Macrophage-Specific Expression of IL-37 in Hyperlipidemic Mice Attenuates Atherosclerosis

This information is current as Sara McCurdy, Yvonne Baumer, Emma Toulmin, Bog-Hieu of September 29, 2021. Lee and William A. Boisvert J Immunol published online 13 October 2017 http://www.jimmunol.org/content/early/2017/10/13/jimmun ol.1601907 Downloaded from

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 13, 2017, doi:10.4049/jimmunol.1601907 The Journal of Immunology

Macrophage-Specific Expression of IL-37 in Hyperlipidemic Mice Attenuates Atherosclerosis

Sara McCurdy,* Yvonne Baumer,* Emma Toulmin,* Bog-Hieu Lee,† and William A. Boisvert*,‡

Atherosclerosis, the progressive buildup of plaque within arterial vessels, can lead to fatal downstream events, such as attack or stroke. A key event contributing to the development of atherosclerosis is the infiltration of 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 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 , 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 –derived macrophages via retroviral transduc- Downloaded from tion. Macrophage IL-37 expression in vitro resulted in decreased mRNA (e.g., IL-1B, IL-6, and IL-12) and secreted 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 http://www.jimmunol.org/ 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.

he first pathological stage of atherosclerosis begins with Inhibiting inflammation has been a central tenet in the pursuit of the attraction of immune cells, specifically monocytes, to innovative therapies to treat or prevent atherosclerosis development. T the inflamed endothelial lining of medium and large ar- IL-37 is a recently discovered anti-inflammatory cytokine be- teries. Monocytes transmigrate through the endothelium to the longing to the IL-1 family. There are five known splice variants, of by guest on September 29, 2021 intima (1). Chronic inflammation and dysregulation of cholesterol which IL-37b is the best-characterized isoform, found predomi- metabolism by macrophages within the plaque are the major nantly in cells of the immune system (6, 7). The precursor IL-37b driving forces of atherosclerosis progression (2). Macrophages isoform is expressed at low levels under steady-state conditions encounter and take up modified lipoproteins (3), which leads to (8), and its expression is upregulated under inflammatory condi- activation of pattern recognition receptors and TLR expression, tions induced by LPS and other TLR ligands (9). IL-37 expression promoting the production of inflammatory immune mediators (4, 5). has been strongly associated with many inflammatory human diseases (10–15), including acute coronary syndrome (16). Al- *Center for Cardiovascular Research, John A. Burns School of Medicine, University though no murine homolog for IL-37 has been identified, the † of Hawaii, Honolulu, HI 96813; Department of Food and Nutrition, College of human protein is functional in the mouse, and studies have been Biotechnology and Natural Resources, Chung-Ang University, Anseong 17546, Re- public of Korea; and ‡Institute of Fundamental Medicine and Biology, Kazan Federal carried out using an IL-37b–expressing transgenic mouse model University, Kazan 420008, Russia (9). These mice have been shown to be strongly protected from ORCID: 0000-0001-7400-0773 (Y.B.). various inflammatory conditions (9, 17, 18), defining a protective Received for publication November 8, 2016. Accepted for publication September 12, role for IL-37 in the suppression of pathogenic inflammation. 2017. Because macrophages are central to disease pathogenesis in var- This work was supported by Chung-Ang University research grants in 2015. S.M. ious ways from the early to late stages of atherosclerosis, they provide was supported by a predoctoral grant from the American Heart Association. a valuable delivery system for expression of anti-inflammatory me- Address correspondence and reprint requests to Dr. William A. Boisvert or Dr. Bog- Hieu Lee, Center for Cardiovascular Research, University of Hawaii, John A. Burns diators, such as IL-37, to alter the plaque microenvironment (19). The School of Medicine, 651 Ilalo Street, BSB 311-C, Honolulu, HI 96813 (W.A.B.) or anti-inflammatory benefits of IL-37 have been widely accepted, and Department of Food and Nutrition, College of Biotechnology and Natural Resources, its potential as a therapeutic measure for reducing the pathogen- Chung-Ang University, 72-1, Naeri, Daedeok, Anseong 17546, Republic of Korea (B.-H.L.). E-mail addresses: [email protected] (W.A.B.) or [email protected] (B.-H.L.) esis of atherosclerosis has been discussed (20), yet exploration of The online version of this article contains supplemental material. its effects on plaque development has been limited to systemic Abbreviations used in this article: AcLDL, acetylated low-density lipoprotein; treatment with rIL-37 (21, 22). The delivery of IL-37 directly to BMDM, bone marrow-derived macrophage; BMT, bone marrow transplantation; the plaque microenvironment via macrophage-specific expression EdU, 5-ethynyl-29-deoxyuridine; EGFP, enhanced GFP; EV, empty vector; HFD, has not been investigated. The aims of this study were to test the high-fat diet; HSC, hematopoietic stem cell; LDL, low-density lipoprotein; LN, lymph node; OxLDL, oxidative low-density lipoprotein; PFA, paraformaldehyde; effects of IL-37b in vitro with regard to macrophage inflammatory Ph-E, Phoenix Ecotropic; RT, room temperature; RT-qPCR, real-time quantitative response and cholesterol homeostasis, as well as in vivo via bone PCR. marrow transplantation (BMT) of transduced hematopoietic stem 2/2 Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 cells (HSCs) to atherosclerosis-prone Ldlr mice to determine

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601907 2 IL-37 ATTENUATES ATHEROSCLEROSIS the potential role of macrophage-expressed IL-37 in the preven- sonicated using a probe sonicator (Thermo Fisher) and centrifuged at tion of atherogenesis. 400 3 g for 5 min at 4˚C to pellet cell debris. Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Thermo Fisher). Precast NuPAGE Novex 4–12% Bis-Tris Protein Gels (Invitrogen) were Materials and Methods used for SDS-PAGE. SDS loading buffer (43) was added to 10–20 mgof Retroviral vector generation protein and heated to 95˚C for 5 min. Gels were run in 13 NuPAGE MES SDS Running Buffer (Invitrogen). The protein was then transferred to a The CD68S–HA–EGFP retroviral vector was a generous gift from low-fluorescence polyvinylidene difluoride membrane using Efficient Dr. E. Raines (University of Washington, Seattle, WA). The vector was Western Transfer Buffer (catalog number 786-019; G-Biosciences). The constructed as described previously (19) and uses a segment of the human membranes were blocked in LI-COR blocking buffer for 1 h. EGFP CD68 promoter to drive macrophage-specific expression of an en- (Abcam) or IL-37 (R&D Systems) primary Abs were diluted 1:1000 in LI- hanced GFP (EGFP) reporter gene. CD68–HA–EGFP or CD68 empty COR blocking buffer and incubated at room temperature (RT) for 1 h. LI- vector (EV) were used as the control for all transduction experiments, as COR secondary Abs were diluted in LI-COR blocking buffer at RT for 1 h. described. The cDNA encoding human IL-37b was obtained from a After three additional 5-min washes with 0.1% PBST, the membranes were commercial CMV6 plasmid purchased from OriGene (Rockville, MD), visualized on a LI-COR Odyssey infrared scanner, and the images were and the restriction enzymes NotI and HindIII (New England Biolabs) were analyzed using LI-COR Image Studio software (both from LI-COR Bio- used to subclone IL-37b–Myc–DDK cDNA in the place of HA-EGFP sciences). cDNA. Detecting IL-37 protein by ELISA Transfection of Phoenix Ecotropic cells A DuoSet ELISA for human IL-37 (R&D Systems) was used to analyze High-titer retroviral supernatants were generated by transfecting Phoenix cell culture supernatants or mouse serum samples for human IL-37 protein. Ecotropic (Ph-E) packaging cells (American Type Culture Collection) with One hundred microliters of undiluted cell culture supernatant or mouse 3 Downloaded from the retroviral constructs CD68S-EGFP or CD68S–IL-37b using 2 HEPES- serum samples diluted 1:4 with 1% BSA in PBS were added to the plate in buffered saline and 2 M CaCl2, as described previously (23), with the ex- duplicate, and the assay was performed according to the manufacturer’s ception that Ph-E cells were not selected using puromycin, and retrovirus- instructions. containing cell supernatants were collected at 48 and 72 h for use in HSC transduction. Ph-E cells were grown in DMEM/F12 supplemented with 10% RNA extraction, cDNA synthesis, and real FBS and 1% penicillin/streptomycin. time–quantitative PCR Isolation and culture of HSCs from bone marrow Total RNA was recovered from cells or tissues with TRIzol Reagent (Life http://www.jimmunol.org/ Bone marrow was isolated from 8–12-wk-old male C57BL/6 mice. The Technologies). A volume of chloroform equal to 20% of the volume of femur and tibia bones were flushed with ice-cold PBS, and the cells were TRIzol was added, and samples were inverted vigorously to mix. The passed through a 40-mm cell strainer. RBCs were lysed using multispecies samples were centrifuged at 12,400 rpm for 15 min in a precooled centrifuge RBC lysis buffer (eBioscience), according to the manufacturer’s instruc- set to 4˚C. The aqueous phase was carefully pipetted into a new tube, and tions, and cells were plated in stem cell compete medium (DMEM/F12 an equal volume of nuclease-free 70% ethanol was added. Samples were supplemented with 15% FBS, 1% penicillin/streptomycin, 100 ng/ml mixed well, and RNA was isolated using the purification columns provided recombinant mouse , 20 ng/ml recombinant human IL-6, with the QIAGEN RNeasy RNA extraction kit. An on-column DNA digest and 10 ng/ml recombinant mouse IL-3 [all cytokines were from Pepro- was performed for 15 min at RT. RNA concentrations and quality were Tech]). The stem cells were plated at a density of 25 3 106 cells per 10 ml measured on a NanoDrop 2000. One microgram of total RNA per sample of medium in 10-cm petri dishes (non– culture treated) and allowed was transcribed to cDNA using the qScript cDNA Synthesis Kit (Quanta by guest on September 29, 2021 to cycle for 2 d. Biosciences). Real-time quantitative PCR (RT-qPCR) was performed using SYBR Green 23 master mix with ROX (Roche), and samples were plated Retroviral transduction of HSCs in triplicate in a 384-well quantitative PCR plate and run on an Applied Biosystems 7900HT Fast Real-Time PCR System. Retroviral transduction of HSCs was performed as described previously (23). In brief, the cycling HSCs in complete stem cell medium were Macrophage proliferation in vitro using combined with the appropriate Ph-E retroviral supernatant, plated into 5-ethynyl-29-deoxyuridine fibronectin-coated six-well plates, and centrifuged for 2 h at 37˚C. The following day, the procedure was repeated using fresh retroviral superna- Transduced macrophages (EV versus IL-37b) from C57BL/6 male mice tant. The cells were then collected from the six-well plates, washed with were plated at 2 3 105 cells per well of an eight-well chamber glass slide. PBS, and differentiated in bone marrow–derived macrophage (BMDM) Cells were treated with 5-ethynyl-29-deoxyuridine (EdU) for 6 h in parallel medium (DMEM/F12 GlutaMAX [Invitrogen], supplemented with 10% with FBS supplementation at low (2%) and normal (10%) levels, LPS and heat-inactivated FBS [HyClone], 20% L929-conditioned medium as a IFN-g (10 ng/ml each), or M-CSF (20 ng/ml). Cells were then fixed with source of M-CSF, and 1% penicillin/streptomycin [Life Technologies]) for 4% paraformaldehyde (PFA) for 10 min and parallelized using 0.1% Triton use in vitro, or were resuspended in PBS at 1 3 106 cells/0.1 ml for tail X-100 in PBS for 10 min, and detection of EdU was performed according vein injection into irradiated Ldlr2/2 recipient mice for the in vivo BMT to the manufacturer’s instructions using a Click-iT EdU Alexa Fluor 488 atherosclerosis study. Imaging Kit. Cells were washed with PBS, and nuclei were stained with DAPI. In vitro BMDM culture Macrophage apoptosis in vitro For in vitro culture of transduced BMDMs, the cells were plated at a density of 1 3 106 cells per milliliter in 15-cm tissue culture–treated plates BMDMs from C57BL/6 mice were plated in 12-well plates at 7.5 3 105 (Corning) in 25 ml of medium each. Five to ten milliliters of medium was cells per well. Cells were transfected with EV or IL-37b expression plas- added to each plate every other day for a total of 7 d. Adherent differen- mids using Lipofectamine 2000. Twenty-four hours after transfection, cells tiated macrophages were detached using Cell Stripper (Life Technologies), were challenged with camptothecin (2 mg/ml) or an equivalent volume of counted, and replated into the appropriate wells for in vitro experiments. DMSO (1:500) for 6 h. Cells were then harvested using Cell Stripper and L929-conditioned medium was produced by culturing 4.7 3 105 L929 stained with an Annexin V–FITC Apoptosis Detection Kit (catalog number cells (American Type Culture Collection) per T-75 flask in 50 ml of me- BMS500FI-100; eBioscience), according to the manufacturer’s directions. dium (DMEM/F12, 10% FBS, 1% HEPES [Life Technologies], and 1% Cells were then analyzed for Annexin V–FITC and propidium iodide (PI) penicillin/streptomycin) for 1 wk before collecting, sterile filtering, and fluorescence on an Attune NxT Flow Cytometer (Thermo Fisher) to measure storing the supernatant at 280˚C. apoptosis. Western blot Cholesterol uptake using DiI-labeled cholesterol RIPA buffer (10 mM Tris-Cl [pH 7.6], 1 mM EDTA, 1% Triton X-100, 0.1% For cholesterol uptake, transduced macrophages (EV versus IL-37b) from sodium deoxycholate, 0.1% SDS, 140 mM NaCl) was used for all lysate C57BL/6 male mice were plated at 2.5 3 105 cells per well in an 8-well preparations. Cells grown in six-well plates were washed with PBS and chamber glass slide for analysis by microscopy or were plated at 1 3 106 lysed with 100 ml of ice-cold lysis buffer supplemented with protease and cells per well in a 12-well plate for analysis by flow cytometry. The phosphatase inhibitor cocktails (both diluted 1:100; Roche). Samples were macrophages were treated or not with LPS to induce IL-37 expression and The Journal of Immunology 3 were also treated with 20 mg/ml DiI-labeled acetylated low-density lipo- to the company’s instructions. The mouse serum samples were also ana- protein (AcLDL) or oxidized low-density lipoprotein (OxLDL) for 4 h. lyzed for triglyceride levels using a Triglyceride Colorimetric Assay Kit The cells in the 12-well plates were detached with Cell Stripper and fixed (item number 10010303; Cayman Chemical), according to the manufac- for 10 min with 4% PFA before being analyzed by flow cytometry for turer’s instructions. intensity of DiI fluorescence. The cells treated in the glass slides were also fixed for 10 min with 4% PFA and then analyzed with an epifluorescence Luminex magnetic bead assay microscope using a Rhodamine filter. A custom multiplex magnetic bead assay (R&D Systems) was used to Cholesterol uptake using AcLDL and BODIPY staining measure 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, and Differentiated macrophages from the bone marrow of C57BL/6 male mice TNF-a. The assay was performed according to the manufacturer’s instruc- were transfected using Lipofectamine 2000 (EV versus IL-37b) and plated tions, and the median fluorescence intensity was recorded on a Luminex at 2 3 105 cells per well in an 8-well chamber glass slide for analysis by analyzer with a threshold of 50 events per bead and a flow rate of 60 ml/min. microscopy or were plated at 7.5 3 105 cells per well in a 12-well plate for analysis by flow cytometry. The macrophages were treated or not with LPS Analysis of atherosclerotic plaque and IFN-g (10 ng/ml each) and were also treated or not with 40 mg/ml Whole aortas isolated from the study mice were cleaned of the adventitial fat AcLDL for 6, 12, or 24 h. The cells in the 12-well plates were detached and any branching arteries, cut open longitudinally, excised, and pinned to a with Cell Stripper and fixed for 10 min with 4% PFA. Cells were then wax-lined dissecting tray. A working solution of Oil Red O was prepared centrifuged and resuspended in PBS containing the lipid stain BODIPY fresh for each stain, as described previously (23). The pinned aortas were (1:500; Invitrogen) for 30 min at RT. After washing cells twice with PBS, washed once with 60% isopropanol and then incubated completely covered they were analyzed for green fluorescence intensity, representing lipid with Oil Red O working solution for 15 min. The aortas were then washed content, by flow cytometry using an Attune NxT Flow Cytometer (Thermo with 60% isopropanol three or four times, photographed, and analyzed for Fisher). The cells treated in the glass slides were also fixed for 10 min with plaque area using ImageJ. Downloaded from 4% PFA and then analyzed with an epifluorescence microscope using an The fixed collected from the study mice at sacrifice were mounted FITC filter. in O.C.T. (Tissue-Tek) and frozen at 280˚C. The O.C.T.-embedded hearts Transmigration of macrophages were sectioned sagittally using a cryostat through the aortic valve. Serial sections of 5 mm thickness were made, with five representative sections Using a 5-mm-pore filter (Costar), transduced and differentiated BMDMs spanning the aortic root of each heart selected, and stained with Oil Red O were plated in the upper chamber of the Transwell filter (100,000 cells per to visualize the lesion area. The cryosections were incubated in PBS for 5 min well of a 24-well filter plate) in DMEM/F12 + 0.2% BSA medium. Cells and air dried. Sections were dipped 10 times in 60% isopropanol and in- were given 2 h to become adherent before the medium in the lower chamber cubated in fresh Oil Red O working solution for 15 min. Excess solution was http://www.jimmunol.org/ was replaced with new medium containing the chemoattractant MCP-1 removed, and the slides were dipped 10 times in isopropanol, rinsed for 5 min (25 ng/ml) to stimulate the transmigration of cells through the filter. After in running tap water, and embedded using mounting media (Sigma). 3 overnight incubation in a humidified incubator at 37˚C with 5% CO2, the top Photos of the sections taken at 10 magnification were analyzed for lesion filter chamber was carefully and completely swiped with a Q-tip to remove area using ImageJ and expressed as the percentage of total aortic area. nonmigrated cells. The filter was fixed with 4% PFA for 10 min and stained with the nuclear stain DAPI to visualize and quantify the number of cells Immunohistochemistry of aortic root sections that had migrated through the filter. Photographs of the lower side of the Aortic root sections were stained for MOMA-2 (product code ab33451; filter (three photos per well) were analyzed with ImageJ. Final counts were Abcam). Trypsin Ag retrieval was performed for 15 min at 37˚C, sections compared between control and IL-37–expressing macrophages. All condi- were blocked in 10% normal donkey serum in 2% BSA/PBS, and primary tions were run in triplicate with n = 3 separate experiments. Ab incubation was performed overnight at 4˚C. The sections were washed by guest on September 29, 2021 Mouse atherosclerosis study: irradiation of donor with 0.1% PBS-Tween and incubated with a Cy3-labeled secondary Ab (Abcam) for 1 h at RT. Primary Abs against IL-37 (catalog number mice and BMT AF1975; R&D Systems) or EGFP (product code ab290; Abcam) were used Seven- to eight-week-old male recipient Ldlr2/2 mice (on the C57BL/6 to stain aortic root sections using Donkey anti-Rabbit or Donkey anti-Goat background) were subjected to 1000 rad of whole-body irradiation to de- HRP-conjugated secondary Abs with an AEC detection kit (all from stroy their endogenous HSCs. Half of the irradiated mice (n = 15) received Thermo Fisher), according to the manufacturer’s directions. 6 1–2 3 10 HSCs transduced with CD68S-EGFP and the other half received Statistical analyses HSCs transduced with CD68S–IL-37b via tail vein injections. Injected mice were allowed a 4-wk recovery period for full reconstitution of their immune For the in vivo mouse study comparing two groups with an SD within groups systems before initiation of an atherogenic diet containing 15.8% fat (w/w) , 20% from the mean, which is typical for the plaque and other analyses, a and 1.25% cholesterol (w/w) (Harlan Teklad) for an additional 10 wk. After minimum of 12–14 mice per group is necessary to detect an effect size $ the 4-wk recovery period, 100 ml of blood was collected by submandibular 20% between groups. To account for the potential loss of one or more mice bleeding to confirm immune cell reconstitution by flow cytometry of blood during the study and the unknown effects of IL-37b expression on mice, 15 leukocyte populations and to check for circulating IL-37 protein. mice per group were used for the in vivo study. At the end of the study, the animals were euthanized by CO2 asphyx- For some experimental measurements from the study mice, including iation, and total blood was collected by cardiac puncture of the left ven- circulating leukocyte analysis, BMDM analysis for expression of IL-37 or tricle. The mice were then perfused with ice-cold PBS, followed by 4% EGFP by Western blot, as well as lymph node (LN) transcript analysis, the PFA/5% sucrose. The hearts and aortas were collected and analyzed for SD was small enough to detect significant differences using seven biological plaque, as described below. One hundred microliters of whole blood per replicates per study group, although all other assays were run with n = 14, as mouse was analyzed for immune populations by flow cytometry on a indicated. The Student t test was used to compare differences between FACSAria flow cytometer (BD Biosciences), whereas the remaining blood control and IL-37 groups with significance set at p , 0.05, and one-way was centrifuged at 1500 3 g for 15 min to obtain plasma for the purpose of ANOVA was used for analysis of three or more groups. Statistics calcula- measuring lipid levels, as well as proinflammatory and anti-inflammatory tions for all experiments were done using Prism (GraphPad), with *p , 0.05, cytokine levels by ELISA or Luminex bead array. With regard to the gating **p , 0.001, and ***p , 0.0001. strategy for analysis of blood leukocytes, events were gated for singlets and then dead cells were gated out using a fixable live/dead stain. Next, + + + Results CD45 cells were gated to include all leukocytes. Of all CD45 /CD3 Macrophage-expressed IL-37b reduces proinflammatory gene 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 to and protein expression + + identify monocytes, whereas GR1 /Ly6G cells were considered neutrophils. Expression of human IL-37b in mouse BMDMs was achieved via Analysis of serum cholesterol and triglyceride levels retroviral transduction of HSCs. A macrophage-specific retroviral vector was used to create the CD68S–IL-37b construct (Supplemental Blood cholesterol and triglyceride measurements were performed at the end of the BMT atherosclerosis study from serum samples of all mice from each Fig. 1A, 1B). Retroviral transduction was performed as described group (n = 14). Total cholesterol was measured using a Cholesterol Fluo- previously (19, 23), with alterations as described in Materials and rometric Assay Kit (item number 10007640; Cayman Chemical), according Methods. 4 IL-37 ATTENUATES ATHEROSCLEROSIS

The effect of IL-37 expression on the macrophage inflammatory that OxLDL had a similar inhibitory effect as AcLDL on the response was tested by challenging the transduced BMDMs with expression of IL-1b and IL-6 (data not shown). This provides new various inflammatory stimuli relevant to atherosclerosis. EGFP- or insight into the role of IL-37 in regulating the macrophage in- IL-37–expressing macrophages were left unstimulated or treated flammatory response to modified LDL uptake. with AcLDL, IFN-g,orTNF-a overnight. RT-qPCR analysis revealed reduced expression of inflammatory , such as IL-1a, IL-1b, IL-37b inhibits macrophage transmigration IL-6, IL-12, MIP-1b, and TNF-a, compared with EGFP control The migratory response of monocytes and macrophages toward cells (Fig. 1A). Under conditions of inflammation that would be chemoattractants is a central feature in the pathogenesis of ath- typical of the plaque microenvironment, macrophage IL-37 ex- erosclerosis. To determine whether IL-37 expression effects the pression effectively suppressed inflammatory . migration of macrophages toward the chemoattractant MCP-1, a To determine whether the anti-inflammatory effects of IL-37 on transwell filter assay was performed. As shown in Fig. 1C, IL-37 macrophage gene expression were also applicable for the synthesis expression significantly reduced macrophage transmigration and secretion of inflammatory cytokines, cell supernatants from compared with EV controls. Interestingly, IFN-g pretreatment EGFP- and IL-37–transduced BMDMs, which were left unsti- increased EV control macrophage transmigration, but it did not mulated or were treated with AcLDL, were analyzed by Ab array. have a significant effect on IL-37 macrophages. IL-37 expression resulted in the suppression of inflammatory pro- tein production under basal conditions (Fig. 1B, left panel). In- Expression of IL-37b suppresses proliferation and flammatory mediators, such as ICAM1, IL-6, M-CSF, and MIP-1a, apoptosis of macrophages were all downregulated by IL-37 expression in untreated mac- Because macrophage proliferation and apoptosis are important Downloaded from rophages. A more potent anti-inflammatory effect of IL-37 ex- features of atherosclerosis, we sought to determine whether IL-37b pression is seen after AcLDL treatment (Fig. 1B, right panel), in has an impact on these cellular properties. To determine the effect which a majority of cytokines tested were downregulated com- of IL-37 expression or treatment with rIL-37 on macrophage pared with EGFP control, including IL-1a, IL-6, IFN-g,CXCL9, proliferation in vitro, BMDMs were treated with EdU for 6 h in MIP-1a, and M-CSF. We also wanted to determine whether parallel with FBS supplementation at low (2%) and normal (10%)

treating the BMDMs with a more physiologically relevant modi- levels, LPS and IFN-g (10 ng/ml), or M-CSF (20 ng/ml). Under http://www.jimmunol.org/ fied low-density lipoprotein (LDL), OxLDL, would have similar different conditions, including low and normal serum supple- effects as treatment with AcLDL. We were able to confirm mentation, inflammatory stimulus (IFN-g+LPS), and stimulation by guest on September 29, 2021

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. The Journal of Immunology 5 of proliferation (M-CSF), we found that IL-37 suppresses mac- rophage proliferation (Fig. 2A). An additional experiment was performed using EdU in vivo to determine whether the observed reduction 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, the mice were sacrificed, and tissues were isolated, digested, and stained for analysis by flow cytometry. We found strong trends of reduced EdU incorporation in CD45+/CD11b+ cells, as well as myeloid cells (using ROSA22/LysM-Cre reporter mice) from the blood and myeloid cells in the aorta, although the differences were not 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 a 1:500 dilution of 1 mg/ml camptothecin for 6 h to induce apo- ptosis. Cells were then harvested, stained with Annexin V–FITC and PI, and analyzed by flow cytometry. Compared with controls,

IL-37–transfected BMDMs showed reduced apoptosis at baseline, Downloaded from as well as when challenged with DMSO or the apoptosis-inducing chemical camptothecin (Fig. 2B). Effect of IL-37b expression on macrophage cholesterol processing

The uptake of modified lipoprotein within the plaque is central to http://www.jimmunol.org/ foam cell formation. Inhibiting cholesterol uptake or increasing cholesterol efflux is beneficial in preventing foam cell formation and protects against plaque growth and development. To investigate whether IL-37 affects macrophage cholesterol uptake, we treated EV- or IL-37–transduced macrophages with DiI-labeled AcLDL and 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 LDL FIGURE 2. IL-37b expression suppresses proliferation and apoptosis of compared with controls, with a significant decrease in AcLDL and BMDM. (A) Primary BMDMs from C57BL/6 mice were plated in eight- by guest on September 29, 2021 OxLDL uptake, implying a protective role for IL-37 in preventing well chamber slides at 2 3 105 cells per well. To determine the effect of foam cell formation. This was confirmed by performing BODIPY IL-37 expression or treatment with rIL-37 on macrophage proliferation staining of EV- or IL-37b–transfected BMDMs incubated with in vitro, cells were treated with EdU for 6 h in parallel with FBS supple- AcLDL for 6, 12, or 24 h and treated or not with LPS/IFN-g. mentation at low (2%) and normal (10%) levels, LPS and IFN-g (10 ng/ml), Although 12 h of treatment with AcLDL+ IFN-g/LPS was the or M-CSF (20 ng/ml). Cell were fixed, and EdU was detected using a only condition that resulted in a significant increase in lipid content 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 compared with AcLDL treatment alone, there was a trend toward 12-well plates at 7 3 105 cells per well. After transfection with EV or IL-37 increased lipid uptake with IFN-g/LPS at 6 and 24 h (Fig. 4A). The expression plasmids, the BMDMs were challenged with DMSO or a 1:500 significant decrease in lipid content seen in IL-37–transfected dilution of 1 mg/ml camptothecin for 6 h to induce apoptosis. Cells were macrophages after 6 h, with or without IFN-g/LPS stimulation, was then harvested, stained with Annexin V–FITC and PI, and analyzed by no longer apparent after 12 or 24 h, although there was a trend flow cytometry, with quadrants used to determine whether cells were toward less lipid at 12 h (Fig. 4B). live (AnnexinV2/PI2), early apoptotic (AnnexinV+/PI2), late apoptotic 2 In assessing the effects of IL-37 on lipid efflux, we found that (AnnexinV+/PI+), or dead (AnnexinV /PI+). Results are shown as propor- ABCA1 and PPARg were upregulated with IL-37 overexpression. tions of total cells analyzed (n = 3). *p , 0.05, ***p , 0.001, one-way However, actual cholesterol efflux at multiple time points, whether ANOVA with the Tukey post hoc test. using ApoA1 or high-density lipoprotein as the cholesterol ac- ceptor, was not influenced by IL-37 (data not shown). protein, respectively (Supplemental Fig. 1C, 1D). Various circulating Taken together, the in vitro findings that IL-37 expression re- immune cell populations were analyzed by flow cytometry to con- duces macrophage inflammation and migration and inhibits firm successful repopulation with the donor marrow (Supplemental modified LDL uptake support a strong athero-protective role for Fig. 2A). Circulating leukocytes were analyzed again after the study macrophage-expressed IL-37. To test whether these in vitro find- mice had been on a high-fat diet (HFD) for 10 wk (Supplemental ings hold true in the complex setting of atherosclerosis, an in vivo Fig. 2B), with no differences observed between groups before and 2/2 study using atherosclerosis-prone Ldlr mice was performed. after HFD. Serum cholesterol and triglyceride levels, as well as 2 2 body weight before and after HFD, also were not different be- BMT of EGFP or IL-37b HSCs into Ldlr / mice tween groups (Supplemental Fig. 2C, 2D). Transplantation of EGFP- or IL-37b–transduced HSCs into lethally irradiated Ldlr2/2 recipient mice was performed as previously de- EGFP and IL-37 expression in BMT mice scribed (23). A portion of transduced HSCs was differentiated into ELISA analysis of serum samples for the presence of IL-37 protein macrophages in vitro and analyzed by PCR, as well as by Western revealed a range of concentrations in the IL-37 BMT mice, with no blot and flow cytometry, for the presence of IL-37 transcript and expression detected in the EGFP control mice (Fig. 5A). IL-37 6 IL-37 ATTENUATES ATHEROSCLEROSIS

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)(DiIinred, nuclei stained with DAPI in blue) and quantified by flow cytometry (B and C) for DiI-labeled AcLDL and DiI-labeled OxLDL, respec- Downloaded from tively (n =3).*p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/ serum levels were measured following 10 wk on an HFD, with At sacrifice, the femurs from seven mice per group were IL-37 BMT sera concentrations in a similar range as pre-HFD, harvested, and the bone marrow was cultured for analysis of whereas EGFP mice did not show detectable IL-37 protein BMDMs. As seen in Fig. 5B, clear expression of EGFP or IL-37 (Fig. 5A). was detectable by Western blot in each group. by guest on September 29, 2021

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 7 3 105 cells per well in a 12-well plate for analysis by flow cytometry or at 2 3 105 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 BODIPY at 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. The Journal of Immunology 7

Finally, aortic root sections were analyzed for the presence of athero-protective effect of macrophage-expressed IL-37b in EGFP or IL-37 in the plaque areas using immunohistochemistry lesion-prone Ldlr2/2 mice. Aortic root sections from EGFP or IL-37 (Fig. 5E). The staining of EGFP and IL-37 within the BMT mice were also analyzed for plaque area following staining respective BMT mice confirmed the presence of transduced mac- with Oil Red O. As seen in Fig. 6B, aortic root plaque area was rophages within the atherosclerotic plaque. significantly reduced in IL-37 BMT mice compared with EGFP BMT mice, further supporting the athero-protective role of IL-37 Systemic inflammation is reduced by macrophage IL-37 in vivo. expression in vivo To gain a general view of the inflammatory state of the BMT mice, The presence of macrophages and smooth muscle cells in the lumbar LNs collected at sacrifice were analyzed for inflammatory atheroma is not affected by IL-37 gene expression by RT-qPCR. LN transcripts from IL-37 mice Aortic root sections were subjected to immunofluorescent staining showed a significant reduction in IL-6 and IL-1b expression to detect macrophages or smooth muscle cells within the plaque. compared with LNs from EGFP mice (Fig. 5C), indicating that Each aortic root section was imaged at 203 magnification and IL-37 expression had a systemic anti-inflammatory effect in vivo. analyzed with ImageJ to determine macrophage-positive areas. Additionally, serum samples from EGFP and IL-37 BMT mice MOMA-2 staining is shown for EGFP and IL-37 sections in were analyzed with a Luminex magnetic bead assay. The absolute Fig. 6C, along with the macrophage-positive area, expressed as a quantities of circulating inflammatory cytokine levels measured percentage of total plaque for quantification. Although the total were not significantly different between IL-37 and EGFP BMT plaque area was reduced in IL-37 mice, there was no difference mice (Supplemental Fig. 3). However, linear regression analysis observed in macrophage content per plaque area between the Downloaded from revealed a significant negative correlation between IL-37 protein EGFP and IL-37 groups, indicating that the composition of the abundance and M-CSF levels in the serum of IL-37 BMT mice plaque was very similar in the two groups. (R = 20.7071, p = 0.0047) (Fig. 5D), implying a systemic anti- inflammatory role for IL-37 in vivo. There was no significant correlation for each of the other cytokines analyzed, although Discussion The experiments detailed in this study demonstrate that

there was a positive trend between IL-37 and IL-4 serum con- http://www.jimmunol.org/ centrations (Supplemental Fig. 3B). macrophage-specific IL-37 expression leads to suppression of inflammation, cholesterol uptake, cell proliferation, and apoptosis, Macrophage-expressed IL-37 reduces total plaque area in 2/2 as well as macrophage transmigration in vitro. Furthermore, BMT Ldlr mice of IL-37–transduced HSCs resulted in reduced plaque develop- To analyze the atherosclerotic plaque development in EGFP and ment and decreased systemic inflammation in atherosclerosis- IL-37 BMT mice, the entire aortas were opened and stained en face prone Ldlr2/2 mice compared with controls. To our knowledge, with Oil Red O to visualize atherosclerotic plaque. Aortas from this is the first study elucidating the role of macrophage-expressed IL-37 BMT mice displayed significantly less plaque area compared IL-37 in the context of atherosclerosis, and it provides key evi-

with EGFP control mice (Fig. 6A), clearly demonstrating the dence for future investigation into its potential therapeutic value in by guest on September 29, 2021

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. 8 IL-37 ATTENUATES ATHEROSCLEROSIS Downloaded from

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. http://www.jimmunol.org/ the treatment and prevention of human atherosclerosis, as well as importance of limiting its expression to prevent the disease. other chronic inflammatory diseases. M-CSF protein production was reduced by IL-37 expression in Expression of various inflammatory cytokines known to be unstimulated and AcLDL-stimulated macrophages in vitro. In detrimental in the context of atherosclerosis (24) was reduced by addition, circulating levels of M-CSF in IL-37–transduced BMT macrophage IL-37 expression in vitro. This protective effect of IL-37 mice were negatively correlated with serum IL-37 protein con- was observed after classical inflammatory stimuli, such as IFN-g centration. and TNF-a, as well as AcLDL treatment, indicating that IL-37 The observation that IL-37 expression reduces macrophage by guest on September 29, 2021 also protects against a type of inflammation caused by modified transmigration toward MCP-1 corresponds with the reduction in LDL that has not previously been reported. IL-37 retains its anti- inflammatory cytokines and chemoattractant molecules seen in IL- inflammatory properties, which is crucial for effective function in 37–expressing macrophages. MCP-1 secretion from IL-37–expressing a state of hyperlipidemia. macrophages was reduced in vitro, although the serum levels were Inflammatory mediators implicated in atherogenesis that were not detectable. Thus, it is unclear whether they differed between reduced by IL-37 in vitro and in vivo include IL-1b,IL-6,and study groups in vivo. Although not explored in this study, a pos- M-CSF. IL-6 is associated with unstable angina in humans (25) sible mechanism for IL-37 in reducing macrophage migration and is known to stimulate production of matrix-degrading enzymes could involve decreased phosphorylation of various kinases and by macrophages (26). IL-37 expression in macrophages consistently transcription factors, as shown by Nold-Petry et al. (31) to phys- reduced IL-6 transcript and protein expression in vitro and in vivo, ically decrease the chemotactic response. as evidenced by reduced IL-6 transcripts in LNs isolated from IL-37 In addition, the reduced proliferation of macrophages by IL-37 mice compared with controls. Serum IL-6 levels were undetect- could be a major contributing factor to the attenuated plaque size able by Luminex analysis (data not shown); thus, it remains un- found in IL-37 BMT mice compared with controls, because the vast clear whether IL-37 had an effect on circulating levels of IL-6 majority of the plaque is composed of macrophages at this early protein. stage. Recently, Robbins et al. (32), using a symbiosis model of Macrophage expression of IL-37 led to reduced IL-1a and IL-1b atherosclerosis-prone CD45.1 and CD45.2 mice put on an HFD, gene and protein expression in vitro, as well as decreased IL-1b showed that the majority of BrdU+ proliferating macrophages transcripts in the LNs of IL-37 BMT mice compared with con- within developed lesions were from the host parabiont rather than trols. It has been shown that, within minutes of macrophage in- from the donor, indicating that proliferation is a significant con- flammatory activation, IL-37 expression closely follows that of tributor to the macrophage content of lesions. Because rIL-37 also IL-1a and IL-1b (9), with the gene regulatory regions of all three strongly inhibited macrophage proliferation, it is very likely that genes found in the IL-1 coming into close proximity fol- the IL-37–expressing macrophages within the plaque microenvi- lowing LPS stimulation (27). This programmed expression of IL-37 ronment secrete IL-37 that could act to reduce the proliferation of during the inflammatory response likely acts to modulate exces- nearby macrophages, even if they did not express IL-37. The sive inflammation. observation that macrophage apoptosis is decreased by IL-37 in- M-CSF gene expression has long been known to be linked to dicates that necrotic core formation may be attenuated in IL-37– atherosclerosis development (28), and its expression within the expressing mice, because macrophage death is associated with the plaque leads to differentiation of macrophages and expression of presence of a necrotic core. scavenger receptors (29). M-CSF deficiency in ApoE-knockout Of note, the macrophage content of the aortic root plaque did not mice results in reduced plaque formation (30), emphasizing the differ between EGFP and IL-37 BMT mice, indicating that the The Journal of Immunology 9 composition of the plaques was similar. However, the total plaque References area was reduced in IL-37 BMT mice, reflecting an overall re- 1. Moore, K. J., and I. Tabas. 2011. Macrophages in the pathogenesis of athero- duction in macrophage infiltration. 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