of October 13, 2017.This information is current as

AtherosclerosisHyperlipidemic Mice Attenuates Macrophage-Specific Expression of IL-37 in

Lee and William A. BoisvertSara McCurdy, Yvonne Baumer, Emma Toulmin, Bog-Hieu

ol.1601907http://www.jimmunol.org/content/early/2017/10/13/jimmun

published online 13 October 2017J Immunol 

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The Journal of Immunology

Macrophage-Specific Expression of IL-37 in HyperlipidemicMice Attenuates Atherosclerosis

Sara McCurdy,* Yvonne Baumer,* Emma Toulmin,* Bog-Hieu Lee,† and

William A. Boisvert*,‡

Atherosclerosis, the progressive buildup of plaque within arterial blood vessels, can lead to fatal downstream events, such as heart

attack or stroke. A key event contributing to the development of atherosclerosis is the infiltration of monocytes and its associated

inflammation, as well as the formation of lipid-laden macrophage foam cells within the vessel wall. IL-37 is recognized as an

important anti-inflammatory cytokine expressed especially by immune cells. This study was undertaken to elucidate the role of

macrophage-expressed IL-37 in reducing the production and effects of proinflammatory cytokines, preventing foam cell forma-

tion, and reducing the development of atherosclerosis. Expression of human IL-37 was achieved with a macrophage-specific

overexpression system, using the CD68 promoter in mouse primary bone marrow–derived macrophages via retroviral transduc-

tion. Macrophage IL-37 expression in vitro resulted in decreased mRNA (e.g., IL-1B, IL-6, and IL-12) and secreted protein

production (e.g., IL-6, M-CSF, and ICAM-1) of key inflammatory mediators. IL-37 expression also inhibited macrophage pro-

liferation, apoptosis, and transmigration, as well as reduced lipid uptake, compared with controls in vitro. The in vivo effects of

macrophage-expressed IL-37 were investigated through bone marrow transplantation of transduced hematopoietic stem cells into

irradiated atherosclerosis-prone Ldlr2/2 mice. After 10 wk on a high-fat/high-cholesterol diet, mice with IL-37–expressing mac-

rophages showed reduced disease pathogenesis, which was demonstrated by significantly less arterial plaque development and

systemic inflammation compared with control mice. The athero-protective effect of macrophage-expressed IL-37 has implications

for development of future therapies to treat atherosclerosis, as well as other chronic inflammatory diseases. The Journal of

Immunology, 2017, 199: 000–000.

The first pathological stage of atherosclerosis begins withthe attraction of immune cells, specifically monocytes, tothe inflamed endothelial lining of medium and large ar-

teries. Monocytes transmigrate through the endothelium to theintima (1). Chronic inflammation and dysregulation of cholesterolmetabolism by macrophages within the plaque are the majordriving forces of atherosclerosis progression (2). Macrophagesencounter and take up modified lipoproteins (3), which leads toactivation of pattern recognition receptors and TLR expression,promoting the production of inflammatory immune mediators (4, 5).

Inhibiting inflammation has been a central tenet in the pursuit ofinnovative therapies to treat or prevent atherosclerosis development.IL-37 is a recently discovered anti-inflammatory cytokine be-

longing to the IL-1 family. There are five known splice variants, of

which IL-37b is the best-characterized isoform, found predomi-

nantly in cells of the immune system (6, 7). The precursor IL-37b

isoform is expressed at low levels under steady-state conditions

(8), and its expression is upregulated under inflammatory condi-

tions induced by LPS and other TLR ligands (9). IL-37 expression

has been strongly associated with many inflammatory human

diseases (10–15), including acute coronary syndrome (16). Al-

though no murine homolog for IL-37 has been identified, the

human protein is functional in the mouse, and studies have been

carried out using an IL-37b–expressing transgenic mouse model

(9). These mice have been shown to be strongly protected from

various inflammatory conditions (9, 17, 18), defining a protective

role for IL-37 in the suppression of pathogenic inflammation.Because macrophages are central to disease pathogenesis in var-

ious ways from the early to late stages of atherosclerosis, they provide

a valuable delivery system for expression of anti-inflammatory me-

diators, such as IL-37, to alter the plaque microenvironment (19). The

anti-inflammatory benefits of IL-37 have been widely accepted, and

its potential as a therapeutic measure for reducing the pathogen-

esis of atherosclerosis has been discussed (20), yet exploration of

its effects on plaque development has been limited to systemic

treatment with rIL-37 (21, 22). The delivery of IL-37 directly to

the plaque microenvironment via macrophage-specific expression

has not been investigated. The aims of this study were to test the

effects of IL-37b in vitro with regard to macrophage inflammatory

response and cholesterol homeostasis, as well as in vivo via bone

marrow transplantation (BMT) of transduced hematopoietic stem

cells (HSCs) to atherosclerosis-prone Ldlr2/2 mice to determine

*Center for Cardiovascular Research, John A. Burns School of Medicine, Universityof Hawaii, Honolulu, HI 96813; †Department of Food and Nutrition, College ofBiotechnology and Natural Resources, Chung-Ang University, Anseong 17546, Re-public of Korea; and ‡Institute of Fundamental Medicine and Biology, Kazan FederalUniversity, Kazan 420008, Russia

ORCID: 0000-0001-7400-0773 (Y.B.).

Received for publication November 8, 2016. Accepted for publication September 12,2017.

This work was supported by Chung-Ang University research grants in 2015. S.M.was supported by a predoctoral grant from the American Heart Association.

Address correspondence and reprint requests to Dr. William A. Boisvert or Dr. Bog-Hieu Lee, Center for Cardiovascular Research, University of Hawaii, John A. BurnsSchool of Medicine, 651 Ilalo Street, BSB 311-C, Honolulu, HI 96813 (W.A.B.) orDepartment of Food and Nutrition, College of Biotechnology and Natural Resources,Chung-Ang University, 72-1, Naeri, Daedeok, Anseong 17546, Republic of Korea(B.-H.L.). E-mail addresses: wab@hawaii.edu (W.A.B.) or lbheelb@cau.ac.kr (B.-H.L.)

The online version of this article contains supplemental material.

Abbreviations used in this article: AcLDL, acetylated low-density lipoprotein;BMDM, bone marrow-derived macrophage; BMT, bone marrow transplantation;EdU, 5-ethynyl-29-deoxyuridine; EGFP, enhanced GFP; EV, empty vector; HFD,high-fat diet; HSC, hematopoietic stem cell; LDL, low-density lipoprotein; LN,lymph node; OxLDL, oxidative low-density lipoprotein; PFA, paraformaldehyde;Ph-E, Phoenix Ecotropic; RT, room temperature; RT-qPCR, real-time quantitativePCR.

Copyright� 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00

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the potential role of macrophage-expressed IL-37 in the preven-tion of atherogenesis.

Materials and MethodsRetroviral vector generation

The CD68S–HA–EGFP retroviral vector was a generous gift fromDr. E. Raines (University of Washington, Seattle, WA). The vector wasconstructed as described previously (19) and uses a segment of the humanCD68 gene promoter to drive macrophage-specific expression of an en-hanced GFP (EGFP) reporter gene. CD68–HA–EGFP or CD68 emptyvector (EV) were used as the control for all transduction experiments, asdescribed. The cDNA encoding human IL-37b was obtained from acommercial CMV6 plasmid purchased from OriGene (Rockville, MD),and the restriction enzymes NotI and HindIII (New England Biolabs) wereused to subclone IL-37b–Myc–DDK cDNA in the place of HA-EGFPcDNA.

Transfection of Phoenix Ecotropic cells

High-titer retroviral supernatants were generated by transfecting PhoenixEcotropic (Ph-E) packaging cells (American Type Culture Collection) withthe retroviral constructs CD68S-EGFP or CD68S–IL-37b using 23 HEPES-buffered saline and 2 M CaCl2, as described previously (23), with the ex-ception that Ph-E cells were not selected using puromycin, and retrovirus-containing cell supernatants were collected at 48 and 72 h for use in HSCtransduction. Ph-E cells were grown in DMEM/F12 supplemented with 10%FBS and 1% penicillin/streptomycin.

Isolation and culture of HSCs from bone marrow

Bone marrow was isolated from 8–12-wk-old male C57BL/6 mice. Thefemur and tibia bones were flushed with ice-cold PBS, and the cells werepassed through a 40-mm cell strainer. RBCs were lysed using multispeciesRBC lysis buffer (eBioscience), according to the manufacturer’s instruc-tions, and cells were plated in stem cell compete medium (DMEM/F12supplemented with 15% FBS, 1% penicillin/streptomycin, 100 ng/mlrecombinant mouse stem cell factor, 20 ng/ml recombinant human IL-6,and 10 ng/ml recombinant mouse IL-3 [all cytokines were from Pepro-Tech]). The stem cells were plated at a density of 25 3 106 cells per 10 mlof medium in 10-cm petri dishes (non–tissue culture treated) and allowedto cycle for 2 d.

Retroviral transduction of HSCs

Retroviral transduction of HSCs was performed as described previously(23). In brief, the cycling HSCs in complete stem cell medium werecombined with the appropriate Ph-E retroviral supernatant, plated intofibronectin-coated six-well plates, and centrifuged for 2 h at 37˚C. Thefollowing day, the procedure was repeated using fresh retroviral superna-tant. The cells were then collected from the six-well plates, washed withPBS, and differentiated in bone marrow–derived macrophage (BMDM)medium (DMEM/F12 GlutaMAX [Invitrogen], supplemented with 10%heat-inactivated FBS [HyClone], 20% L929-conditioned medium as asource of M-CSF, and 1% penicillin/streptomycin [Life Technologies]) foruse in vitro, or were resuspended in PBS at 1 3 106 cells/0.1 ml for tailvein injection into irradiated Ldlr2/2 recipient mice for the in vivo BMTatherosclerosis study.

In vitro BMDM culture

For in vitro culture of transduced BMDMs, the cells were plated at a densityof 1 3 106 cells per milliliter in 15-cm tissue culture–treated plates(Corning) in 25 ml of medium each. Five to ten milliliters of medium wasadded to each plate every other day for a total of 7 d. Adherent differen-tiated macrophages were detached using Cell Stripper (Life Technologies),counted, and replated into the appropriate wells for in vitro experiments.L929-conditioned medium was produced by culturing 4.7 3 105 L929cells (American Type Culture Collection) per T-75 flask in 50 ml of me-dium (DMEM/F12, 10% FBS, 1% HEPES [Life Technologies], and 1%penicillin/streptomycin) for 1 wk before collecting, sterile filtering, andstoring the supernatant at 280˚C.

Western blot

RIPA buffer (10 mMTris-Cl [pH 7.6], 1 mMEDTA, 1%Triton X-100, 0.1%sodium deoxycholate, 0.1% SDS, 140 mM NaCl) was used for all lysatepreparations. Cells grown in six-well plates were washed with PBS andlysed with 100 ml of ice-cold lysis buffer supplemented with protease andphosphatase inhibitor cocktails (both diluted 1:100; Roche). Samples were

sonicated using a probe sonicator (Thermo Fisher) and centrifuged at400 3 g for 5 min at 4˚C to pellet cell debris. Protein concentrations weredetermined with a Pierce BCA Protein Assay Kit (Thermo Fisher).

Precast NuPAGE Novex 4–12% Bis-Tris Protein Gels (Invitrogen) wereused for SDS-PAGE. SDS loading buffer (43) was added to 10–20 mg ofprotein and heated to 95˚C for 5 min. Gels were run in 13 NuPAGE MESSDS Running Buffer (Invitrogen). The protein was then transferred to alow-fluorescence polyvinylidene difluoride membrane using EfficientWestern Transfer Buffer (catalog number 786-019; G-Biosciences). Themembranes were blocked in LI-COR blocking buffer for 1 h. EGFP(Abcam) or IL-37 (R&D Systems) primary Abs were diluted 1:1000 in LI-COR blocking buffer and incubated at room temperature (RT) for 1 h. LI-COR secondary Abs were diluted in LI-COR blocking buffer at RT for 1 h.After three additional 5-min washes with 0.1% PBST, the membranes werevisualized on a LI-COR Odyssey infrared scanner, and the images wereanalyzed using LI-COR Image Studio software (both from LI-COR Bio-sciences).

Detecting IL-37 protein by ELISA

A DuoSet ELISA kit for human IL-37 (R&D Systems) was used to analyzecell culture supernatants or mouse serum samples for human IL-37 protein.One hundred microliters of undiluted cell culture supernatant or mouseserum samples diluted 1:4 with 1% BSA in PBS were added to the plate induplicate, and the assay was performed according to the manufacturer’sinstructions.

RNA extraction, cDNA synthesis, and realtime–quantitative PCR

Total RNA was recovered from cells or tissues with TRIzol Reagent (LifeTechnologies). A volume of chloroform equal to 20% of the volume ofTRIzol was added, and samples were inverted vigorously to mix. Thesamples were centrifuged at 12,400 rpm for 15min in a precooled centrifugeset to 4˚C. The aqueous phase was carefully pipetted into a new tube, andan equal volume of nuclease-free 70% ethanol was added. Samples weremixed well, and RNAwas isolated using the purification columns providedwith the QIAGEN RNeasy RNA extraction kit. An on-column DNA digestwas performed for 15 min at RT. RNA concentrations and quality weremeasured on a NanoDrop 2000. One microgram of total RNA per samplewas transcribed to cDNA using the qScript cDNA Synthesis Kit (QuantaBiosciences). Real-time quantitative PCR (RT-qPCR) was performed usingSYBR Green 23 master mix with ROX (Roche), and samples were platedin triplicate in a 384-well quantitative PCR plate and run on an AppliedBiosystems 7900HT Fast Real-Time PCR System.

Macrophage proliferation in vitro using5-ethynyl-29-deoxyuridine

Transduced macrophages (EV versus IL-37b) from C57BL/6 male micewere plated at 2 3 105 cells per well of an eight-well chamber glass slide.Cells were treated with 5-ethynyl-29-deoxyuridine (EdU) for 6 h in parallelwith FBS supplementation at low (2%) and normal (10%) levels, LPS andIFN-g (10 ng/ml each), or M-CSF (20 ng/ml). Cells were then fixed with4% paraformaldehyde (PFA) for 10 min and parallelized using 0.1% TritonX-100 in PBS for 10 min, and detection of EdU was performed accordingto the manufacturer’s instructions using a Click-iT EdU Alexa Fluor 488Imaging Kit. Cells were washed with PBS, and nuclei were stained withDAPI.

Macrophage apoptosis in vitro

BMDMs from C57BL/6 mice were plated in 12-well plates at 7.5 3 105

cells per well. Cells were transfected with EV or IL-37b expression plas-mids using Lipofectamine 2000. Twenty-four hours after transfection, cellswere challenged with camptothecin (2 mg/ml) or an equivalent volume ofDMSO (1:500) for 6 h. Cells were then harvested using Cell Stripper andstained with an Annexin V–FITC Apoptosis Detection Kit (catalog numberBMS500FI-100; eBioscience), according to the manufacturer’s directions.Cells were then analyzed for Annexin V–FITC and propidium iodide (PI)fluorescence on an Attune NxT Flow Cytometer (Thermo Fisher) to measureapoptosis.

Cholesterol uptake using DiI-labeled cholesterol

For cholesterol uptake, transduced macrophages (EV versus IL-37b) fromC57BL/6 male mice were plated at 2.5 3 105 cells per well in an 8-wellchamber glass slide for analysis by microscopy or were plated at 1 3 106

cells per well in a 12-well plate for analysis by flow cytometry. Themacrophages were treated or not with LPS to induce IL-37 expression and

2 IL-37 ATTENUATES ATHEROSCLEROSIS

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were also treated with 20 mg/ml DiI-labeled acetylated low-density lipo-protein (AcLDL) or oxidized low-density lipoprotein (OxLDL) for 4 h.The cells in the 12-well plates were detached with Cell Stripper and fixedfor 10 min with 4% PFA before being analyzed by flow cytometry forintensity of DiI fluorescence. The cells treated in the glass slides were alsofixed for 10 min with 4% PFA and then analyzed with an epifluorescencemicroscope using a Rhodamine filter.

Cholesterol uptake using AcLDL and BODIPY staining

Differentiated macrophages from the bone marrow of C57BL/6 male micewere transfected using Lipofectamine 2000 (EV versus IL-37b) and platedat 2 3 105 cells per well in an 8-well chamber glass slide for analysis bymicroscopy or were plated at 7.53 105 cells per well in a 12-well plate foranalysis by flow cytometry. The macrophages were treated or not with LPSand IFN-g (10 ng/ml each) and were also treated or not with 40 mg/mlAcLDL for 6, 12, or 24 h. The cells in the 12-well plates were detachedwith Cell Stripper and fixed for 10 min with 4% PFA. Cells were thencentrifuged and resuspended in PBS containing the lipid stain BODIPY(1:500; Invitrogen) for 30 min at RT. After washing cells twice with PBS,they were analyzed for green fluorescence intensity, representing lipidcontent, by flow cytometry using an Attune NxT Flow Cytometer (ThermoFisher). The cells treated in the glass slides were also fixed for 10 min with4% PFA and then analyzed with an epifluorescence microscope using anFITC filter.

Transmigration of macrophages

Using a 5-mm-pore filter (Costar), transduced and differentiated BMDMswere plated in the upper chamber of the Transwell filter (100,000 cells perwell of a 24-well filter plate) in DMEM/F12 + 0.2% BSA medium. Cellswere given 2 h to become adherent before the medium in the lower chamberwas replaced with new medium containing the chemoattractant MCP-1(25 ng/ml) to stimulate the transmigration of cells through the filter. Afterovernight incubation in a humidified incubator at 37˚C with 5% CO2, the topfilter chamber was carefully and completely swiped with a Q-tip to removenonmigrated cells. The filter was fixed with 4% PFA for 10 min and stainedwith the nuclear stain DAPI to visualize and quantify the number of cellsthat had migrated through the filter. Photographs of the lower side of thefilter (three photos per well) were analyzed with ImageJ. Final counts werecompared between control and IL-37–expressing macrophages. All condi-tions were run in triplicate with n = 3 separate experiments.

Mouse atherosclerosis study: irradiation of donormice and BMT

Seven- to eight-week-old male recipient Ldlr2/2 mice (on the C57BL/6background) were subjected to 1000 rad of whole-body irradiation to de-stroy their endogenous HSCs. Half of the irradiated mice (n = 15) received1–2 3 106 HSCs transduced with CD68S-EGFP and the other half receivedHSCs transduced with CD68S–IL-37b via tail vein injections. Injected micewere allowed a 4-wk recovery period for full reconstitution of their immunesystems before initiation of an atherogenic diet containing 15.8% fat (w/w)and 1.25% cholesterol (w/w) (Harlan Teklad) for an additional 10 wk. Afterthe 4-wk recovery period, 100 ml of blood was collected by submandibularbleeding to confirm immune cell reconstitution by flow cytometry of bloodleukocyte populations and to check for circulating IL-37 protein.

At the end of the study, the animals were euthanized by CO2 asphyx-iation, and total blood was collected by cardiac puncture of the left ven-tricle. The mice were then perfused with ice-cold PBS, followed by 4%PFA/5% sucrose. The hearts and aortas were collected and analyzed forplaque, as described below. One hundred microliters of whole blood permouse was analyzed for immune populations by flow cytometry on aFACSAria flow cytometer (BD Biosciences), whereas the remaining bloodwas centrifuged at 15003 g for 15 min to obtain plasma for the purpose ofmeasuring lipid levels, as well as proinflammatory and anti-inflammatorycytokine levels by ELISA or Luminex bead array. With regard to the gatingstrategy for analysis of blood leukocytes, events were gated for singletsand then dead cells were gated out using a fixable live/dead stain. Next,CD45+ cells were gated to include all leukocytes. Of all CD45+/CD3+

T cells, CD4+ and CD8+ cells were gated to identify each T cell sub-population. CD45+/CD11b+/CD11c2 cells were gated for GR-1+/Ly6G2 toidentify monocytes, whereas GR1+/Ly6G+ cells were considered neutrophils.

Analysis of serum cholesterol and triglyceride levels

Blood cholesterol and triglyceride measurements were performed at the endof the BMTatherosclerosis study from serum samples of all mice from eachgroup (n = 14). Total cholesterol was measured using a Cholesterol Fluo-rometric Assay Kit (item number 10007640; Cayman Chemical), according

to the company’s instructions. The mouse serum samples were also ana-lyzed for triglyceride levels using a Triglyceride Colorimetric Assay Kit(item number 10010303; Cayman Chemical), according to the manufac-turer’s instructions.

Luminex magnetic bead assay

A custom multiplex magnetic bead assay (R&D Systems) was used tomeasure EGFP or IL-37 BMT mouse serum samples for MCP-1, CCL3,CXCL2, IFN-g, IL-1a, IL-1b, IL-10, IL-12 p70, IL-4, IL-6, M-CSF, andTNF-a. The assay was performed according to the manufacturer’s instruc-tions, and the median fluorescence intensity was recorded on a Luminexanalyzer with a threshold of 50 events per bead and a flow rate of 60 ml/min.

Analysis of atherosclerotic plaque

Whole aortas isolated from the studymicewere cleaned of the adventitial fatand any branching arteries, cut open longitudinally, excised, and pinned to awax-lined dissecting tray. A working solution of Oil Red O was preparedfresh for each stain, as described previously (23). The pinned aortas werewashed once with 60% isopropanol and then incubated completely coveredwith Oil Red O working solution for 15 min. The aortas were then washedwith 60% isopropanol three or four times, photographed, and analyzed forplaque area using ImageJ.

The fixed hearts collected from the study mice at sacrifice were mountedin O.C.T. (Tissue-Tek) and frozen at 280˚C. The O.C.T.-embedded heartswere sectioned sagittally using a cryostat through the aortic valve. Serialsections of 5 mm thickness were made, with five representative sectionsspanning the aortic root of each heart selected, and stained with Oil Red Oto visualize the lesion area. The cryosections were incubated in PBS for 5 minand air dried. Sections were dipped 10 times in 60% isopropanol and in-cubated in fresh Oil Red O working solution for 15 min. Excess solution wasremoved, and the slides were dipped 10 times in isopropanol, rinsed for 5 minin running tap water, and embedded using mounting media (Sigma).Photos of the sections taken at 103 magnification were analyzed for lesionarea using ImageJ and expressed as the percentage of total aortic area.

Immunohistochemistry of aortic root sections

Aortic root sections were stained for MOMA-2 (product code ab33451;Abcam). Trypsin Ag retrieval was performed for 15 min at 37˚C, sectionswere blocked in 10% normal donkey serum in 2% BSA/PBS, and primaryAb incubation was performed overnight at 4˚C. The sections were washedwith 0.1% PBS-Tween and incubated with a Cy3-labeled secondary Ab(Abcam) for 1 h at RT. Primary Abs against IL-37 (catalog numberAF1975; R&D Systems) or EGFP (product code ab290; Abcam) were usedto stain aortic root sections using Donkey anti-Rabbit or Donkey anti-GoatHRP-conjugated secondary Abs with an AEC detection kit (all fromThermo Fisher), according to the manufacturer’s directions.

Statistical analyses

For the in vivomouse study comparing two groups with an SDwithin groups, 20% from the mean, which is typical for the plaque and other analyses, aminimum of 12–14 mice per group is necessary to detect an effect size $20% between groups. To account for the potential loss of one or more miceduring the study and the unknown effects of IL-37b expression on mice, 15mice per group were used for the in vivo study.

For some experimental measurements from the study mice, includingcirculating leukocyte analysis, BMDM analysis for expression of IL-37 orEGFP by Western blot, as well as lymph node (LN) transcript analysis, theSDwas small enough to detect significant differences using seven biologicalreplicates per study group, although all other assays were run with n = 14, asindicated. The Student t test was used to compare differences betweencontrol and IL-37 groups with significance set at p , 0.05, and one-wayANOVA was used for analysis of three or more groups. Statistics calcula-tions for all experiments were done using Prism (GraphPad), with *p, 0.05,**p , 0.001, and ***p , 0.0001.

ResultsMacrophage-expressed IL-37b reduces proinflammatory geneand protein expression

Expression of human IL-37b in mouse BMDMs was achieved viaretroviral transduction of HSCs. A macrophage-specific retroviralvector was used to create the CD68S–IL-37b construct (SupplementalFig. 1A, 1B). Retroviral transduction was performed as describedpreviously (19, 23), with alterations as described in Materials andMethods.

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The effect of IL-37 expression on the macrophage inflammatoryresponse was tested by challenging the transduced BMDMs withvarious inflammatory stimuli relevant to atherosclerosis. EGFP- orIL-37–expressing macrophages were left unstimulated or treatedwith AcLDL, IFN-g, or TNF-a overnight. RT-qPCR analysis revealedreduced expression of inflammatory genes, such as IL-1a, IL-1b,IL-6, IL-12, MIP-1b, and TNF-a, compared with EGFP controlcells (Fig. 1A). Under conditions of inflammation that would betypical of the plaque microenvironment, macrophage IL-37 ex-pression effectively suppressed inflammatory gene expression.To determine whether the anti-inflammatory effects of IL-37 on

macrophage gene expression were also applicable for the synthesisand secretion of inflammatory cytokines, cell supernatants fromEGFP- and IL-37–transduced BMDMs, which were left unsti-mulated or were treated with AcLDL, were analyzed by Ab array.IL-37 expression resulted in the suppression of inflammatory pro-tein production under basal conditions (Fig. 1B, left panel). In-flammatory mediators, such as ICAM1, IL-6, M-CSF, and MIP-1a,were all downregulated by IL-37 expression in untreated mac-rophages. A more potent anti-inflammatory effect of IL-37 ex-pression is seen after AcLDL treatment (Fig. 1B, right panel), inwhich a majority of cytokines tested were downregulated com-pared with EGFP control, including IL-1a, IL-6, IFN-g, CXCL9,MIP-1a, and M-CSF. We also wanted to determine whethertreating the BMDMs with a more physiologically relevant modi-fied low-density lipoprotein (LDL), OxLDL, would have similareffects as treatment with AcLDL. We were able to confirm

that OxLDL had a similar inhibitory effect as AcLDL on theexpression of IL-1b and IL-6 (data not shown). This provides newinsight into the role of IL-37 in regulating the macrophage in-flammatory response to modified LDL uptake.

IL-37b inhibits macrophage transmigration

The migratory response of monocytes and macrophages towardchemoattractants is a central feature in the pathogenesis of ath-erosclerosis. To determine whether IL-37 expression effects themigration of macrophages toward the chemoattractant MCP-1, atranswell filter assay was performed. As shown in Fig. 1C, IL-37expression significantly reduced macrophage transmigrationcompared with EV controls. Interestingly, IFN-g pretreatmentincreased EV control macrophage transmigration, but it did nothave a significant effect on IL-37 macrophages.

Expression of IL-37b suppresses proliferation andapoptosis of macrophages

Because macrophage proliferation and apoptosis are importantfeatures of atherosclerosis, we sought to determine whether IL-37bhas an impact on these cellular properties. To determine the effectof IL-37 expression or treatment with rIL-37 on macrophageproliferation in vitro, BMDMs were treated with EdU for 6 h inparallel with FBS supplementation at low (2%) and normal (10%)levels, LPS and IFN-g (10 ng/ml), or M-CSF (20 ng/ml). Underdifferent conditions, including low and normal serum supple-mentation, inflammatory stimulus (IFN-g+LPS), and stimulation

FIGURE 1. In vitro effects of macrophage-expressed IL-37 on inflammation and lipid homeostasis. (A) EGFP- or IL-37–expressing macrophages were

left unstimulated or were treated with 20 mg/ml AcLDL, 25 ng/ml IFN-g, or 20 ng/ml TNF-a overnight, followed by RNA extraction and RT-qPCR

analysis. Fold changes are relative to EGFP control cells for each treatment (n = 3). (B) The pooled supernatants (n = 3, in duplicate) of EGFP- or IL-37–

expressing macrophages that were left untreated (open bars) or were treated with 20 mg/ml AcLDL (pattered bars) for 48 h were tested for secreted cytokine

expression by Ab array. (C) EV- or IL-37–transduced macrophages (untreated or treated overnight with 25 ng/ml IFN-g) were allowed to migrate overnight

toward MCP-1–containing medium (25 ng/ml) in a Transwell filter assay. Transmigrated cells were stained with DAPI, and three random locations per well

were analyzed with ImageJ (n = 3, in triplicate). *p , 0.05, **p , 0.01, ***p , 0.001.

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of proliferation (M-CSF), we found that IL-37 suppresses mac-rophage proliferation (Fig. 2A). An additional experiment wasperformed using EdU in vivo to determine whether the observedreduction in macrophage proliferation also occurred in the blood,spleen, or aorta of mice treated short-term with rIL-37. After i.p.injection with EdU, with or without rIL-37 (40 ng/g) for 12 h, themice were sacrificed, and tissues were isolated, digested, andstained for analysis by flow cytometry. We found strong trends ofreduced EdU incorporation in CD45+/CD11b+ cells, as well asmyeloid cells (using ROSA22/LysM-Cre reporter mice) from theblood and myeloid cells in the aorta, although the differences werenot statistically significant (Supplemental Fig. 4).To check for apoptosis, BMDMs were plated in 12-well plates at

7 3 105 cells per well. After transfection with EV or IL-37 ex-pression plasmids, the BMDMs were challenged with DMSO or a1:500 dilution of 1 mg/ml camptothecin for 6 h to induce apo-ptosis. Cells were then harvested, stained with Annexin V–FITCand PI, and analyzed by flow cytometry. Compared with controls,IL-37–transfected BMDMs showed reduced apoptosis at baseline,as well as when challenged with DMSO or the apoptosis-inducingchemical camptothecin (Fig. 2B).

Effect of IL-37b expression on macrophagecholesterol processing

The uptake of modified lipoprotein within the plaque is central tofoam cell formation. Inhibiting cholesterol uptake or increasingcholesterol efflux is beneficial in preventing foam cell formationand protects against plaque growth and development. To investigatewhether IL-37 affects macrophage cholesterol uptake, we treatedEV- or IL-37–transduced macrophages with DiI-labeled AcLDLand OxLDL and visualized the uptake using fluorescence mi-croscopy (Fig. 3A) and flow cytometry (Fig. 3B, 3C). IL-37 ex-pression had a suppressive effect on the uptake of modified LDLcompared with controls, with a significant decrease in AcLDL andOxLDL uptake, implying a protective role for IL-37 in preventingfoam cell formation. This was confirmed by performing BODIPYstaining of EV- or IL-37b–transfected BMDMs incubated withAcLDL for 6, 12, or 24 h and treated or not with LPS/IFN-g.Although 12 h of treatment with AcLDL+ IFN-g/LPS was theonly condition that resulted in a significant increase in lipid contentcompared with AcLDL treatment alone, there was a trend towardincreased lipid uptake with IFN-g/LPS at 6 and 24 h (Fig. 4A). Thesignificant decrease in lipid content seen in IL-37–transfectedmacrophages after 6 h, with or without IFN-g/LPS stimulation, wasno longer apparent after 12 or 24 h, although there was a trendtoward less lipid at 12 h (Fig. 4B).In assessing the effects of IL-37 on lipid efflux, we found that

ABCA1 and PPARg were upregulated with IL-37 overexpression.However, actual cholesterol efflux at multiple time points, whetherusing ApoA1 or high-density lipoprotein as the cholesterol ac-ceptor, was not influenced by IL-37 (data not shown).Taken together, the in vitro findings that IL-37 expression re-

duces macrophage inflammation and migration and inhibitsmodified LDL uptake support a strong athero-protective role formacrophage-expressed IL-37. To test whether these in vitro find-ings hold true in the complex setting of atherosclerosis, an in vivostudy using atherosclerosis-prone Ldlr2/2 mice was performed.

BMT of EGFP or IL-37b HSCs into Ldlr2/2 mice

Transplantation of EGFP- or IL-37b–transduced HSCs into lethallyirradiated Ldlr2/2 recipient mice was performed as previously de-scribed (23). A portion of transduced HSCs was differentiated intomacrophages in vitro and analyzed by PCR, as well as by Westernblot and flow cytometry, for the presence of IL-37 transcript and

protein, respectively (Supplemental Fig. 1C, 1D). Various circulatingimmune cell populations were analyzed by flow cytometry to con-firm successful repopulation with the donor marrow (SupplementalFig. 2A). Circulating leukocytes were analyzed again after the studymice had been on a high-fat diet (HFD) for 10 wk (SupplementalFig. 2B), with no differences observed between groups before andafter HFD. Serum cholesterol and triglyceride levels, as well asbody weight before and after HFD, also were not different be-tween groups (Supplemental Fig. 2C, 2D).

EGFP and IL-37 expression in BMT mice

ELISA analysis of serum samples for the presence of IL-37 proteinrevealed a range of concentrations in the IL-37 BMT mice, with noexpression detected in the EGFP control mice (Fig. 5A). IL-37

FIGURE 2. IL-37b expression suppresses proliferation and apoptosis of

BMDM. (A) Primary BMDMs from C57BL/6 mice were plated in eight-

well chamber slides at 2 3 105 cells per well. To determine the effect of

IL-37 expression or treatment with rIL-37 on macrophage proliferation

in vitro, cells were treated with EdU for 6 h in parallel with FBS supple-

mentation at low (2%) and normal (10%) levels, LPS and IFN-g (10 ng/ml),

or M-CSF (20 ng/ml). Cell were fixed, and EdU was detected using a

Click-It kit (Thermo Fisher) with an Alexa Fluor 488 azide. Five to eight

images per well were analyzed and averaged. (B) BMDMs were plated in

12-well plates at 7 3 105 cells per well. After transfection with EVor IL-37

expression plasmids, the BMDMs were challenged with DMSO or a 1:500

dilution of 1 mg/ml camptothecin for 6 h to induce apoptosis. Cells were

then harvested, stained with Annexin V–FITC and PI, and analyzed by

flow cytometry, with quadrants used to determine whether cells were

live (AnnexinV2/PI2), early apoptotic (AnnexinV+/PI2), late apoptotic

(AnnexinV+/PI+), or dead (AnnexinV2/PI+). Results are shown as propor-

tions of total cells analyzed (n = 3). *p , 0.05, ***p , 0.001, one-way

ANOVA with the Tukey post hoc test.

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serum levels were measured following 10 wk on an HFD, withIL-37 BMT sera concentrations in a similar range as pre-HFD,whereas EGFP mice did not show detectable IL-37 protein(Fig. 5A).

At sacrifice, the femurs from seven mice per group wereharvested, and the bone marrow was cultured for analysis ofBMDMs. As seen in Fig. 5B, clear expression of EGFP or IL-37was detectable by Western blot in each group.

FIGURE 3. Macrophage-expressed

IL-37 reduces lipid uptake in vitro.

Transduced and differentiated IL-

37b–expressing or EV control mac-

rophages were treated with DiI-la-

beled AcLDL or DiI-labeled OxLDL

(20 mg/ml), with or without parallel

IFN-g (20 ng/ml) treatment. After 4 h,

lipid uptake was visualized with fluo-

rescence microscopy (original mag-

nification 3100) (A) (DiI in red,

nuclei stained with DAPI in blue)

and quantified by flow cytometry

(B and C) for DiI-labeled AcLDL

and DiI-labeled OxLDL, respec-

tively (n = 3). *p, 0.05, **p, 0.01,

***p , 0.001.

FIGURE 4. Macrophage expression of IL-37b reduces neutral lipid content in BMDMs. EV- or IL-37–transfected BMDMs were treated with 25 mg/ml AcLDL,

with or without IFN-g/LPS for 6, 12, and 24 h. BMDMs were plated at 73 105 cells per well in a 12-well plate for analysis by flow cytometry or at 23 105 cells per

well in an 8-well chamber slide for analysis by fluorescent microscopy. Following treatment, cells were fixed in 2% PFA for 10 min and stained with BODIPYat 1:500

dilution of 1 mg/ml in PBS for 30 min. (A) AcLDL uptake is shown in green with DAPI-stained nuclei in blue (original magnification 3100). (B) Quantification of

BODIPY-stained lipids by flow cytometry. Mean fluorescence intensity is shown for green fluorescence in the FITC channel (n = 3). *p , 0.05, **p , 0.01.

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Finally, aortic root sections were analyzed for the presence ofEGFP or IL-37 in the plaque areas using immunohistochemistry(Fig. 5E). The staining of EGFP and IL-37 proteins within therespective BMT mice confirmed the presence of transduced mac-rophages within the atherosclerotic plaque.

Systemic inflammation is reduced by macrophage IL-37expression in vivo

To gain a general view of the inflammatory state of the BMT mice,lumbar LNs collected at sacrifice were analyzed for inflammatorygene expression by RT-qPCR. LN transcripts from IL-37 miceshowed a significant reduction in IL-6 and IL-1b expressioncompared with LNs from EGFP mice (Fig. 5C), indicating thatIL-37 expression had a systemic anti-inflammatory effect in vivo.Additionally, serum samples from EGFP and IL-37 BMT mice

were analyzed with a Luminex magnetic bead assay. The absolutequantities of circulating inflammatory cytokine levels measuredwere not significantly different between IL-37 and EGFP BMTmice (Supplemental Fig. 3). However, linear regression analysisrevealed a significant negative correlation between IL-37 proteinabundance and M-CSF levels in the serum of IL-37 BMT mice(R = 20.7071, p = 0.0047) (Fig. 5D), implying a systemic anti-inflammatory role for IL-37 in vivo. There was no significantcorrelation for each of the other cytokines analyzed, althoughthere was a positive trend between IL-37 and IL-4 serum con-centrations (Supplemental Fig. 3B).

Macrophage-expressed IL-37 reduces total plaque area inLdlr2/2 mice

To analyze the atherosclerotic plaque development in EGFP andIL-37 BMTmice, the entire aortas were opened and stained en facewith Oil Red O to visualize atherosclerotic plaque. Aortas fromIL-37 BMTmice displayed significantly less plaque area comparedwith EGFP control mice (Fig. 6A), clearly demonstrating the

athero-protective effect of macrophage-expressed IL-37b inlesion-prone Ldlr2/2 mice. Aortic root sections from EGFP or IL-37BMT mice were also analyzed for plaque area following stainingwith Oil Red O. As seen in Fig. 6B, aortic root plaque area wassignificantly reduced in IL-37 BMT mice compared with EGFPBMT mice, further supporting the athero-protective role of IL-37in vivo.

The presence of macrophages and smooth muscle cells in theatheroma is not affected by IL-37

Aortic root sections were subjected to immunofluorescent stainingto detect macrophages or smooth muscle cells within the plaque.Each aortic root section was imaged at 203 magnification andanalyzed with ImageJ to determine macrophage-positive areas.MOMA-2 staining is shown for EGFP and IL-37 sections inFig. 6C, along with the macrophage-positive area, expressed as apercentage of total plaque for quantification. Although the totalplaque area was reduced in IL-37 mice, there was no differenceobserved in macrophage content per plaque area between theEGFP and IL-37 groups, indicating that the composition of theplaque was very similar in the two groups.

DiscussionThe experiments detailed in this study demonstrate thatmacrophage-specific IL-37 expression leads to suppression ofinflammation, cholesterol uptake, cell proliferation, and apoptosis,as well as macrophage transmigration in vitro. Furthermore, BMTof IL-37–transduced HSCs resulted in reduced plaque develop-ment and decreased systemic inflammation in atherosclerosis-prone Ldlr2/2 mice compared with controls. To our knowledge,this is the first study elucidating the role of macrophage-expressedIL-37 in the context of atherosclerosis, and it provides key evi-dence for future investigation into its potential therapeutic value in

FIGURE 5. Macrophage-expressed IL-37 in vivo quells inflammation. EGFP- or IL-37b–transduced HSCs were transplanted into lethally irradiated

Ldlr2/2 recipient mice, followed by a 4-wk recovery and a 10-wk HFD to induce atherosclerosis. (A) IL-37 protein was measured by ELISA in the sera of

all study mice before and after HFD. (B) BMDMs cultured from the femurs of study mice at sacrifice were analyzed by Western blot for IL-37 or EGFP

protein expression. (C) LNs collected at sacrifice were analyzed by RT-qPCR for inflammatory gene expression (n = 14). (D) Serum M-CSF concentration

in EGFP or IL-37 study mice was measured by Luminex assay (left panel). Linear regression analysis with IL-37 serum concentration reveals a significant

negative correlation (right panel). (E) Five-micrometer-thick aortic sinus sections were stained using primary Abs against EGFP or IL-37 and detected with

an HRP secondary Ab, followed by staining with an AEC kit. EGFP and IL-37 protein (both shown in red) were detected in the plaques of EGFP and IL-37

BMT study mice, respectively. Background control staining is shown in the insets (original magnification 3200) (n = 14). *p , 0.05. ND, not detected.

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the treatment and prevention of human atherosclerosis, as well asother chronic inflammatory diseases.Expression of various inflammatory cytokines known to be

detrimental in the context of atherosclerosis (24) was reduced bymacrophage IL-37 expression in vitro. This protective effect of IL-37was observed after classical inflammatory stimuli, such as IFN-gand TNF-a, as well as AcLDL treatment, indicating that IL-37also protects against a type of inflammation caused by modifiedLDL that has not previously been reported. IL-37 retains its anti-inflammatory properties, which is crucial for effective function ina state of hyperlipidemia.Inflammatory mediators implicated in atherogenesis that were

reduced by IL-37 in vitro and in vivo include IL-1b, IL-6, andM-CSF. IL-6 is associated with unstable angina in humans (25)and is known to stimulate production of matrix-degrading enzymesby macrophages (26). IL-37 expression in macrophages consistentlyreduced IL-6 transcript and protein expression in vitro and in vivo,as evidenced by reduced IL-6 transcripts in LNs isolated from IL-37mice compared with controls. Serum IL-6 levels were undetect-able by Luminex analysis (data not shown); thus, it remains un-clear whether IL-37 had an effect on circulating levels of IL-6protein.Macrophage expression of IL-37 led to reduced IL-1a and IL-1b

gene and protein expression in vitro, as well as decreased IL-1btranscripts in the LNs of IL-37 BMT mice compared with con-trols. It has been shown that, within minutes of macrophage in-flammatory activation, IL-37 expression closely follows that ofIL-1a and IL-1b (9), with the gene regulatory regions of all threegenes found in the IL-1 locus coming into close proximity fol-lowing LPS stimulation (27). This programmed expression of IL-37during the inflammatory response likely acts to modulate exces-sive inflammation.M-CSF gene expression has long been known to be linked to

atherosclerosis development (28), and its expression within theplaque leads to differentiation of macrophages and expression ofscavenger receptors (29). M-CSF deficiency in ApoE-knockoutmice results in reduced plaque formation (30), emphasizing the

importance of limiting its expression to prevent the disease.M-CSF protein production was reduced by IL-37 expression inunstimulated and AcLDL-stimulated macrophages in vitro. Inaddition, circulating levels of M-CSF in IL-37–transduced BMTmice were negatively correlated with serum IL-37 protein con-centration.The observation that IL-37 expression reduces macrophage

transmigration toward MCP-1 corresponds with the reduction ininflammatory cytokines and chemoattractant molecules seen in IL-37–expressing macrophages. MCP-1 secretion from IL-37–expressingmacrophages was reduced in vitro, although the serum levels werenot detectable. Thus, it is unclear whether they differed betweenstudy groups in vivo. Although not explored in this study, a pos-sible mechanism for IL-37 in reducing macrophage migrationcould involve decreased phosphorylation of various kinases andtranscription factors, as shown by Nold-Petry et al. (31) to phys-ically decrease the chemotactic response.In addition, the reduced proliferation of macrophages by IL-37

could be a major contributing factor to the attenuated plaque sizefound in IL-37 BMTmice compared with controls, because the vastmajority of the plaque is composed of macrophages at this earlystage. Recently, Robbins et al. (32), using a symbiosis model ofatherosclerosis-prone CD45.1 and CD45.2 mice put on an HFD,showed that the majority of BrdU+ proliferating macrophageswithin developed lesions were from the host parabiont rather thanfrom the donor, indicating that proliferation is a significant con-tributor to the macrophage content of lesions. Because rIL-37 alsostrongly inhibited macrophage proliferation, it is very likely thatthe IL-37–expressing macrophages within the plaque microenvi-ronment secrete IL-37 that could act to reduce the proliferation ofnearby macrophages, even if they did not express IL-37. Theobservation that macrophage apoptosis is decreased by IL-37 in-dicates that necrotic core formation may be attenuated in IL-37–expressing mice, because macrophage death is associated with thepresence of a necrotic core.Of note, the macrophage content of the aortic root plaque did not

differ between EGFP and IL-37 BMT mice, indicating that the

FIGURE 6. Macrophage-expressed IL-37 in vivo reduces atherosclerosis development. (A) Whole aortas from the aortic arch to the iliac bifurcation were

stained en face with Oil Red O to visualize neutral lipids (red). Representative aortas from each group (upper panels), with quantification of plaque area as

the percentage of total area (lower panel). (B) Aortic sinus sections were stained with Oil Red O and quantified for plaque area. (C) Immunofluorescent

staining with MOMA-2 was used to detect macrophages in the plaque of aortic sinus sections. Original magnification 350. n = 14. *p , 0.05, **p , 0.01.

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composition of the plaques was similar. However, the total plaquearea was reduced in IL-37 BMT mice, reflecting an overall re-duction in macrophage infiltration. Therewas noticeably less IL-37+

staining in IL-37 mice than EGFP staining in EGFP BMT mice,possibly as a result of reduced infiltration of IL-37–expressingmacrophages. Taken together, the data described above supportthe anti-inflammatory function of IL-37 that was likely a signifi-cant contributing factor in the reduction in atherosclerosis devel-opment in vivo.In terms of lipid metabolism, IL-37 expression inmacrophages led

to a reduction in modified LDL uptake, as shown by the reduced DiI-labeled AcLDL and OxLDL content of the cells after 4 h of in-cubation, as well as by reduced BODIPY staining in IL-37–expressing macrophages, indicating lower neutral lipid content.Aside from the obvious benefit that this would have in reducingfoam cell formation, it may have anti-inflammatory benefits, be-cause activated plaque macrophages become especially pathogenicwhen they take up modified LDL and become lipid-laden foamcells (2, 33). Although we observed decreased uptake of modifiedLDL in macrophages as a result of IL-37 expression, we did notobserve changes in cholesterol efflux to the acceptors ApoA1 orhigh-density lipoprotein at multiple time points (data not shown).Taken together, the reduction in modified lipid uptake by IL-37expression, coupled with its well-established role in preventinginflammation, are likely the two key components of its protectivemechanism against the development of atherosclerosis in vivo.Although the goal of expressing IL-37 specifically in macro-

phages to deliver the anti-inflammatory cytokine to the plaquemicroenvironment was successful, the relatively high concentrationof IL-37 protein in the sera of IL-37 mice at the 4- and 14-wk timepoints implicates a possible systemic role for IL-37 in reducingatherosclerosis as well. Circulating IL-37 protein could haveinfluenced the function of other immune cells, thereby also re-ducing systemic inflammation. The number of circulating mono-cytes, CD4+ and CD8+ T cells, neutrophils, and B cells betweengroups was not significantly different, although it is possible thatthe inflammatory state or infiltration of other leukocytes couldhave been affected by circulating IL-37. Although the systemicreduction in inflammation was not pronounced, the reduction inM-CSF likely played an important role in preventing the recruit-ment of monocytes/macrophages to the plaque and, in turn, re-ducing atherosclerosis development.IL-37, similar to other IL-1 family cytokines, is processed by

caspase-1 and can then be secreted to act extracellularly, or it cantranslocate to the nucleus and act as a transcription factor (34, 35).It has recently been discovered that a complex consisting of IL-37,IL-18Ra, and the decoy receptor SIGIRR is necessary for IL-37 tofunction extracellularly to inhibit inflammatory signaling (31).The separate mechanisms of action were not investigated in thisstudy, and the particular contribution of each in preventing ath-erosclerosis development remains to be determined. The results ofthis study warrant further investigation of IL-37 as a potentialagent for therapy against atherosclerosis, as well as various otherinflammatory diseases.

AcknowledgmentsWe thank E. Raines from the University of Washington for generously do-

nating the CD68S–HA–EGFP retroviral vector and for many helpful tips.

We also thank Monica Montgomery, Whitney Regan, and Martin Alcala for

excellent technical support. This work was performed within the Russian

Government Program of Competitive Growth of Kazan Federal University.

DisclosuresThe authors have no financial conflicts of interest.

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