Macrophage FC content is positively associated with lipid loaded with cholesterol by ﬂ ow cytometry. We used two raft content and proinﬂ ammatory response types of raft markers: fPEG-chol ( 26, 31, 32 ), which parti- To directly examine the effect of FC on macrophage tions into membrane raft microdomains in direct relation proinﬂ ammatory activation, we incubated WT mac- to their cholesterol content, and Alexa ﬂ uor 488-CT-B, rophages with 10 mM M CD (cholesterol depletion) or which binds to ganglioside GM1 located in lipid rafts. As M CD loaded with 40 or 80 g/ml of cholesterol (choles- shown in Fig. 1C , cellular ﬂ uorescent intensity of fPEG- terol overloading) before cells were challenged with Kdo2- chol increased with increasing macrophage FC content. lipid A, a speciﬁ c TLR4 agonist. Figure 1A shows that Incubation of cells with M CD also resulted in decreased incubating macrophages with M CD or M CD-cholesterol CT-B binding, presumably due to the disruption of lipid resulted in a 2-fold variation in cellular FC content. Cho- raft structure by M CD, but unlike fPEG-chol, cholesterol lesteryl ester content in macrophages was negligible with overloading did not result in an increase in CT-B binding all treatments (data not shown). Compared with no treat- on the cell surface, suggesting a rapid increase of FC on Fig. 1. Manipulation of cellular FC content alters proinﬂ ammatory status and cell surface lipid raft, but not TLR4, content. A: WT BMDMs were incubated at 37°C with M CD for 0.5 h or with M CD-chol for 1 h before FC measurement by GLC. B: WT BMDMs were incubated with Kdo2-lipid A for 6 h after in- cubation with M CD or M CD-chol, and cytokine expression was measured by ELISA. Cytokine con- centration was plotted against FC content of mac- rophages. The line of best ﬁ t for IL-6 ( r = 0.92) and IL-12p40 ( r = 0.75) is shown. C–E: WT-elicited PMs were treated with M CD or M CD-chol before stained with fPEG-chol (C), Alexa ﬂ uor CT-B (D), or APC-TLR4/MD2 (MTS510) (E). Cells were analyzed using ﬂ ow cytometry. Histogram of individual sam- ples is shown in the left panel and mean ﬂ uorescence intensity is shown in the right panel. Values with unlike letters are signiﬁ cantly different ( P < 0.05). Results represent mean ± SEM of 2 independent experiments; n = 4–5 dishes of cells/group. Chol: M CD-cholesterol. Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3199 -M/-M the cell surface does not alter the distribution of ganglio- Abca1 macrophages are hypersensitive to stimulation by various TLR agonists side GM1 ( Fig. 1D ). Furthermore, disrupting lipid rafts us- ing M CD resulted in a slight decrease of TLR4 surface In addition to TLR4, we also investigated the effect of expression compared with control, but cholesterol over- macrophage ABCA1 deﬁ ciency on the activation of other loading did not signiﬁ cantly change the TLR4 surface ex- TLRs. PMs were treated with speciﬁ c TLR agonists Pam SCK 3 4 pression ( Fig. 1E ). (TLR2), poly (I:C) (TLR3), Loxoribine (TLR7), or CpG -M/-M Fig. 2. Abca1 macrophages are hypersensitive to TLR2, 7, and 9, but not to TLR3 stimulation. A–C: Elicited PMs were treated with SCK (TLR2), Loxoribine (TLR7), or TLR9 for 0–12 h. The secretion of inﬂ ammatory factors was measured by ELISA. D, E: PMs were Pam 3 4 treated with poly (I:C) (TLR3) for 0–12 h. Cytokine or chemokine secretion was measured by ELISA. Results represent mean ± SEM of at +/+ -M/-M : WT; -M/-M: Abca1 . least two independent experiments; n = 3 dishes of cells/group. * P <0.05, compared with WT. 3200 Journal of Lipid Research Volume 51, 2010 (TLR9) for 3–12 h and cytokine secretion from the stim- ing raft (caveolin-1 and CD14) and nonraft (clathrin) ulated cells was measured by ELISA. Compared with markers, we designated the top eight fractions (nos. 1–7) -M/-M WT, Abca1 macrophages secreted more IL-6, IL-12p40, as lipid rafts and the bottom fractions (nos. 9–12) as non- -M/-M and RANTES over the time course after stimulation with rafts ( Fig. 4A ). Interestingly, we found that in Abca1 Pam SCK , Loxoribine, or CpG ( Fig. 2A –C ). A similar pat- cells, each lipid raft fraction contained more FC ( Fig. 4B ), 3 4 tern was observed for TNF- secretion (data not shown). resulting in an average 23.01 ± 2.12% greater FC content However, TLR3 stimulation by poly (I:C) resulted in for the whole lipid raft fraction (i.e., fractions 1–7) com- similar secretion of inﬂ ammatory factors from WT and pared with the same fractions from WT cells ( Fig. 4C ). On -M/-M Abca1 macrophages ( Fig. 2D, E). Similar results were the other hand, FC content was similar in nonraft fractions -M/-M obtained with BMDMs (data not shown). TLR3 is the only of WT and Abca1 cells ( Fig. 4B, C ) . Taken together, member of the TLR family whose signal transduction is our data suggested that ABCA1 deﬁ ciency results in an in- independent of MyD88. The data in Fig. 2 suggest that crease in lipid raft content, likely due to a striking increase ABCA1 deﬁ ciency mainly affects TLR pathways that signal in FC content of this fraction. We also tested an alternative through MyD88. hypothesis that sphingomyelin (SM) content in lipid rafts may be increased in the absence of ABCA1, resulting in -M/-M Lipid rafts from Abca1 macrophages are speciﬁ cally increased lipid raft content (supplementary Figs. II–IV). enriched in FC However, SM content and PL fatty acyl molecular species To determine whether the increase in lipid rafts previ- in raft and nonraft fractions were similar among the three -M/-M ously described in Abca1 macrophages ( 24 ) is the di- genotypes of mice. rect result of increased FC accumulation in lipid rafts, we performed two experiments. First, we examined the inten- Increased lipid raft TLR4 content in -M/-M Abca1 macrophages sity of FC-enriched microdomains by staining macrophages with fPEG-chol. As shown in Fig. 3A , D, compared with TLR4 signaling is initiated by ligand binding and tran- -M/-M WT, Abca1 macrophages had a slight but signiﬁ cantly sient trafﬁ cking of TLR4 to lipid rafts after macrophages brighter staining of fPEG-chol, indicating more FC- are activated ( 20–23 ). Our data suggested that total cellu- enriched microdomains located on the cell surface. Further- lar ( 24 ) and surface expression of TLR4 were indistin- -M/-M more, cholesterol depletion using M CD normalized the guishable between WT and Abca1 macrophages difference in fPEG-chol staining between the two geno- (supplementary Fig. V; see more details in supplementary types ( Fig. 3B , D), whereas the difference of fPEG-chol Results). To investigate whether ABCA1 deﬁ ciency results staining was maintained when cells were overloaded with in enhanced TLR4 recruitment to lipid rafts, we isolated cholesterol ( Fig. 3C, D). To determine whether the in- rafts and nonrafts from BMDMs ± 100 ng/ml LPS and im- -M/-M creased FC in Abca1 macrophages is located exclusively munoblotted for TLR4. In resting macrophages, the ma- in lipid rafts, we isolated lipid rafts and nonrafts from mac- jority of TLR4 was observed in nonraft fractions ( Fig. 5A ). rophages using a nondetergent isolation method ( 27 ). Us- However, a portion of TLR4 was also detected in raft frac- Fig. 3. ABCA1 deﬁ ciency in macrophages resulted in increased membrane lipid raft content. Elicited PMs were incubated with or without M CD for 0.5 h or M CD-chol (80 µg/ml) for 1 h before staining with fPEG-chol. Cell ﬂ uorescence was analyzed using ﬂ ow cytometry. A–C : Histograms show macrophage fPEG-chol staining in the absence (A) or presence (B) of M CD or presence of M CD-chol (C), respec- tively. D: Data from panels A–C are presented as mean ± SEM. Statistical signiﬁ cant differences be- tween groups are indicated (*, P < 0.05; **, P < 0.01). Results are representative of two independent ex- +/+ periments with at least three mice per group. : -M/-M WT; -M/-M: Abca1 . Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3201 Nuclear factor- B pathway and lipid rafts are involved with the enhanced TLR9 signaling in -M/-M Abca1 macrophages TLR7 or TLR9 was shown to translocate from ER to the endosome/lysosome upon stimulation by speciﬁ c agonists, resulting in activation of the nuclear factor (NF)- B and mitogen-activated protein kinase (MAPK) pathways ( 33 ). To examine whether the NF- B pathway is upregulated in -M/-M TLR9 agonist (CpG)-treated Abca1 macrophages, we -M/-M stimulated WT and Abca1 BMDMs with 1 M CpG for 0–3 h before isolating cell lysates for immunoblotting. As shown in Fig. 6A , within 30 min in both genotypes of mac- rophages, CpG induced I B degradation, which is indic- ative of NF B activation. Interestingly, compared with WT, -M/-M Abca1 BMDMs showed a greater and prolonged I B degradation during 3 h of TLR9 stimulation. Because CpG stimulation was shown not only to induce translocation of TLR9 to endosomes but also to induce trafﬁ cking of TLR9 to plasma membrane lipid rafts ( 20, 34, 35 ), we next tested the hypothesis that CpG may induce more TLR9 transloca- -M/-M tion to lipid rafts in Abca1 versus WT macrophages. To test this hypothesis, we isolated lipid rafts and nonrafts -M/-M from WT and Abca1 BMDMs using a simpliﬁ ed nonde- tergent isolation method ( 28 ). The majority of TLR9 was Fig. 4. Macrophage ABCA1 deﬁ ciency results in an increase in located in nonraft fractions in resting BMDMs ( Fig. 6B ). lipid raft FC. A: Lipid raft and nonraft fractions were isolated using Next, we pooled raft (fractions 1–8) and nonraft (fractions a nondetergent method. Each fraction was analyzed by Western 10–13) fractions isolated from BMDMs ± CpG treatment blotting for the indicated proteins. B: FC and protein concentra- and performed Western-blot analysis ( Fig. 6C ) or immu- tions in each fraction in panel A were measured and the results noprecipitation ( Fig. 6D ) to visualize and quantify the traf- were plotted as µg cholesterol/mg protein. Mean ± range (n = 2). Results are representative of two independent experiments. C: ﬁ cking of TLR9 to lipid rafts. As shown in Fig. 6C–E , we Data from panel B are presented as raft (fractions 1–7) and nonraft observed a trend of increased basal lipid raft TLR9 con- -M/-M (fractions 9–12) cholesterol content for each genotype. * P < 0.05, tent in resting Abca1 cells ( P = 0.08). More interest- +/+ -M/-M compared with WT. : WT; -M/-M: Abca1 . ingly, CpG stimulation signiﬁ cantly triggered increased translocation of TLR9 from nonraft to lipid raft fractions -M/-M tions, colocalized with CD14. Interestingly, TLR4 was more in Abca1 vs. WT BMDMs ( Fig. 6C–E ). These combined diffusely distributed in the lipid raft fractions (fractions results suggest that the increased lipid raft content in -M/-M -M/-M Abca1 cells increases MyD88-dependent activation of 1–7) of Abca1 BMDMs ( Fig. 5B ) compared with that in plasma membrane and intracellular TLRs through aug- WT cells ( Fig. 5A ). To visualize the trafﬁ cking of TLR4 induced by LPS stimulation, we pooled raft and nonraft mented translocation or trafﬁ cking to lipid rafts, resulting fractions isolated from BMDMs ± LPS treatment and per- in hyper-activation of downstream signaling transduction formed Western-blot analysis. Lipid raft and nonraft frac- and exaggerated proinﬂ ammatory cytokine expression. tions were identiﬁ ed based on the distribution of raft markers (caveolin-1, CD14, and ﬂ otillin-1) and the non- DISCUSSION raft marker, clathrin ( Fig. 5C ). The majority of TLR4 was The well-known function of ABCA1 is to transport FC distributed in the nonraft membrane fraction in both WT -M/-M and PLs out of cells, protecting cells from excessive choles- and Abca1 BMDMs. However, we observed a signiﬁ - cantly higher TLR4 content in lipid rafts from resting (i.e., terol accumulation. Interestingly, studies from our labora- -M/-M nontreated) Abca1 BMDMs compared with WT cells tory and others suggest an additional antiinﬂ ammatory ( Fig. 5C, D). Upon LPS stimulation, a trend toward in- role of macrophage ABCA1 ( 17, 24, 25, 36 ), but the mech- anism still remains unclear. The present study provides creased translocation of TLR4 into lipid rafts was observed -M/-M in Abca1 relative to WT macrophages ( Fig. 5C, D). As direct evidence showing that ABCA1 deﬁ ciency in mac- a control, another lipid raft protein, caveolin-1, was exam- rophages leads to selective FC accumulation in lipid rafts ined. Unlike TLR4, caveolin-1 content in lipid rafts was without alteration of PL composition. ABCA1 deﬁ ciency unaffected by LPS treatment of macrophages or by the in macrophages also results in more TLR content in lipid genotype of macrophages (supplementary Fig. VI). Col- rafts, consequently leading to enhanced TLR activation. lectively, these data suggest that ABCA1 deﬁ ciency in mac- To our knowledge, this is the ﬁ rst study to directly show rophages results in a signiﬁ cant increase in FC-enriched that ABC transporters downregulate TLR (TLR2, 4, 7, and lipid rafts, which concentrate and recruit more TLR4 in 9) signaling by reducing FC enrichment in lipid rafts and resting and potentially in LPS-activated macrophages. trafﬁ cking of TLRs into rafts. 3202 Journal of Lipid Research Volume 51, 2010 -M/-M Fig. 5. Increased TLR4 in lipid rafts of Abca1 macrophages. A and B: Lipid raft and nonraft mem- brane regions were isolated using a nondetergent method and analyzed for TLR4, clathrin (non-raft marker), and CD14 (raft marker) distribution by Western blotting. C: BMDMs were treated with 100 ng/ml LPS for 1 h and then lipid raft (fractions 1–8) and nonraft (fractions 10–13) fractions were pooled, and the distribution of the indicated proteins was analyzed by Western blotting. Results are representa- tive of two independent experiments. D: TLR4 con- tent of macrophage lipid rafts was calculated as a percentage of total membrane TLR4 (lipid rafts + nonrafts). Data are presented as mean ± range (n = 2). Statistically signiﬁ cant differences between +/+ genotypes are indicated (* P < 0.05). : WT, -M/-M: M/-M Abca1 . Lipid rafts are liquid-ordered membrane microdomains plasma membrane ﬂ uidity but not sufﬁ cient to activate enriched in FC and SM ( 19 ). ABCA1 expression results in TLR signaling without speciﬁ c agonist stimulation. redistribution of SM and FC from lipid raft to nonraft frac- Of the 13 members in the TLR family discovered so far, tions in baby hamster kidney cells ( 18 ). Our previous study TLR2 and TLR4 on the cell surface recognize bacterial suggested that ABCA1 deﬁ ciency in macrophages was as- lipoteichoic acid and LPS, respectively, whereas TLR3, 7, sociated with an increase in lipid rafts, presumably due to and 9, located in intracellular compartments, detect nu- the increase in FC accumulation ( 24 ). To test an alterna- cleic acids derived from pathogens ( 33 ). Beside their criti- -M/-M tive hypothesis that the increase in lipid rafts in Abca1 cal role in innate immunity, TLRs are also involved in the macrophages was the result of increased SM enrichment, development of chronic inﬂ ammatory diseases, such as we analyzed the PL proﬁ le of lipid rafts from macrophages atherosclerosis ( 38, 39 ) and insulin resistance ( 40 ). In ad- using MS. Our data suggested that the relative distribution dition, TLR9 was shown to be involved in macrophage of SM and phosphatidylcholine fatty acyl species in lipid foam cell formation ( 41 ). We previously showed that -M/-M rafts was similar for WT and Abca1 macrophages (sup- ABCA1 deﬁ ciency resulted in an enhanced MyD88-depen- M/-M plementary Fig. IV). Thus, the hypersensitivity of Abca1 dent TLR4 signal ( 24 ). Here, we further demonstrated macrophages to TLR activation was not due to alterations that ABCA1 deﬁ ciency also resulted in enhanced activa- in SM or phosphatidylcholine content of lipid rafts. tion of TLR2, 7, and 9, but not TLR3. Despite having in -M/-M On the other hand, Abca1 macrophages contained common an intracellular localization, TLR7 and TLR9 more FC-enriched lipid rafts, demonstrated by fPEG-chol employ MyD88, whereas TLR3 recruits TRIF to propagate staining and cholesterol quantiﬁ cation in isolated lipid downstream signal transduction. The mechanism by which rafts. ABCA1 deﬁ ciency favors MyD88-dependent TLR pathways Excessive FC in late endosomes or in the plasma mem- remains unknown. / / brane activates the p38 MAPK pathway through TLR3 or WT and Abca1 Abcg1 macrophages had similar TLR4, respectively, without speciﬁ c agonist stimulation cytokine gene expression when cells were treated with ( 37 ), indicating that FC overloading alone is sufﬁ cient to TLR2, TLR7, or TLR9 agonists ( 25 ). In contrast, when / / activate TLRs in certain situations. In our study, when FC cholesterol loaded using M CD-chol, Abca1 Abcg1 content of WT macrophages was varied over a 2-fold range, macrophages were hyper-sensitive to TLR4 stimulation / / we observed a signiﬁ cant positive association among mac- compared with WT cells. In addition, Abca1 Abcg1 rophage FC content, lipid raft content, and the inﬂ amma- macrophages were also hyper-responsive to TLR3 stimula- tory response to TLR4 agonist ( Fig. 1 ). However, in tion with or without cholesterol loading ( 25 ). The differ- M/-M Abca1 macrophages, FC content was only slightly in- ence between our study and the study of Yvan-Charvet / / creased ( 10%) compared with WT cells, and no signiﬁ - et al. ( 25 ) is that Abca1 Abcg1 macrophages (without cant difference in the activation of NF- B and MAPK cholesterol overloading) were hyper-responsive to TLR3 pathways or inﬂ ammatory gene expression was observed and TLR4, but not to TLR2, 7, or 9 stimulation, whereas -M/-M -M/-M between WT and Abca1 macrophages in the absence of our Abca1 macrophages were hyper-responsive to speciﬁ c ligand stimulation ( 24, 25 ), suggesting that a sub- TLR2, 4, 7, or 9 stimulation but not to TLR3 activation. / / tle increase in FC content alone is sufﬁ cient to alter the Notably, Abcg1 Ldlr macrophages had a 60% Macrophage ABCA1, lipid rafts, and TLR trafﬁ cking 3203 Fig. 6. NF- B pathway and lipid rafts are involved -M/-M with enhanced TLR9 signaling in Abca1 mac- rophages. A: BMDMs were incubated with 1 M CpG for 0–3 h. Cell lysates were harvested and the pro- teins were analyzed by immunoblotting with the indi- cated antibodies. I B was quantiﬁ ed and normalized to -actin. The degradation of I B induced by CpG stimulation is presented as fraction of basal protein level in control WT cells. B: BMDMs were fractioned into raft and nonraft fractions using a simpliﬁ ed non- detergent method. The protein distribution in each fraction was analyzed by Western blotting using the indicated antibodies. C: BMDMs were treated with 1 M CpG for 1 h and then lipid rafts (fractions 1–8) and nonrafts (fractions 10–13) were pooled, and the distribution of the indicated proteins was analyzed by Western blotting. D: Lipid raft and nonraft fractions isolated as described in panel C were pooled and im- munoprecipitated with anti-TLR9 polyclonal anti- body, followed by immunoblotting with anti-TLR9 polyclonal antibody. Results are representative of two independent experiments. E: Lipid raft TLR9 con- tent in macrophages was calculated as a percentage of total TLR9 (lipid rafts + nonrafts). Data are pre- sented as mean ± SEM; n = 3. Statistically signiﬁ cant differences between groups are indicated (* P < 0.05). +/+ -M/-M : WT, -M/-M: Abca1 . increase in FC compared with Ldlr cells ( 25 ). Although and potentially after LPS stimulation compared with WT macrophages ( Fig. 5 ). Evidence suggests that recognition Yvan-Charvet et al. ( 25 ) did not show evidence of FC en- / / of CpG by TLR9 is likely to occur not only in endosomes, richment in endosomes, the excess FC in Abca1 Abcg1 macrophages might accumulate in intracellular compart- but also in plasma membrane lipid rafts ( 20, 34 ). By con- ments, resulting in activation of TLR3 ( 37 ). In our study, trast, TLR3 activation does not occur in the rafts ( 43, 44 ). -M/-M FC content in Abca1 macrophages was only increased With these ﬁ ndings in mind, we hypothesized that the in- -M/-M creased lipid raft content in Abca1 macrophages might 10% compared with WT macrophages, and the FC enrich- ment was selective for lipid rafts. Therefore, the difference also result in enhanced TLR9 trafﬁ cking into rafts and our / / in FC content between Abca1 Abcg1 macrophages results supported this hypothesis ( Fig. 6 ). Because TLR2 -M/-M and Abca1 cells as well as the differential compartmen- and TLR4 (cell surface receptors) or TLR7 and TLR9 (in- tracellular receptors) share many properties and similar tal enrichment may account for the different responsive- ness to TLRs stimulation. activation mechanisms, respectively ( 22, 45, 46 ), we as- TLR2 or TLR4 signaling is thought to be initiated after sume that ABCA1 deﬁ ciency in macrophages results in TLRs rapidly trafﬁ c to lipid rafts in response to speciﬁ c li- increased translocation or recruitment of all MyD88- gands or saturated fatty acids ( 22, 23, 42 ). Based on these dependent TLRs ( 2, 4, 7 and 9 ) into lipid rafts, which leads ﬁ ndings, we hypothesized that the enhanced TLR4 activa- to enhanced downstream signal transduction and augmented -M/-M -M/-M tion in Abca1 macrophages may be the result of an in- proinﬂ ammatory cytokine production in Abca1 creased amount of TLR4 on the plasma membrane or macrophages. increased TLR4 trafﬁ cking into lipid rafts. Our data sug- It is intriguing to speculate that an important physiolog- gested that total cellular and surface expression of TLR4 ical and antiinﬂ ammatory role of macrophage ABCA1 is to -M/-M was indistinguishable between WT and Abca1 mac- suppress activation of TLRs by endogenous (saturated rophages (supplementary Fig. V). Despite similar surface fatty acids) and exogenous (bacterial and viral products) expression of TLR4, there was greater expression of TLR4 stimuli by decreasing trafﬁ cking of TLRs into lipid rafts. 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Journal

Journal of Lipid Research – American Society for Biochemistry and Molecular Biology

Published: Nov 1, 2010

Keywords: free cholesterol; cytokines; immunology; lipid droplets; lymphocytes; retinoids

References

ABCA1. The gatekeeper for eliminating excess tissue cholesterol

R.M. Lawn
Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis

M.R. Hayden
The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease

et al.
Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency

et al.
Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1

et al.
ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease

J.W. Heinecke
A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I

A.R. Tall
Intestinal ABCA1 directly contributes to HDL biogenesis in vivo

et al.
Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I

et al.
Beta-cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment

et al.
Specific loss of brain ABCA1 increases brain cholesterol uptake and influences neuronal structure and function

et al.
Atherosclerosis. the road ahead

J.L. Witztum
Atherosclerosis

A.J. Lusis
Obesity is associated with macrophage accumulation in adipose tissue

A.W. Ferrante
Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels

R.J. Aiello
Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues

et al.
Increased cholesterol deposition, expression of scavenger receptors, and response to chemotactic factors in Abca1-deficient macrophages

R.J. Aiello
ATP-binding cassette transporter A1 expression disrupts raft membrane microdomains through its ATPase-related functions

X. Zha
Lipid rafts and signal transduction

D. Toomre
Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts

et al.
Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages

O.D. Rotstein
Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation

K. Triantafilou
Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane

K. Triantafilou
Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages

et al.
Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions

A.R. Tall
Dyslipidemia induces opposing effects on intrapulmonary and extrapulmonary host defense through divergent TLR response phenotypes

M.B. Fessler
A detergent-free method for purifying caveolae membrane from tissue culture cells

R.G. Anderson
A simplified method for the preparation of detergent-free lipid rafts

L.J. Pike
Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil

G.S. Worthen
A rapid method of total lipid extraction and purification

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Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol [S]

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol [S]

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol [S]

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol [S]

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