IL-19 Halts Progression of Atherosclerotic Plaque, Polarizes, and Increases Cholesterol Uptake and Efflux in Macrophages

Abstract

Atherosclerosis regression is an important clinical goal, and treatments that can reverse atherosclerotic plaque formation are actively being sought. Our aim was to determine whether administration of exogenous IL-19, a Th2 cytokine, could attenuate progression of preformed atherosclerotic plaque and to identify molecular mechanisms. LDLR(-/-) mice were fed a Western diet for 12 weeks, then administered rIL-19 or phosphate-buffered saline concomitant with Western diet for an additional 8 weeks. Analysis of atherosclerosis burden showed that IL-19-treated mice were similar to baseline, in contrast to control mice which showed a 54% increase in plaque, suggesting that IL-19 halted the progression of atherosclerosis. Plaque characterization showed that IL-19-treated mice had key features of atherosclerosis regression, including a reduction in macrophage content and an enrichment in markers of M2 macrophages. Mechanistic studies revealed that IL-19 promotes the activation of key pathways leading to M2 macrophage polarization, including STAT3, STAT6, Kruppel-like factor 4, and peroxisome proliferator-activated receptor γ, and can reduce cytokine-induced inflammation in vivo. We identified a novel role for IL-19 in regulating macrophage lipid metabolism through peroxisome proliferator-activated receptor γ-dependent regulation of scavenger receptor-mediated cholesterol uptake and ABCA1-mediated cholesterol efflux. These data show that IL-19 can halt progression of preformed atherosclerotic plaques by regulating both macrophage inflammation and cholesterol homeostasis and implicate IL-19 as a link between inflammation and macrophage cholesterol metabolism.

Figure 1

IL-19 administration induces plaque stasis. A: Representative photomicrograph of aortic arch from LDLR^−/− mice after consuming the atherogenic diet for 12 weeks, injected with either PBS or 10 ng/g IL-19 per day. Surface lesion en face stained with Sudan IV. B: Graphic depiction of atherosclerotic lesion size quantitated from en face-stained aortic arches as depicted in panel A. IL-19 does not modify serum lipids or weight. C: Representative photomicrographs of aortic root stained with Oil Red O from mice treated with 10 ng/g IL-19 or PBS per day. D: Quantitation of lesion area from four transverse serial sections from the aortic sinus to disappearance of valve cusps per aortic root from mice were stained with Oil Red O, and positive-stained areas were quantitated. E: Cholesterol and triglycerides in mice fed an atherogenic diet for 12 weeks receiving either PBS or 10 ng/g IL-19 per day do not statistically differ between groups at time of euthanasia. F: Weight gain does not statistically differ between control and IL-19 groups. G: IL-19 reduces macrophage infiltrate in vivo. Aortic roots were recovered from mice, and lesion areas from transverse serial sections from the aortic sinus to disappearance of valve cusps per aortic root from mice were immunostained with antibody to CD68. H: Positively stained areas were quantified as a percentage of total lesion area by quantitative morphometry. Data are expressed as means ± SEM. n = 7 each (B, E, and F); n = 8 mice per group, using at least three sections per aortic root (H). ^∗∗P < 0.01. LDLR^−/−, low-density lipoprotein receptor knockout; PBS, phosphate-buffered saline.

Figure 2

IL-19 polarizes plaque macrophage to the M2 phenotype. A: RT-qPCR on RNA extracted from aorta of mice removed at the end of the study which received either PBS or rIL-19. RNA was amplified with the primer pairs for the macrophage phenotypes shown. B: RT-qPCR on RNA extracted from spleen from mice receiving either PBS or 10 ng/g IL-19 per day. Spleens were removed at the end of the study, RNA was extracted, reverse transcribed, and amplified with the primer pairs shown. C and D: Increased expression of M2 macrophage markers (C) and Th2 lymphocyte markers (D) in spleen of IL-19–treated mice as detected by flow cytometry. White bars, PBS; black bars, rIL-19. Data are expressed as means ± SEM. n = 6 aorta per group (A); n = 6 spleen each group (C and D). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. PBS, phosphate-buffered saline; RT-qPCR, quantitative RT-PCR.

Figure 3

IL-19 reduces macrophage inflammation. A: IL-19 reduces expression of inflammatory cytokines in stimulated BMDMs. BMDMs were pretreated with IL-19 for 8 hours, then TNF-α to induce expression of proinflammatory cytokines. RNA was extracted and subject to quantitative RT-PCR with the use of primers for the indicated proinflammatory cytokines. B: Representative immunoblot of lysates from BMDMs treated as described above, blotted with antibody for the indicated cytokines. C: Densiometric quantification of three independent Western blot analyses of BMDMs stimulated with TNF-α, IL-19, or together. Data are expressed as means ± SEM. n = 3 Western blot analyses (C). ^∗P < 0.05, ^∗∗P < 0.01. BMDM, bone marrow-derived macrophage; MCP, monocyte chemoattractant protein; TNF, tumor necrosis factor.

Figure 4

IL-19 polarization of BMDMs to M2 phenotype is KLF4 dependent. A: BMDMs were treated with IL-19 for 24 hours, RNA was subjected to RT-qPCR with the use of the primer pairs shown. B: Representative Western blot analysis of IL-19 induction of KLF4 expression. Lysates from BMDMs stimulated with IL-19 for the indicated time was immunoblotted with anti-KLF4 antibody. C: Densiometric quantification of independent Western blot analyses of BMDMs stimulated with IL-19 blotted for KLF4 protein expression. D: IL-19 expression is induced in human macrophages stimulated with M2 stimuli. Representative immunoblot showing lysates from human macrophages treated with IFN-γ and LPS to induce M1, or IL-4 to induce the M2 phenotype, blotted with the indicated antibody. E: Densiometric quantification of independent Western blot analyses of BMDMs treated with IFN-γ and LPS to induce M1, or IL-4 to induce the M2 phenotype, and blotted for IL-19 and KLF4 expression. KLF4 knockdown reduces IL-19–driven M2 polarization. F: KLF4 siRNA reduces IL-19–induced KLF4 protein abundance. G: BMDMs transfected with KLF4 siRNA were then stimulated with IL-19, and mRNA abundance for M2 associated genes were quantitated by RT-PCR. H: Percentage of inhibition was calculated from scrambled controls stimulated with IL-19. Data are expressed as means ± SEM. n = 3 independent Western blot analyses (C and E), ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. BMDM, bone marrow-derived macrophage; C, control; IFN, interferon; KLF, Kruppel-like factor; LPS, lipopolysaccharide; PPAR, peroxisome proliferator-activated receptor; RT-qPCR, quantitative RT-PCR; unstim, unstimulated.

Figure 5

IL-19 activates STAT proteins and PPARγ. A: Representative immunoblot of BMDMs stimulated with IL-19 for the indicated times. Extracts were blotted with anti-Phospho STAT3 (Tyr705), total STAT3, anti-phospho STAT6, or total STAT6. B: Densiometry was performed and values were normalized to total protein. C: IL-19 increases PPARγ mRNA expression. RNA isolated from BMDMs treated with IL-19 for 24 hours was subjected to quantitative RT-PCR with the use of PPARγ-specific primers. D: IL-19 increases PPARγ protein expression. Lysates from BMDMs treated with IL-19 underwent Western blot analysis with the use of anti-PPARγ antibody. E: Lysates from human macrophages were treated with IL-19 and underwent Western blot analysis with the use of anti-PPARγ antibody. F: STAT inhibition reduces IL-19–driven PPARγ protein expression. Lysates from inhibitor-treated BMDMs were blotted with the indicated antibody. G: STAT inhibition reduces IL-19–driven Ym1, Arg1, and PPARγ mRNA abundance. BMDMs treated with STAT-specific inhibitors were stimulated with IL-19, and mRNA abundance for M2 associated genes was quantitated by RT-PCR. H: Percentage of inhibition calculated from IL-19–stimulated vehicle controls. I: IL-19 activates PPARγ transcriptional activity. BMDMs were transfected with a PPARγ luciferase reporter plasmid and stimulated with IL-19, the PPARγ activator Rosiglitazone, the PPARγ inhibitor GW9664, or combinations thereof. PPARγ response element activation was quantitated by luciferase activity. Data are expressed as means ± SEM. n = 3 or more experiments (B); n = 3 or more Western blot analyses (D–F). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. BMDM, bone marrow-derived macrophage; PPAR, peroxisome proliferator-activated receptor; unstim, unstimulated.

Figure 6

IL-19 induces cholesterol uptake and expression of lipid scavenger receptors. A: BMDMs isolated from wild-type C57B/6 mice were incubated with rIL-19, then with fluorescent Dil-oxLDL, and were washed, and cholesterol uptake was quantitated by flow cytometry. B–D: BMDMs were incubated with rIL-19 for the indicated times, and RNA was extracted and subjected to quantitative RT-PCR with the use of primers for the shown lipid scavenger receptors. E: Representative Western blot analysis of BMDMs treated with IL-19. Lysates made 24 hours after stimulation were subjected to Western blot analysis with the use of antibody for the shown lipid scavenger receptors. F: Densiometry was performed, and values were normalized to total protein G: Representative immunoblot of lysates from human macrophages treated with IL-19 blotted with antibody for the indicated lipid scavenger receptors. H: IL-19–driven increase in oxLDL uptake does not increase apoptosis. No significant difference in Casp3/7 luminescence is noted between IL-19–treated and untreated groups. Data are expressed as means ± SEM. n = 3 experiments or more (F). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. BMDM, bone marrow-derived macrophage; Casp3/7, caspase 3/7; MFI, mean fluorescence intensity; oxLDL, oxidized low-density lipoprotein.

Figure 7

IL-19 regulates cholesterol metabolism via PPARγ. A: BMDMs transfected with PPARγ siRNA effectively reduce PPARγ protein expression, and PPARγ knockdown reduces IL-19–mediated expression of cholesterol uptake receptors. BMDMs transfected with PPARγ siRNA were treated with rIL-19 for 24 hours, then underwent Western blot analysis for the indicated cholesterol scavenger receptors with the use of specific antibody. B: Quantitation of protein expression by densiometry. C: PPARγ knockdown reduces IL-19–mediated cholesterol uptake. BMDMs were transfected with PPARγ siRNA, then treated with rIL-19, then with fluorescent DiL-oxLDL, washed, and uptake was quantitated by flow cytometry. Data are expressed as means ± SEM. n = 3 or more Western blot analyses. ^∗P < 0.05 for scrambled control versus siRNA; ^∗∗P < 0.01. BMDM, bone marrow-derived macrophage; MFI, mean fluorescence intensity; oxLDL, oxidized low-density lipoprotein; PPAR, peroxisome proliferator-activated receptor.

Figure 8

IL-19 induces cholesterol uptake and expression of cholesterol transport proteins. A and B: BMDMs was incubated with rIL-19 for the indicated times, RNA was extracted and subjected to quantitative RT-PCR with the use of primers for the shown cholesterol transport proteins. C: Representative Western blot analysis of BMDMs treated with IL-19. Lysates made 24 hours after stimulation were subjected to Western blot analysis with the use of antibody for the shown cholesterol transport proteins. D: BMDMs transfected with PPARγ siRNA effectively reduces IL-19–mediated expression of cholesterol transport proteins. E: Densiometry was performed, and values were normalized to total protein F: IL-19 induces cholesterol efflux. BMDMs were incubated with acLDL, then 16 hours with or without IL-19. Cells were incubated with apoA1, in the presence or absence of IL-19, and efflux to ApoA1 was quantitated. Data are expressed as means ± SEM. n = 3 or more experiments (E). ^∗P < 0.05, ^∗∗P < 0.01. apoA1, apolipoprotein A1; BMDM, bone marrow-derived macrophage; LDL, low-density lipoprotein.