IL-33 Reduces Foam Cell Formation James E. McLaren, Daryn R. Michael, Rebecca C. Salter, Tim G. Ashlin, Claudia J. Calder, Ashley M. Miller, Foo Y. This information is current as Liew and Dipak P. Ramji of September 28, 2021. J Immunol 2010; 185:1222-1229; Prepublished online 11 June 2010; doi: 10.4049/jimmunol.1000520

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-33 Reduces Macrophage Foam Cell Formation

James E. McLaren,* Daryn R. Michael,* Rebecca C. Salter,* Tim G. Ashlin,* Claudia J. Calder,† Ashley M. Miller,‡ Foo Y. Liew,‡ and Dipak P. Ramji*

The development of , a chronic inflammatory disease characterized by the formation of arterial fibrotic plaques, has been shown to be reduced by IL-33 in vivo. However, whether IL-33 can directly affect macrophage foam cell formation, a key feature of atherosclerotic plaques, has not been determined. In this study, we investigated whether IL-33 reduces macrophage foam cell accumulation in vivo and if IL-33 reduces their formation in vitro using THP-1 and primary human -derived . In Apolipoprotein E2/2 mice fed on a high diet, IL-33 treatment significantly reduced the accumulation of macrophage-derived foam cells in atherosclerotic plaques. IL-33 also reduced macrophage foam cell formation in vitro by decreasing acetylated and oxidized low-density uptake, reducing intracellular total and esterified content and enhancing cholesterol efflux. These changes were associated with IL-33–mediated reduction in the expression of genes involved in modified low-density lipoprotein uptake, such as CD36, and simultaneous increase in genes involved in Downloaded from cholesterol efflux, including , thereby providing a mechanism for such an action for this cytokine. IL-33 also decreased the expression of key genes implicated in cholesterol esterification and triglyceride storage, including Acyl-CoA: cholesterol acyltransferase 1 and Adipocyte differentiation-related protein. Furthermore, using bone marrow-derived macro- phages from ST22/2 mice, we demonstrate that the IL-33 receptor, ST2, is integral to the action of IL-33 on macrophage foam cell formation. In conclusion, IL-33 has a protective role in atherosclerosis by reducing macrophage foam cell formation suggesting that IL-33 maybe a potential therapeutic agent against atherosclerosis. The Journal of Immunology, 2010, 185: http://www.jimmunol.org/ 1222–1229.

recently discovered member of the IL-1 cytokine family switch and by stimulating the production of IL-5 and oxidized is IL-33 (IL-33, IL-1F11), which signals through ST2, the low-density lipoprotein (OxLDL)-specific Abs (7). The IL-33 de- A IL-1–receptor superfamily member (1). The ST2 gene coy receptor, sST2, reversed the atheroprotective actions of IL-33 encodes two full length protein isoforms: the membrane-bound and even exacerbated the disease in vivo (7) and sST2 serum ST2L and a soluble form, sST2, which acts as a decoy receptor levels were markedly increased after (8). for IL-33. IL-33 induces the production of the Th2 cytokines, Atherosclerosis is a progressive, Th1-driven chronic inflamma- by guest on September 28, 2021 IL-4 and IL-13, and enhances serum Ig synthesis (1). Consistent tory disease of the vasculature characterized by the formation of with these observations, IL-33 is a promoter of Th2-dependent fibrotic plaques in the major (9, 10). The chronic inflam- inflammatory diseases (2, 3). IL-33 activates a number of cell mation characteristic of atherosclerosis is believed to be due to types, including Th2 cells, mast cells and basophils (reviewed in the persistent recruitment of immune cells, including Refs. 2, 3), and mediates anaphylactic shock (4) and promotes and CD4+ Th1 T lymphocytes, to atherosclerotic lesions. Macro- systemic sclerosis (5). Conversely, IL-33 has been found to be phages, which differentiate from infiltrating monocytes in response cardioprotective through ST2 (6) and can inhibit the development to endothelial cell released M-CSF, are capable of ingesting mod- of atherosclerosis in vivo by inducing a phenotypical Th1-to-Th2 ified forms of LDL, such as OxLDL, which accumulate in the subendothelial compartment to form cholesterol-loaded foam cells (9–11). Macrophage-derived foam cells form the foundation of *Cardiff School of Biosciences; †Department of Infection, Immunity, and Biochem- atherosclerotic plaques (9, 10) and their formation is sensitive to istry, School of Medicine, Cardiff University, Cardiff; and ‡Division of Immunology, Infection, and Inflammation, Glasgow Biomedical Research Centre, University of cytokines, like IFN-g (11). Glasgow, Glasgow, United Kingdom In this report, we demonstrate for the first time that IL-33 mark- Received for publication February 15, 2010. Accepted for publication May 6, 2010. edly reduces foam cell accumulation in the atherosclerotic plaques 2/2 This work was supported by British Heart Foundation Grant PG/07/031/22716 (to of Apolipoprotein E (ApoE) mice. In addition, we show that D.P.R) and by British Heart Foundation Intermediate Basic Science Research Fel- IL-33 significantly reduces macrophage foam cell formation lowship FS/08/035/25309 (to A.M.M.). in vitro in THP-1 macrophages and primary human monocyte- Address correspondence and reprint requests to Dr. Dipak P. Ramji, Cardiff School of derived macrophages (HMDMs) by decreasing acetylated and ox- Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, U.K. E-mail address: [email protected] idized LDL uptake, reducing intracellular total and esterified cho- The online version of this article contains supplemental material. lesterol content and by enhancing cholesterol efflux. Furthermore, we delineate a potential mechanism for the action of IL-33 in- Abbreviations used in this paper: ABCA-1, ATP-binding cassette transporter A-1; ABCG-1, ATP-binding cassette transporter G-1; ACAT-1, Acyl-CoA:cholesterol acyl- volving decreased expression of key genes involved in modified transferase-1; AcLDL, acetylated LDL; ADRP,adipocyte differentiation-related protein; LDL uptake, cholesterol esterification and triglyceride storage and Apo, Apolipoprotein; BMM, bone marrow-derived macrophage; CPT-1, carnitine palmitoyltransferase-1; DiI, 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyane per- simultaneously increased expression of those involved in the traf- chlorate; HMDM, human monocyte-derived macrophage; LDL, low-density lipoprotein; ficking and efflux of cholesterol. Finally, using bone-marrow de- NCEH, neutral cholesteryl ester hydrolase; NPC, Niemann Pick type C; OxLDL, oxi- rived macrophages (BMMs) from ST22/2 mice, we demonstrate dized LDL; SR,scavenger receptor; VSMC, cell; WT,wild-type. that the IL-33 receptor, ST2, is integral to the action of IL-33 on Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 macrophage foam cell formation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000520 The Journal of Immunology 1223

Materials and Methods ABCA-1, and ABCG-1. Quantitative PCR was performed using the DNA Reagents and cell culture Engine Opticon 2 real-time PCR detection system (MJ Research, Cam- bridge, MA) and transcript levels were determined using the comparative Human THP-1 and mouse BMM cell cultures were maintained in complete Ct method and normalized to GAPDH (human) and b-actin (mouse) tran- RPMI 1640 or DMEM medium, respectively, supplemented with 10% (v/v) script levels. Human GAPDH and mouse b-actin were used as normalizing heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 mg/ml), and genes because they displayed stable expression levels in all samples. All L-glutamine (2 mmol/L) (all Invitrogen, Paisley, U.K.), at 37˚C in a humid- PCRs were performed in duplicate and cDNAs, cloned into pGEM-T vector, ified atmosphere containing 5% (v/v) CO2. HMDMs were isolated from were used as standards for quantitation and to verify specificity by DNA buffy coats supplied by the Welsh Blood service using Ficoll-Hypaque sequencing. purification. Briefly, blood from the buffy coat was layered over Lympho- prep (Nycomed Pharmaceuticals, Oslo, Norway) in Accuspin tubes Western blotting 3 (Sigma-Aldrich, Poole, U.K.) and centrifuged at 800 g for 30 min. Total cell lysates (THP-1 cells and HMDMs) were size-fractionated on The resultant mononuclear cell interface was collected and washed six NuPAGE 4–12% SDS-polyacrylamide gels (Invitrogen) and then analyzed to eight times in PBS containing 0.4% (v/v) trisodium citrate to remove by Western blotting using a previously described method (14). Abs against platelets. Monocytes were allowed to adhere to tissue culture plates in CD36 (sc-9154), SR-A (sc-20660), SR-B1 (sc-32342), and ABCG-1 (sc- complete RPMI 1640 medium containing 5% (v/v) FCS for 7 d to enable 11150) were from Santa Cruz Biotechnology (Santa Cruz, CA). Abs to ApoE macrophage differentiation and permit removal of nonadherent lympho- (0650-1904) and ABCA-1 (NB400-105) were from Biogenesis (Poole, U.K.), cytes. Recombinant human and murine IL-33 was from Peprotech (Lon- and Novus Biologicals (Cambridge, U.K.), respectively. The Ab to b-actin don, U.K.) and R&D Systems (Abingdon, U.K.), respectively. PMA was was from Sigma-Aldrich. Semiquantitative measurement of Western blots from Sigma-Aldrich. THP-1 cells were differentiated into macrophages was performed by densitometric analysis using the Gene Tools software using 160 nM PMA for 24 h. Cytokines were made up in PBS (murine (GRI, Braintree, U.K.). IL-33) or PBS/0.1% BSA (human IL-33). PBS/0.1% BSA was used as a vehicle control for in vitro human IL-33 stimulations. Quantification of macrophage total and esterified cholesterol Downloaded from Mice content 2 2 Macrophage (THP-1, HMDMs, and mouse BMMs) total and esterified cho- ST2 / mice, on a BALB/c background, were generated as previously lesterol content was quantified using the Amplex Red Cholesterol Assay Kit described (12) BMMs were isolated from 7- to 8-wk-old female ST2 wild- 2 2 2 2 (Molecular Probes, Eugene, OR), according to an adaptation of a previously type (WT) and ST2 / mice as previously described (13). Male ApoE / described method (15). Macrophages were fixed in 2% (v/v) paraformal- mice on the C57BL/6 background (backcrossed 103, Charles River, U.K.) dehyde for 15 min, washed three times with PBS before incubation with were bred inhouse in a pathogen-free facility, weaned at 6 wk old and fed http://www.jimmunol.org/ 200 ml 3:2 (v/v) hexane:isopropanol for 30 min at 4˚C to extract cellular an atherogenic diet ad libitum (0.15% cholesterol and 21% lard, Special lipids. Total cholesterol and cholesterol ester content was determined by Diet Services, Essex, U.K.). At 12 wk mice were randomly grouped and incubating 50 ml extracted lipids, diluted in 13 Reaction buffer supplied injected i.p. twice per week for 6 wk with PBS (as control) or murine IL-33 2 2 with the kit, with 50 ml Amplex Red Assay reagent containing cholesterol (1 mg/injection). ApoE / mice were euthanized at 18 wk of age prior to esterase (total cholesterol) or with 50 ml Amplex Red Assay reagent lack- analysis. All mice were maintained at the Joint Animal Facilities, Univer- ing cholesterol esterase (free cholesterol) for 30 min at 37˚C in the dark sity of Glasgow, Glasgow, U.K., and all experiments were conducted in and then measuring fluorescence (BMG Labtech [Aylesbury, U.K.] accordance to the animal guidelines of the Home Office, United Kingdom, FLUOstar Optima microplate reader; 535 nm excitation, 595 nm emis- under the Home Office Project License 60/3605. sion). Total and free cholesterol content of each sample was calculated DiI-AcLDL and DiI-OxLDL uptake assays using a cholesterol standard curve. Cholesterol ester content was calcu- lated by subtracting levels of free cholesterol from levels of total choles- by guest on September 28, 2021 Cells were incubated for 24 h unless otherwise stated with 1,19-dioctadecyl- terol for each sample. Lipid-extracted macrophages were incubated with 3,3,39,39-tetramethylindocarbocyane perchlorate (DiI)-labeled acetylated 0.1% (weight/volume ratio [w/v]) SDS/0.2 M NaOH for 30 min at room LDL (DiI-AcLDL) or oxidized LDL (DiI-OxLDL) (10 mg/ml; both Intra- temperature to extract cellular protein. Total cellular protein levels were cel, Frederick, MD) in RPMI 1640 or DMEM (mouse BMMs) containing determined using the Pierce BCA assay (Pierce, Cramlington, U.K.). Total 0.2% (v/v) fatty-acid free BSA (Sigma-Aldrich) at 37˚C. DiI-AcLDL or cholesterol and cholesteryl ester levels were represented as nanogram of DiI-OxLDL uptake was analyzed by flow cytometry on a FACSCanto (BD total cholesterol or cholesteryl ester per microgram of protein. Biosciences, Oxford, U.K.) flow cytometer with at least 10,000 events acquired for each sample. DiI-AcLDL or DiI-OxLDL uptake is represented Quantification of foam cells in vivo as a percentage with the untreated control indicated as 100%. Quantification of macrophage-derived foam cell content in vivo was per- Cholesterol efflux assay formed using F4/80 staining of atherosclerotic plaques with an adaptation Macrophages (THP-1, HMDMs, and mouse BMMs) were converted into foam cells by incubation with AcLDL (50 mg/ml; Intracel) in media con- taining 0.2% (v/v) fatty-acid free BSA and [4-14C]cholesterol (0.5 mCi/ml; Amersham, Buckinghamshire, U.K.) for 24 h. Foam cell formation was confirmed by Oil Red O staining (data not shown). Subsequently, cells were treated for 24 h in media containing 0.2% (v/v) fatty acid-free BSA and Apolipoprotein A-I (ApoA-I) (10 mg/ml; Sigma-Aldrich). After this, the media was collected and the remaining cells were solubilized in 0.2 M NaOH. Cholesterol efflux was calculated as the percentage of [14C] counts in the medium versus total (cells and medium) as measured by a liquid scintillation counter. Fold change in cholesterol efflux was the fold difference in efflux between control and treated samples. Real-time quantitative PCR Total RNA was prepared using the RNeasy Plus Mini kit (Qiagen, Crawley, U.K.) and was reverse transcribed into cDNA using M-MLV reverse tran- scriptase (Promega, Southampton, U.K.) and random hexamer primers. FIGURE 1. IL-33 reduces foam cell accumulation in ApoE2/2 mice Real-time quantitative PCR analysis was performed using SYBR Green in vivo. Male ApoE2/2 mice were treated with PBS (open bars) or IL-33 Taq + JumpStart ReadyMix (Sigma-Aldrich) and primers, detailed in Supple- (filled bars). A, Representative photomicrographs of F4/80 macrophages mental Table 1, specific for human scavenger receptor A (SR-A), CD36, SR- 2 2 (brown, original magnification 340) staining in plaques of ApoE / mice BI, ApoE, ATP-binding cassette transporter A-1 (ABCA-1), ATP-binding 2 cassette transporter G-1 (ABCG-1), Niemann Pick type C (NPC)-1, NPC- treated with PBS or IL-33 (n = 8–9 mice). B, Quantification (cells/mm )of + 2, carnitine palmitoyltransferase-1 (CPT-1), adipocyte differentiation-related the number of plaque foam cells (F4/80 macrophages with intracellular protein (ADRP), Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), neutral lipid droplets, n = 8–9 mice). Data represents mean plaque foam cell con- cholesteryl ester hydrolase (NCEH) and mouse SR-A, CD36, SR-BI, ApoE, tent 6 SD. pppp , 0.001 by Student t test. 1224 IL-33 REDUCES MACROPHAGE FOAM CELL FORMATION of a method described previously (16). Briefly, each heart from ApoE2/2 IL-33 reduces AcLDL and OxLDL uptake and enhances mice was sectioned parallel to the atria and sections fixed, processed and cholesterol efflux in human macrophages in vitro embedded in paraffin wax. Five 7-mm sections starting from the three valve cusps of the at 35-mm intervals were cut and stained with anti- An imbalance in the uptake of modified LDL and the efflux of F4/80 (clone CI:A3-1, Serotec, Kidlington, U.K.) for macrophages. Stain- cholesterol by macrophages is known to drive their formation into ing was visualized as previously described (7). The mean number of foam foam cells (9–11). Proatherogenic cytokines, such as IFN-g, pro- cells (F4/80+ macrophages that contained intracellular lipid droplets) were counted per millimeter squared of plaque in five sections from each mouse mote macrophage foam cell formation by enhancing modified (from each group, n = 8–9 mice) using Scion Image software (Scion, LDL uptake, and ablating cholesterol efflux (11). To investigate Frederick, MD). the role of IL-33 in modified LDL uptake and cholesterol efflux, Statistical analysis we first measured the impact of exogenous IL-33 stimulation on the uptake of AcLDL [a type of LDL extensively used in the All data are mean 6 SD with n indicating the number of independent in vitro foam cell formation assays (17–19)] and OxLDL [a key experiments. Statistical analysis was performed using Student t test. form of modified LDL found in atherosclerotic plaques (9, 10, 17, 18)] and on the efflux of [14C]cholesterol by both 24 h PMA- Results differentiated THP-1 macrophages and 7 d differentiated HMDMs. IL-33 reduces macrophage foam cell accumulation in vivo PMA-differentiation of THP-1 macrophages for 24 h was used to We first investigated the effect of IL-33 on the accumulation of ensure high expression levels of scavenger receptors (20). ST2L macrophage-derived foam cells into atherosclerotic plaques expression was confirmed on both THP-1 macrophages and in vivo. To do this, we analyzed the same sections that were used HMDMs (Supplemental Fig. 1). DiI-AcLDL and DiI-OxLDL up- Downloaded from to identify that IL-33–reduced macrophage accumulation in aortic take and [14C]cholesterol efflux by both THP-1 macrophages and sinus plaques of ApoE2/2 mice (7) to ascertain whether IL-33 HMDMs was assayed after 24 h stimulation with or without IL-33. altered the number of foamy macrophages (F4/80+ macrophages Using a range of concentrations (1–20 ng/ml) and length of stim- containing lipid droplets). Fig. 1 shows that, in these sections, ulation period (1–24 h), it was found that IL-33 produced a signif- IL-33 treatment significantly reduced the number of lesion- icant decrease in DiI-AcLDL uptake by THP-1 macrophages with associated, F4/80+ macrophage foam cells by 74% (6116 6 1711 versus 1634 6 316 cells/mm2, p , 0.001). These results http://www.jimmunol.org/ demonstrate that not only does IL-33 reduce the percentage of macrophages residing in atherosclerotic plaques in mice as de- scribed previously (7), it can effectively reduce the number of macrophages that have been converted into foam cells. To inves- tigate the mechanism by which IL-33 reduces macrophage foam cell formation and potential clinical relevance, we studied the effect of IL-33 on human macrophage cells as detailed below. by guest on September 28, 2021

FIGURE 3. IL-33 regulates the expression of key genes implicated in FIGURE 2. IL-33 decreases the uptake of AcLDL and OxLDL and enhan- the uptake and efflux of cholesterol in THP-1 macrophages and HMDMs. ces cholesterol efflux by THP-1 macrophages and HMDMs. DiI-AcLDL (A) Real-time quantitative PCR for SR-A, CD36, SR-BI (A), ApoE, ABCA-1, and DiI-OxLDL (B) uptake were measured in 24 h 160 nM PMA- and ABCG-1 (B) was performed on cDNA from 24 h PMA-differentiated differentiated THP-1 macrophages (open bars) or 7 d differentiated HMDMs THP-1 macrophages (open bars) or 7 d differentiated HMDMs (filled bars) (filled bars) incubated for 24 h in the presence or absence of IL-33 (10 ng/ml incubated for 24 h in the presence or absence of IL-33 (10 ng/ml) (n = 5).

(n = 4). C, Cholesterol efflux was assayed in foam cells from 24 h PMA- mRNA expression levels were calculated using comparative Ct method and differentiated THP-1 macrophages or 7 d differentiated HMDMs incubated normalized to GAPDH levels with untreated cells given an arbitrary value for 24 h in the presence or absence of IL-33 (10 ng/ml) (n = 6). Data repre- of 1. Data represents mean 6 SD. pp , 0.05; ppp , 0.01; pppp , 0.001 sents mean 6 SD. ppp , 0.01; pppp , 0.001 by Student t test. by Student t test. The Journal of Immunology 1225 the greatest response observed at 24 h by 10 ng/ml (∼15% re- from macrophages by active, reverse cholesterol transport in a pro- duction; Supplemental Fig. 2). This concentration lies within the cess that requires the presence of lipid-free, cholesterol acceptors physiological range of human serum IL-33 levels because they can like ApoE and ApoA-I (9–11). We therefore next investigated reach up to 40 ng/ml depending on health status (4) and has also whether IL-33 can change the expression of these key genes. been widely used by other in vitro studies (1, 6, 21). This concen- IL-33 significantly reduced the mRNA expression of SR-A, CD36, tration and length of stimulation were used subsequently for all and SR-B1 in both THP-1 macrophages and HMDMs (Fig. 3A). future experiments. IL-33 also induced a significant decrease in In contrast, IL-33 elevated the mRNA expression of ApoE, DiI-AcLDL uptake by HMDMs and in DiI-OxLDL uptake by both ABCA-1, and ABCG-1 in THP-1 macrophages and HMDMs (Fig. THP-1 macrophages and HMDMs compared with the untreated 3B). The IL-33–stimulated increases in ApoE, ABCA-1, and control (Fig. 2A,2B). The reduction in DiI-AcLDL and DiI- ABCG-1 mRNA expression were also similarly seen in AcLDL- OxLDL uptake observed contrasts that seen with the proathero- converted foam cells (data not shown). The IL-33–induced genic cytokine IFN-g that is known to increase AcLDL and changes in mRNA expression were confirmed by Western blot OxLDL uptake and promote foam cell formation (11, 22). analysis that showed that IL-33 reduced the expression of all three Furthermore, IL-33 markedly increased cholesterol efflux from scavenger receptor proteins and increased the expression of ApoE, both THP-1 macrophages and HMDMs compared with the un- ABCA-1, and ABCG-1 proteins (Fig. 4). Collectively, these data treated control (Fig. 2C). This change in cholesterol efflux is again suggest that IL-33 decreases AcLDL/OxLDL uptake and enhances the opposite of that seen with IFN-g that is known to decrease cholesterol efflux by altering the mRNA and protein expression of cholesterol efflux from macrophages (23) and is similar to that key genes implicated in the uptake and efflux of cholesterol. seen with the antiatherogenic cytokine TGF-b (24). Downloaded from IL-33 reduces total cholesterol and cholesterol ester content in IL-33 alters the expression of key genes implicated in the human macrophages uptake and efflux of cholesterol Lipid accumulation during macrophage foam cell formation not The uptake of AcLDL and OxLDL into macrophages principally only involves an imbalance in the uptake of modified LDL and involves a family of SRs, including SR-A, SR-B1, and CD36, the efflux of cholesterol but also intracellular mechanisms that

which are known to bind and internalize both forms of LDL promote cytoplasmic storage of cholesteryl esters and triglyceride- http://www.jimmunol.org/ (11, 25). Members of the ABC transporter family, such as rich lipid droplets (26). These mechanisms engage to increase the ABCA-1 and ABCG-1, are involved in directing cholesterol efflux intracellular levels of total and esterified cholesterol in by guest on September 28, 2021

FIGURE 4. IL-33 regulates the expression of key proteins implicated in the uptake and efflux of cholesterol in THP-1 macrophages and HMDMs. Twenty-four–hour PMA-differentiated THP-1 macrophages or 7-d differentiated HMDMs were incubated for 24 h in the presence or absence of IL-33 (10 ng/ml) (n = 4). A, Equal levels of protein from total lysates were analyzed by Western blotting using Abs against CD36, SR-A, SR-B1, ApoE, ABCA-1, ABCG-1, and b-actin. Multiple immunoreactive polypeptides for SR-A, SR-BI, CD36, and ApoE represent different isoforms and/or posttranslationally modified products. B, Semiquantitative analysis of all Western blots was performed by densitometric analysis using the Gene Tools software (GRI) and normalized to b-actin levels. Open bars indicate data for 24-h PMA-differentiated THP-1 macrophages and filled bars indicate data for 7-d differentiated HMDMs. Data represent mean 6 SD. pp , 0.05; ppp , 0.01; pppp , 0.001 by Student t test. 1226 IL-33 REDUCES MACROPHAGE FOAM CELL FORMATION macrophages and, as such, provide an additional indicator of foam the levels of total and esterified cholesterol in AcLDL-converted cell formation. We therefore next investigated whether IL-33 also THP-1– and HMDM-derived foam cells (Fig. 5A,5B). These affects intracellular total and esterified cholesterol levels in results therefore further support the notion that IL-33 reduces THP-1 macrophages and HMDMs. IL-33 significantly reduced macrophage foam cell formation. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. IL-33 reduces total and esterifed cholesterol content in human macrophages. Total cholesterol (A) and esterified (B) cholesterol levels were quantified in AcLDL-converted 24-h PMA-differentiated THP-1(open bars) and 7-d differentiated HMDM foam cells (filled bars) after incubation for 24 h in the presence or absence of IL-33 (10 ng/ml) (n = 4). C, Real-time quantitative PCR for NPC-1, NPC-2, CPT-1, ADRP, NCEH, and ACAT-1 was performed on cDNA from 24-h PMA-differentiated THP-1 macrophages (open bars) or 7-d differentiated HMDMs (filled bars) incubated for 24 h in the presence or absence of IL-33 (10 ng/ml) (n = 4). mRNA expression levels were calculated using comparative Ct method and normalized to GAPDH levels with untreated cells given an arbitrary value of 1. Data represent mean 6 SD. pp , 0.05; ppp , 0.01; pppp , 0.001 by Student t test. The Journal of Immunology 1227

FIGURE 6. ST2 is integral for the actions of IL-33 Downloaded from on macrophage foam cell formation. In vitro DiI- OxLDL uptake (A), total (B) and esterified cholesterol levels (C), cholesterol efflux (D), and mouse CD36, SR-A, SR-BI, ApoE, ABCA-1, and ABCG-1 mRNA expression (E) were measured in WT and ST22/2 BMMs after incubation for 24 h in the presence (filled bars) or absence (open bars) of mouse IL-33 (10 ng/ http://www.jimmunol.org/ ml) (n = 3–4). mRNA expression levels were calcu- lated using comparative Ct method and normalized to mouse b-actin with WT BMMs given an arbitrary value of 1. Data represent mean 6 SD. pp , 0.05; ppp , 0.01; pppp , 0.001 by Student t test. by guest on September 28, 2021

The cellular mechanisms involved in the cytoplasmic storage ADRP, ACAT-1, and NCEH in THP-1 macrophages and HMDMs. of cholesteryl esters and triglyceride-rich lipid droplets are regu- IL-33 significantly increased NPC-1 and NPC-2 mRNA expres- lated by a network of proteins, involved in intracellular cholesterol sion (Fig. 5C). These results are consistent with the higher levels storage and transport, which themselves are cytokine sensitive of cholesterol efflux and mRNA/protein expression of ApoE, (11, 22, 26–28). These include NPC-1, NPC-2, CPT-1, ADRP, ABCA-1 and ABCG-1 in these cells (Figs. 2–4). IL-33 also in- ACAT-1, and NCEH. Both NPC-1 and NPC-2 control the traffick- creased CPT-1 mRNA expression (Fig. 5C) suggesting that IL-33 ing of cholesterol from the late / to the plasma contributes to less cholesterol esterification. Furthermore, IL-33 membrane for cholesterol efflux (26). CPT-1 reduces fatty acid significantly decreased ADRP and ACAT-1 mRNA expression availability for cholesterol esterification and ADRP increases in- (Fig. 5C). The reduction in these two genes may contribute to less tracellular triglyceride storage (26). In addition, ACAT-1, associ- cholesteryl ester accumulation and triglyceride storage. IL-33 also ated with macrophage-derived foam cells in atherosclerotic significantly reduced NCEH mRNA expression although this ob- plaques (11), esterifies cholesterol within the endoplasmic reticu- served effect is not consistent with IL-33–stimulated changes lum and NCEH regulates cholesteryl ester accumulation by con- in cholesteryl ester levels (Fig. 5B) because over-expression of trolling its hydroylsis (26). We therefore investigated whether NCEH reduces cholesteryl esters in human macrophages (29). IL-33 could affect the expression of NPC-1, NPC-2, CPT-1, Collectively, these data indicate that IL-33 reduces the levels of 1228 IL-33 REDUCES MACROPHAGE FOAM CELL FORMATION total and esterified cholesterol content in human macrophages and CD36, SR-A, and SR-B1 mRNA expression in human and murine that this phenotype involves the alteration in the expression of key macrophages by endogenously produced IL-33. In addition, exog- genes associated with cholesterol storage and transport. enously added IL-33 mediates gene suppression in a ST2- dependent manner. IL-33/ST2 signaling can activate several down- ST2 is integral for the actions of IL-33 in reducing macrophage stream pathways leading to AP-1 and NF-kB activation (reviewed foam cell formation in vitro in Ref. 2) and therefore could regulate the expression of NPC- To demonstrate the specificity of IL-33–induced lipid metabolism 1, NPC-2, CPT-1, ADRP, ACAT-1, and NCEH genes. AP-1 and in macrophages, we compared DiI-OxLDL uptake, intracellular NF-kB binding sites exist in the ApoE and ABCA-1 promoters 14 total and esterified cholesterol levels, and [ C]cholesterol efflux (36, 37), suggesting a potential mechanism of IL-33–mediated following exogenous IL-33 stimulation of BMMs isolated from gene regulation through these signaling pathways. 2/2 WT and ST2 mice. IL-33 significantly decreased DiI-OxLDL Analysis of OxLDL uptake by WTand ST22/2 BMMs identified uptake in WT BMMs and intracellular total and esterified choles- a potential autoregulatory role for ST2 involving inhibition of CD36 terol levels in AcLDL-converted WT BMM foam cells (Fig. 6A– expression. In the absence ST2, BMMs produced significantly C), mirroring that seen in human macrophages (Figs. 2, 5). In higher levels of CD36 (Fig. 6E). The ability of cytokine receptors to contrast, IL-33 was unable to induce this change in both DiI- modulate foam cell formation has been previously reported for the 2/2 OxLDL uptake in ST2 BMMs and intracellular total and es- TNF superfamily receptor, TNFR1, which can decrease macro- 2/2 terified cholesterol levels in AcLDL-converted ST2 BMM phage foam cell formation by reducing SR-A expression (38). A foam cells. In addition, the level of DiI-OxLDL uptake in unsti- mechanism by which the autoregulatory role for ST2 is achieved 2/2 mulated ST2 BMMs was significantly higher than that in the could involve autocrine stimulation of ST2 by secreted IL-33. How- Downloaded from WT BMMs, indicating that the IL-33 receptor may autoregulate ever, IL-33, which is expressed on monocytes, is not secreted by OxLDL uptake. IL-33 also significantly enhanced cholesterol ef- these cells unless they are damaged or necrotic (39), and in PMA- 2/2 flux in the WT BMMs but not in the ST2 BMMs (Fig. 6D). differentiated THP-1 macrophages, endogenous IL-33 remains cell Furthermore IL-33 significantly reduced the mRNA expression of associated (40). Alternatively, ST2 could function, in the absence of CD36, SR-A, and SR-B1 and enhanced the expression of ApoE IL-33, as a negative regulator, akin to that reported earlier for 2/2 and ABCA-1 in WT BMMs but not in ST2 BMMs (Fig. 6E). IL-1 synthesis, whereby ST2 competitively binds to MyD88 and http://www.jimmunol.org/ In addition, the levels of mouse CD36 expression were higher in MAL (12). 2/2 unstimulated ST2 BMMs compared with WT BMMs. This In conclusion, data presented in this study demonstrate that could explain why the levels of DiI-OxLDL uptake were higher IL-33 is a potent inhibitor of macrophage foam cell formation 2/2 in unstimulated ST2 BMMs. These data further support the in vivo and in vitro and provide an important, additional molecular notion that IL-33 mediates a reduction in OxLDL uptake and an mechanism by which IL-33 attenuates atherosclerosis. IL-33 enhancement of cholesterol efflux in mouse BMMs by altering the blocks foam cell formation by directly regulating the expression expression of key genes implicated in the uptake of OxLDL and of genes for AcLDL/OxLDL uptake, storage of cholesteryl esters efflux of cholesterol in a ST2-dependent manner. and triglycerides and cholesterol efflux/transport, and by inducing a phenotypical Th1-to-Th2 switch. The potential clinical relevance by guest on September 28, 2021 Discussion of our observation is highlighted by the effect of IL-33 on primary The data reported in this study demonstrate for the first time that human macrophages and suggests that IL-33 represents a promising IL-33 plays an important role in reducing macrophage foam cell therapeutic agent for controlling clinical atherosclerosis. accumulation in atherosclerotic plaques in vivo and in decreasing macrophage foam cell formation in vitro in both human and murine Disclosures macrophages. 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