Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

Larissa Cardilo-Reis; Sabrina Gruber; Sabine M Schreier; Maik Drechsler; Nikolina Papac-Milicevic; Christian Weber; Oswald Wagner; Herbert Stangl; Oliver Soehnlein; Christoph J Binder

doi:10.1002/emmm.201201374

Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

Cardilo-Reis, Larissa; Gruber, Sabrina; Schreier, Sabine M; Drechsler, Maik; Papac-Milicevic, Nikolina; Weber, Christian; Wagner, Oswald; Stangl, Herbert; Soehnlein, Oliver; Binder, Christoph J 2012-10-02 00:00:00 OPEN TRANSPARENT Research Article ACCESS PROCESS IL-13 protects from atherosclerosis Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype 1,2 1,2 3 4 Larissa Cardilo-Reis , Sabrina Gruber , Sabine M. Schreier , Maik Drechsler , 1,2 4 1 3 Nikolina Papac-Milicevic , Christian Weber , Oswald Wagner , Herbert Stangl , 4 1,2 Oliver Soehnlein , Christoph J. Binder Keywords: alternatively activated Atherosclerotic lesions are characterized by the accumulation of oxidized LDL macrophages (M2); atherosclerosis; (OxLDL) and the inﬁltration of macrophages and T cells. Cytokine expression in cytokines; interleukin-13; oxidized LDL the microenvironment of evolving lesions can profoundly contribute to plaque development. While the pro-atherogenic effect of T helper (Th) 1 cytokines, such as IFN-g, is well established, the role of Th2 cytokines is less clear. Therefore, we DOI 10.1002/emmm.201201374 characterized the role of the Th2 cytokine interleukin (IL)-13 in murine athero- sclerosis. Here, we report that IL-13 administration favourably modulated the Received March 14, 2012 Revised July 16, 2012 morphology of already established atherosclerotic lesions by increasing lesional Accepted August 03, 2012 collagen content and reducing vascular cell adhesion molecule-1 (VCAM-1)- dependent monocyte recruitment, resulting in decreased plaque macrophage content. This was accompanied by the induction of alternatively activated (M2) macrophages, which exhibited increased clearance of OxLDL compared to IFN-g- activated (M1) macrophages in vitro. Importantly, deﬁciency of IL-13 results in accelerated atherosclerosis in LDLR mice without affecting plasma cholesterol levels. Thus, IL-13 protects from atherosclerosis and promotes a favourable plaque morphology, in part through the induction of alternatively activated macrophages. INTRODUCTION ﬁbrosis in the vessel wall of arteries (Binder et al, 2002; Hansson & Hermansson, 2011). T cells are a prominent component of the Atherosclerosis is a chronic inﬂammatory disease of the artery atherosclerotic plaque and exhibit signs of activation, including wall, whose pathogenesis is inﬂuenced by dyslipidemia (high the expression of cytokines (Ait-Oufella et al, 2011; Hansson & LDL/low high density lipoprotein, HDL) and inﬂammation Hermansson, 2011). Substantial evidence supports that T helper (Hansson, 2005). Atherosclerotic lesions are characterized by (Th) 1-driven responses, mainly characterized by IFN-g the accumulation of oxidized LDL (OxLDL), the inﬁltration of production, are detrimental and correlate with progression of activated macrophages and T cells, as well as cell death and atherosclerosis (Buono et al, 2003, 2005; Gupta et al, 1997; Tellides et al, 2000). These pro-atherogenic responses have been shown to be dampened by the presence of speciﬁc regulatory (1) Department of Laboratory Medicine, Medical University of Vienna, T cells, which secrete the anti-atherogenic cytokines transform- Vienna, Austria (2) Center for Molecular Medicine (CeMM) of the Austrian Academy of ing growth factor (TGF)-b and interleukin (IL)-10 (Ait-Oufella Sciences, Vienna, Austria et al, 2011). Indeed, IL-10 secreted by T cells decreases (3) Department of Medical Chemistry, Center for Pathobiochemistry and atherogenesis in mice (Mallat et al, 1999; Pinderski Oslund Genetics, Medical University of Vienna, Vienna, Austria et al, 1999). On the other hand, the role of Th2 responses in (4) Institute for Cardiovascular Prevention, Ludwig-Maximilians University lesion formation is more complex. For example, previous Munich, Munich, Germany studies reported a pro-atherogenic role for IL-4 (Davenport & *Corresponding author: Tel: þ43 1 4040073755; Fax: þ43 1 4040073588; Tipping, 2003; King et al, 2002), while a later report found no E-mail: [email protected] 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License (CC BY-NC 3.0), which permits use, distribution 1072 and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. effect (King et al, 2007). We have shown previously that the and reduces macrophage content of existing lesions. The latter / / atheroprotective immunization of LDLR mice with malon- was conﬁrmed in cholesterol-fed ApoE mice, and found to be dialdehyde-modiﬁed LDL (MDA-LDL) induced a Th2-biased a consequence of decreased vascular cell adhesion molecule-1 immune response that was characterized by antigen-speciﬁc (VCAM-1)-dependent monocyte recruitment. In addition, IL-13 production of IL-5 and IL-13 but only small amounts of IL-4 and administration induces a switch of the lesional macrophage IFN-g. In the same study, we demonstrated the capacity of IL-5 phenotype in vivo towards alternative activation. Macrophages, to stimulate natural atheroprotective IgM speciﬁc for OxLDL and which were alternatively activated with IL-13 in vitro, have an its ability to protect from atherosclerosis (Binder et al, 2004). increased capacity of clearing OxLDL by promoting its uptake as In addition to their role in providing help for B cells to secrete well as cholesterol efﬂux without increasing net foam-cell antibodies, another potentially important function of cytokines formation. Moreover, we show that LDLR mice that were in atherogenesis is their capacity to modulate the activation state reconstituted with bone marrow of IL-13-deﬁcient mice develop of macrophages. Continuous recruitment of monocyte/macro- accelerated atherosclerosis. phages into lesions has been shown to be related to plaque progression (Gautier et al, 2009; Peters & Charo, 2001; Potteaux et al, 2011; Swirski et al, 2006). IFN-g (and LPS) can promote RESULTS classical activation of macrophages (M1), which is known to be pro-inﬂammatory and may promote atherogenesis (Khallou- IL-13 administration modulates established atherosclerosis Laschet et al, 2010). On the other hand, IL-4 and/or IL-13 induce To study the capacity of IL-13 to inﬂuence existing athero- alternative macrophage activation (M2), which results in potent sclerosis, we performed an intervention study in which IL-13 anti-inﬂammatory and tissue repair capacities (Gordon & was administered exogenously to mice with established Martinez, 2010). Thus, the cytokine proﬁle of the microenvi- atherosclerotic lesions. LDLR mice were fed an atherogenic ronment can profoundly affect the activation state of macro- diet for 16 weeks and received biweekly intraperitoneal phages and their function, e.g. in foam-cell formation. Recently, injections of IL-13 or phosphate-buffered saline (PBS) during Khallou-Laschet et al (2010) showed that lesion-inﬁltrated the last 5 weeks of diet. Based on our observation that / / macrophages of young ApoE mice exhibit predominantly the atherosclerotic LDLR mice have IL-13 serum levels of M2 phenotype, while M1 macrophages were dominant in more approximately 2.5 ng/ml, we decided to administer 50 ng of advanced lesions of aged mice and their presence correlated IL-13 per LDLR mouse twice per week, which corresponds to with lesion progression. These data suggest a potentially only threefold higher amounts of systemic IL-13 per day. At time atheroprotective role of anti-inﬂammatory M2 macrophages of sacriﬁce, mice were not different with respect to body weight, and the cytokines involved in alternative macrophage activa- total plasma cholesterol (TC), triglycerides (TG), or levels of tion. Importantly, the presence of anti-inﬂammatory M2 total IgG1 or IgG2c antibodies (Table 1). Furthermore, there macrophages has also been documented in human carotid were no differences in the frequencies of splenic T or B cells atherosclerotic lesions (Bouhlel et al, 2007). (Table 1). Stimulated splenocytes from atherosclerotic LDLR IL-13 is exclusively produced by hematopoietic cells, mice treated with PBS or IL-13 produced similar amounts of Th2 including activated Th2 cells and the recently discovered (IL-4, IL-5, IL-10 and IL-13) and Th1 (IFN-g) cytokines, nuocytes (Barlow & McKenzie, 2011; Oliphant et al, 2011). indicating that IL-13 administration at this dose did not alter Because IL-4 and IL-13 share a common receptor signalling the overall Th1/Th2 balance (Fig S1 of Supporting Information). pathway (IL-4Ra/IL-13Ra1/STAT6) similar functions for both In addition, no induction of liver or lung ﬁbrosis due to cytokines have been described (Chomarat & Banchereau, 1998; the continuous IL-13 injection was observed (unpublished Kuperman & Schleimer, 2008). However, IL-13 also possesses observations). As expected with the relatively short time of functions that differ from those of IL-4 (Chiaramonte et al, 1999; administration only during the last 5 weeks of a 16-week feeding Kumar et al, 2002; Liang et al, 2011; McKenzie et al, 1998b; period, there were no differences in the extent of atherosclerosis Oriente et al, 2000). For example, IL-13 can initiate signalling via in the cross-sectional analyses of the aortic origin or in the the alternative IL-13 receptor IL-13Ra2, which exclusively binds entire aorta by en face analyses between atherosclerotic IL-13 and which has been shown to induce TGF-b1 production LDLR mice treated with PBS or IL-13, respectively (Fig 1A in macrophages via a STAT6-independent pathway, leading to and Table 1). collagen deposition in vivo (Fichtner-Feigl et al, 2006). Although As plaque size was similar between the two groups, we were it has been assumed that IL-13 affects atherosclerosis in able to directly compare lesion composition. Morphological the same way as IL-4, no studies are currently available to analyses of the cross-sectional lesions showed no signiﬁcant support this notion (Tedgui & Mallat, 2006). Therefore, we differences in the necrotic core area, suggesting that both groups tested the role of IL-13 in atherosclerotic lesion formation. Based of mice had the same stage of lesion development (Fig S2A of on the prominent functions of IL-13 in inducing ﬁbrosis and Supporting Information). In contrast, the collagen content was alternative macrophage activation, we hypothesized that IL-13 signiﬁcantly higher in atherosclerotic LDLR mice treated might have an atheroprotective role by modulating plaque with IL-13 compared to PBS-treated mice (Fig 1B), while the composition. smooth-muscle cell content remained unchanged between Here, we demonstrate that exogenous administration of IL-13 the groups (Fig S2B of Supporting Information). These results to cholesterol-fed LDLR mice promotes collagen formation suggest that IL-13 injections stimulated the production of EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1073 Research Article www.embomolmed.org IL-13 protects from atherosclerosis Table 1. Overview of experimental data of the IL-13 administration study / / LDLR inj! PBS (n¼ 11) LDLR inj! IL-13 (n¼ 13) Atherosclerosis 4 2 Aortic origin (10 mm /section) 48.16 3.56 47.15 1.22 En face (% of aorta) 6.63 0.94 6.22 0.66 Metabolic parameters Weight (g) 23.6 0.7 22.5 0.5 TC (mg/dl) 1493 49 1362 93 TG (mg/dl) 863 85 777 81 Serum antibody titers Total IgM (mg/ml) 0.97 0.11 0.85 0.11 Total IgG1 (mg/ml) 1.11 0.10 0.97 0.11 Total IgG2c (mg/ml) 1.84 0.16 1.58 0.16 Serum chemokines CCL2/MCP-1 (pg/ml) 59.1 8.3 69.3 8.1 CXCL1/KC (pg/ml) 1709 247 1535 181 Characterization of PEC Total peritoneal cells (10 ) 3.38 0.52 2.22 0.29 CD5 T cells (% of total) 20.40 3.74 15.25 2.71 CD11b Mac cells (% of total) 13.96 2.16 18.78 2.32 CD19 B cells (% of total) 53.25 3.43 51.84 3.29 þ þ CD11b CD5 B1a cells (% of B cells) 27.24 2.71 24.56 3.29 CD11b CD5 B1b cells (% of B cells) 16.95 1.35 19.37 1.47 þ þ CD19 CD23 B2 cells (% of B cells) 28.43 3.39 32.69 3.68 Characterization of PBC Total white blood cells/ml (10 ) 1.40 0.18 1.59 0.25 þ lo CD11b Ly6C monocytes (% of total) 4.11 0.34 4.96 0.43 þ hl CD11b Ly6C monocytes (% of total) 2.59 0.36 2.15 0.36 þ þ CD11b Ly6G neutrophils (% of total) 3.97 0.46 4.51 0.65 Characterization of splenocytes Total spleen cells/ml (10 ) 67.3 11.3 62.3 9.4 CD43 T cells/ml (% of total) 24.83 2.26 23.39 1.42 B220 B cells/ml (% of total) 45.35 3.32 46.67 1.92 þ þ CD43 IgM B1 cells/ml (% of B cells) 16.29 1.27 17.16 1.22 CD43 IgM B2 cells/ml (% of B cells) 81.76 1.21 80.97 1.19 Atherosclerosis in the aortic origin was analyzed by cross-sections through the aortic origin and values represent the average mm /section. En face measurements are given in percent lesion area of the entire aorta. TC, total serum cholesterol; TG, serum triglycerides; PEC, peritoneal exudate cells; PBC, peripheral blood cells. Data are shown as mean SEM. PEC cellular populations as measured by ﬂow cytometry. PBC cellular populations as measured by ﬂow cytometry. Spleen cellular populations were analyzed by ﬂow cytometry. collagen by either macrophages or smooth-muscle cells rather IL-13 administration reduces macrophage content in lesions than the proliferation of these cells, consistent with the pro- of atherosclerotic ApoE mice, independent of macro- ﬁbrotic function of IL-13. In addition, the numbers of T cells were phage egress not different between the two groups (Fig S2C of Supporting The observed morphological changes of atherosclerotic lesions Information). Remarkably, immunohistological analyses of following IL-13 administration suggested the possibility of mac-3 macrophages uncovered a signiﬁcant reduction in macrophage egression. Because CCR7 has been implicated as a lesional macrophages in the IL-13-treated mice (Fig 1C). These key chemokine receptor in this, we ﬁrst evaluated CCR7 differences in macrophage numbers were not due to changes in expression in lesions of the cholesterol-fed LDLR mice that circulating blood cell counts, as total numbers of white blood were treated with either IL-13 or PBS, respectively. There was no hi lo þ cells, monocytes (both classical Ly6C and nonclassical Ly6C ) signiﬁcant difference in the percentage of CCR7 lesion area or neutrophils in the peripheral blood of mice were similar between the two groups, suggesting no major contribution of (Table 1). Moreover, the plasma levels of CCL2/MCP-1 and CCR7-mediated emigration (Fig S3 of Supporting Information). CXCL1/KC, the two major chemokines involved in monocyte We then directly evaluated the ability of IL-13 to induce recruitment, were not reduced in IL-13-injected mice (Table 1). macrophage egress from existing lesions using a method that is Taken together, our data showed that IL-13 modulates based on the tracking of ﬂuorescent bead-labelled monocyte/ established atherosclerotic lesions by inducing a more stable macrophages in ApoE mice (Potteaux et al, 2011). To achieve plaque composition with higher collagen content and fewer comparable lesion development in these mice, they were fed an macrophages. atherogenic diet for a total of 6 weeks and received PBS or IL-13 1074 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. A B C PBS -/- LDLR inj IL-13 *** 70 1500 0 0 -/- -/- 0 50 100 150 200 250 300 LDLR inj PBS IL-13 LDLR inj PBS IL-13 µm PBS IL-13 PBS IL-13 PBS IL-13 Figure 1. IL-13 administration alters plaque morphology of established atherosclerotic lesions. A. LDLR mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n¼ 11) or IL-13 (n¼ 13) during the last / 2 5 weeks. Equal extent of atherosclerotic lesion size in cross-sections of the aortic origin in injected LDLR mice. Values represent mm /section throughout the entire aortic origin (300 mm). Original magnification 50. B. Increased collagen content in lesions of LDLR mice injected with IL-13. Sections were stained with Sirius Red for the presence of collagen, and values represent the percentages of Sirius Red area/total lesion area. p¼ 0.035. Original magnification 100. C. Decreased macrophage content in lesions of LDLR mice injected with IL-13. Sections were stained with the macrophage-specific anti-mac-3 antibody and þ 2 values represent the numbers of mac-3 cells/mm of total lesion area. p¼ 0.0007. Original magnification 100. All data are mean SEM values of all mice of each group. Images show representative examples of the respective stainings. during the last 2 weeks of diet. Following depletion of counts, similar plasma levels of important chemokines, and no monocyte/macrophages by clodronate-liposome injection effect on macrophage egression, we hypothesized that IL-13 20 days after initiation of the atherogenic diet, circulating reduced the recruitment and adhesion of monocytes to the monocytes were labelled with latex-beads to monitor lesional atherosclerotic wall. To elucidate this directly, we assessed the macrophage migration. At day 28, one-third of mice were adhesion of different leukocyte populations by intravital gfp/wt sacriﬁced to obtain a baseline number of beads per plaque microscopy of carotid arteries of Cx cr1 mice that were area in the aortic root. The other mice were divided into available on an ApoE background. Following injection of two groups and received biweekly injections of either IL-13 a ﬂuorescent antibody to GR1 (Ly6C/G), this animal model or PBS, respectively, for the remaining 2 weeks (Fig 2A). At enabled us to differentiate, at the same time, between the time of sacriﬁce, cross-sections of the aortic origin were adhesion of two subtypes of circulating monocytes (nonclassical hiGFP þ loGFP analyzed for the presence of ﬂuorescent beads as well as Gr1 Cx cr1 and classical Gr1 Cx cr1 ) and neutro- 3 3 þ þ mac-2 cells. The numbers of beads per plaque area at phils (Gr1 gfp ). Indeed, the adhesion of circulating monocytes week 6 were not different between PBS- and IL-13-injected to carotid arteries was signiﬁcantly decreased in atherosclerotic animals and comparable to the numbers at baseline mice injected with IL-13 compared to PBS-treated mice (week 4), indicating that IL-13 had no effect on macrophage (Fig 3A), whereas adhesion of circulating neutrophils was egression (Fig 2B). Nevertheless, macrophage content of the similar (Fig 3A). In addition, the adhesion of the two monocyte same lesions was signiﬁcantly lower in IL-13-injected mice subsets was equally reduced (unpublished observation), compared to PBS-injected mice, thereby conﬁrming our initial suggesting that IL-13 administration reduces monocyte observation in a different atherosclerosis-prone mouse strain recruitment through a common pathway. Thus, we hypothe- (Fig 2C). sized that IL-13-induced changes may result in decreased Thus, IL-13 limits lesional macrophage content by a endothelial cell activation. To test this, we performed intravital mechanism other than macrophage emigration. microscopy of carotid arteries of ApoE mice that were fed an atherogenic diet for a total of 6 weeks and received PBS IL-13 administration results in decreased VCAM-1 expression or IL-13 during the last 2 weeks of diet, followed by the and monocyte adhesion in lesions of atherosclerotic ApoE injection of ﬂuorescently labelled beads coupled with anti- mice in vivo VCAM-1 or anti-CCL2 antibodies to analyze the carotid To explain the decreased numbers of macrophages in athero- endothelial activation state in these mice. VCAM-1 expression sclerotic lesions despite unchanged peripheral blood monocyte was signiﬁcantly decreased in IL-13-treated mice (Fig 3B), EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1075 4 2 Lesion area (x10 µµµµm ) Collagen content (%) Macrophage content + 2 (mac-3 cells/mm ) Research Article www.embomolmed.org IL-13 protects from atherosclerosis Figure 2. IL-13 administration has no effect Monocyte Baseline or on macrophage egress from established Clodronate Labeling PBS vs. IL-13 atherosclerotic lesions. ApoE mice were fed an injections (beads) treatment Analysis atherogenic diet for 4 weeks (baseline) or 6 weeks (PBS and IL-13, respectively). After 3 weeks, circulating monocytes of all mice were labelled with fluorescent latex beads following clodronate- 24h liposome depletion 24 h before. One week later, one 0 3 4 6 group of mice was sacrificed for baseline (n¼ 7) Weeks on atherogenic diet measurements of bead and macrophage content and the remaining mice received biweekly B C intraperitoneal injections with either PBS (n¼ 7) or PBS -/- ApoE inj IL-13 (n¼ 8) until sacrifice. 250 IL-13 *** A. Diagram of experimental design. n.s B. Similar content of fluorescent beads per plaque area in the aortic root of PBS and IL-13 admin- istration, respectively, compared to baseline. C. Decreased macrophage content in lesions of ApoE mice injected with IL-13. Sections were 100 stained with the macrophage-specific anti-mac-2 antibody and values represent the percentages of þ þ mac-2 cells/total DAPI cells. p¼ 0.0001. All data are shown as mean SEM values of all mice of each group. -/- ApoE inj PBS IL-13 Weeks on atherogenic diet whereas the endothelial presentation of CCL2 was similar in IL-13 administration skews macrophage phenotype towards both groups (Fig S4 of Supporting Information). alternatively activated macrophages (M2) in vivo These data strongly suggest that the decrease in lesional Because IL-13 is known to induce alternative macrophage macrophage content observed in IL-13-treated mice is activation, we investigated whether IL-13 administration had caused by a reduction in the VCAM-1-dependent monocyte the capacity to generate M2 macrophages in vivo. Indeed, we recruitment. observed a shift within the macrophage population towards A B * *** PBS IL-13 12 35 PBS -/- ApoE inj IL-13 30 2 5 Monocytes Neutrophils PBS IL13 -/- ApoE inj Figure 3. IL-13 administration reduces VCAM-1-dependent monocyte recruitment in established atherosclerotic lesions. gfp/wt / A. Cx3cr1 ApoE mice were fed an atherogenic diet for 6 weeks and received biweekly intraperitoneal injections with PBS (n¼ 8) or IL-13 (n¼ 8) during the last 2 weeks. At the time of sacrifice, PE-conjugated anti-GR1 antibodies were injected intravenously and the number of monocytes (GFP cells) and neutrophils (PE cells) adhering to the carotid bifurcation was assessed by intravital microscopy. Values represent the numbers of cells/optical field. p¼ 0.0185, Mann–Whitney test. B. ApoE mice were fed an atherogenic diet for 6 weeks and received biweekly intraperitoneal injections with PBS (n¼ 8) or IL-13 (n¼ 8) during the last 2 weeks. At time of sacrifice, fluorescent anti-VCAM-1 beads were injected intravenously and the number of beads adhering to the carotid bifurcation was assessed by intravital microscopy. Values represent the numbers of beads/optical field. p¼ 0.0001. Representative microscopy images are shown. Bar: 100 mm. All data are shown as mean SEM values of all mice of each group. 1076 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 Leukocytes adhering to beads/plaque area the carotid bifurcation (cells/field) α α α αVCAM1 beads adhering to Macrophage content + + (%mac-2 cells/DAPI cells) the carotid bifurcation (beads/field) www.embomolmed.org Research Article Larissa Cardilo-Reis et al. þ þ more CD206 macrophages (M2) and less CD80 macrophages fewer M1 macrophages in lesions of IL-13-injected mice (M1) in the peritoneal cavities of IL-13-treated mice as assessed (Fig 4B). Moreover, immunostaining for the expression of by ﬂow cytometry (Fig 4A) and further conﬁrmed by two different M2 markers, Ym-1 and mannose receptor (MR, quantitative polymerase chain reaction (PCR), demonstrating CD206), demonstrated signiﬁcantly higher numbers of M2 a signiﬁcant up-regulation of common M2-related genes, macrophages in lesions of IL-13-injected mice, despite an overall including arginase-1 (Arg-1), chitinase 3-like 3 protein decreased macrophage content (Fig 4C and Fig S5B of (Chi3l3/Ym-1) as well as CCL9, and a concomitant down- Supporting Information, respectively). These changes in regulation of M1-related genes such as inducible nitric oxide macrophage activation states resulted in a signiﬁcantly synthase (iNOS), CD86 and CXCL10 (Fig S5A of Supporting increased M2:M1 ratio in lesions of IL-13-treated mice (Fig 4D). Information). The total numbers of peritoneal macrophages, In summary, we could demonstrate that the decreased T and B cells (including B1 and B2 cells) were not different lesional macrophage content was paralleled by the induction between the two groups (Table 1). Importantly, in analogy to the of M2 macrophages and concomitantly the reduction of M1 activation state of peritoneal macrophages, immunostaining of macrophages in atherosclerotic lesions by exogenous adminis- lesional macrophages for the expression of iNOS demonstrated tration of IL-13. A B * PBS IL-13 -/- LDLR inj PBS IL-13 C D PBS 0.8 *** 0.6 0.4 IL-13 0.2 0.0 -/- -/- LDLR inj PBS IL-13 LDLR inj PBS IL-13 Figure 4. IL-13 administration skews macrophage phenotype towards alternatively activated (M2) macrophages in vivo. A. LDLR mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n¼ 11) or IL-13 (n¼ 13) during the last 5 weeks. Increased frequencies of M2 macrophages and decreased frequencies of M1 macrophages in the peritoneal cavities of LDLR mice injected with IL-13. Peritoneal cells were stained with antibodies against CD80 (M1) and CD206 (M2) and identified by flow cytometry. CD80/CD206 double-positive cells were classified as M1/M2 macrophages. Values represent the percentages of individual macrophage subtypes/total macrophages. p¼ 0.04. B. Decreased numbers of M1 macrophages in lesions of LDLR mice injected with IL-13. Sections were stained with an antibody against iNOS, which is þ 2 specifically expressed by M1 macrophages and values represent the numbers of iNOS cells/mm of total lesion area. p¼ 0.012. C. Increased numbers of M2 macrophages in lesions of LDLR mice injected with IL-13. Sections were stained with an antibody against Ym-1, which is þ 2 specifically expressed by M2 macrophages. Values represent the numbers of Ym-1 cells/mm of total lesion area. p¼ 0.024. D. Increased ratio of M2:M1 macrophages in lesions of LDLR mice injected with IL-13. p¼ 0.0001. All data are shown as mean SEM values of all mice of each group. Images show representative examples of the respective stainings. Original magnification: 400. EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1077 M2 macrophages + 2 (Ym1 cells/mm ) M1 macrophages + 2 (iNOS cells/mm ) M2/M1 ratio Research Article www.embomolmed.org IL-13 protects from atherosclerosis Alternatively activated macrophages by IL-13 show higher stimulated macrophages and to a signiﬁcantly greater extent in clearance of OxLDL in vitro macrophages stimulated with IL-13 (Fig 5A). An increased To study potential effects of differentially activated macro- uptake of CuOx-LDL was further conﬁrmed by Oil Red O (ORO) phages in atherogenesis, we evaluated the ability of IFN-g staining, which revealed a higher percentage of ORO cells in and IL-13 to modulate macrophage foam-cell formation by IL-13-activated macrophages compared to IFN-g-activated promoting either classical (M1) or alternative (M2) macrophage macrophages (Fig S6B of Supporting Information). Consistent activation, respectively. Thioglycollate-elicited macrophages with that, IL-13 stimulation resulted in increased expression of were stimulated with either IFN-g or IL-13, and successful the scavenger receptor CD36, which was further induced after differentiation into M1 or M2 macrophages was conﬁrmed by CuOx-LDL loading (Fig S6C of Supporting Information). the expression of iNOS (M1) and Arg-1 (M2), respectively The expression of scavenger receptor A-1 (SRA-1) and LOX-1 (Fig S6A of Supporting Information). Differentially activated was not different between the two differentially activated macrophages were then incubated with copper-oxidized macrophage foam cells (Fig S6D and S6E of Supporting LDL (CuOx-LDL) to induce foam-cell formation. Subsequent Information). Addition of HDL to the foam-cell cultures analyses of these cells revealed an increase of total cellular signiﬁcantly reduced the increased cellular cholesterol only in cholesterol content in foam-cell cultures derived from IFN-g- IL-13-activated macrophage cultures, thereby abrogating the A B C *** 6 *** *** 50 5 ABCA-1 40 4 n.s 30 3 ABCG-1 ββββ-Actin 0 0 CuOx-LDL 50 µµµµg/mL -+ + - + + CuOx-LDL 50 µµµµg/mL ++ CuOx-LDL 50 µµg/mL µµ -+ - + IFN-γγγγ 100 ng/mL ++ + - - - IFN-γγγγ 100 ng/mL +- IFN-γγγγ 100 ng/mL++ - - IL-13 5 ng/mL --- + + + IL-13 5 ng/mL -+ IL-13 5 ng/mL-- + + HDL 10 µµµµg/mL -- + - - + HDL 10 µµµµg/mL ++ D E F *** 1500 *** 1.75 0.5 *** 1.50 0.4 1000 PBS ** 1.25 0.3 n.s 1.00 IL13 0.75 0.2 0.50 0.1 0.25 0.0 0.00 CuOx-LDL 50 µµµµg/mL -+ - + CuOx-LDL 50 µµµµg/mL -+ - + -/- LDLR inj PBS IL-13 IFN-γγγγ 100 ng/mL ++ - - IFN-γγγγ 100 ng/mL ++ - - IL-13 5 ng/mL IL-13 5 ng/mL -- + + -- + + Figure 5. Alternatively activated macrophages (M2) exhibit increased clearance of OxLDL in vitro. A. Thioglycollate-elicited macrophages were stimulated with IFN-g or IL-13 into classically (M1) or alternatively (M2) activated macrophages, respectively, and then incubated with CuOx-LDL for 24 h to generate foam cells. Increased cellular cholesterol levels in M2-derived foam cells are reduced in the presence of HDL. M1 and M2 macrophages were incubated with CuOx-LDL in the absence or presence of HDL 10 mg/ml. Lipids were extracted from cell lysates and total cholesterol and protein were measured. Data are shown as mean SEM values of two independent experiments performed in quadruplicates and represent mg cholesterol/mg protein. p¼ 0.04, p¼ 0.0001. B. Increased HDL-dependent cholesterol efflux by M2-derived foam cells. M1 and M2 macrophages were incubated with CuOx-LDL plus 1 mMof [ H]-cholesterol and HDL-dependent efflux was assayed as described in Materials and Methods Section. Data represent percentages of HDL-dependent efflux/total efflux. p¼ 0.010, t-test. C. Increased ABCA1 and ABCG1 expression in M2-derived foam cells. Shown is a representative Western blot for the presence of ABCA1, ABCG1 and b-actin in lysates of cells that were treated as indicated. D,E. The quantification of the band intensity of ABCA1 (D) and ABCG1 (E) related to b-actin. p¼ 0.04, p¼ 0.0075, p¼ 0.0001. All data in (B–E) are shown as mean SEM values of three independent experiments performed in triplicates. / / F. Increased ABCA1 expression in lesions of LDLR mice injected with IL-13. LDLR mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n¼ 11) or IL-13 (n¼ 13) during the last 5 weeks. Sections were stained with an antibody against ABCA1 and values þ 2 represent the numbers of ABCA1 cells/mm of total lesion area, p¼ 0.031. Data are shown as mean SEM values of all mice of each group. Images show representative ABCA1 staining. Original magnification 400. 1078 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 ABCA-1 (band intensity) Cholesterol/protein (mg) ABCG-1 (band intensity) HDL-dependent efflux (%) ABCA1 expression + 2 (ABCA1 cells/mm ) www.embomolmed.org Research Article Larissa Cardilo-Reis et al. increased foam-cell formation (Fig 5A). These data suggested a Information). Importantly, cross-sectional analyses of lesions higher cholesterol efﬂux capacity of IL-13-activated compared to in the aortic origin revealed signiﬁcantly accelerated athero- IFN-g-activated foam cells. sclerosis with almost two times larger lesions in IL-13 bone To further test this hypothesis, we performed in vitro marrow recipients, indicating a protective role of IL-13 in cholesterol efﬂux assays using equal amounts of [ H]-choles- atherogenesis (Fig 6A and Table SI of Supporting Information). terol, which demonstrated a signiﬁcantly higher HDL-dependent In addition, en face analyses also showed a trend towards efﬂux of foam-cell cultures derived from IL-13-stimulated increased lesion formation in recipients of IL-13 bone macrophages compared to IFN-g-stimulated macrophages marrow (Table SI of Supporting Information). (Fig 5B). Moreover, to evaluate the ability of IL-13 to directly Lesions of IL-13 bone marrow chimeras displayed increase cholesterol efﬂux in macrophage foam cells, macro- increased necrotic core formation, consistent with advanced phages were ﬁrst loaded with CuOx-LDL plus [ H]-cholesterol plaque progression (Fig 6B). Nevertheless, the relative macro- and then stimulated with IFN-g and IL-13, respectively. phage content was equivalent between the two groups (Fig 6C) Importantly, HDL-dependent efﬂux was signiﬁcantly increased with signiﬁcantly less M2 macrophages in lesions of IL-13 in IL-13-stimulated foam cells using this experimental setup as bone marrow chimeras (Fig 6D). The predominant M1 well (Fig S7 of Supporting Information). We therefore macrophage areas were similar between the two groups investigated the expression levels of the two most important (Fig 6E). transporters responsible for cholesterol efﬂux in macrophages, To investigate potential immunological differences parallel- ATP-binding cassette A1 (ABCA1) and G1 (ABCG1), by ing this increased lesion formation, total splenocytes of mice immunoblotting and found that upon stimulation with CuOx- from both groups were stimulated with anti-CD3 and anti-CD28 LDL the expression of both ABC transporters was signiﬁcantly for 72 h to induce maximal T-cell activation in vitro.As / / up-regulated in foam cells derived from IL-13-activated macro- expected, splenocytes from IL-13 LDLR bone marrow phages compared to IFN-g-activated foam cells (Fig 5C–E) and chimeras produced only minimal amounts of IL-13. Moreover, non-activated foam cells (unpublished observation). This was the production of IL-4 and IL-10, but not IL-5 and IFN-g were also conﬁrmed by quantitative PCR on the messenger RNA signiﬁcantly diminished in these mice (Fig 7A and Table SI of (mRNA) level (Fig S8A and S8B of Supporting Information). In Supporting Information). This selective decrease in Th2 contrast, IFN-g-activated macrophages exhibited only a sig- cytokine production was also reﬂected by a signiﬁcant increase niﬁcant up-regulation of ABCG1 protein following CuOx-LDL in Th1-dependent IgG2c antibodies in serum of the IL-13 stimulation, albeit to a lesser degree than IL-13-activated LDLR bone marrow chimeric mice, while total IgG1 antibody macrophages (Fig 5E). Consistent with that, IL-13 stimulation levels were not different (Fig 7B and Table SII of Supporting also resulted in the increased expression of the nuclear receptor Information). IgM levels were similar between both groups LXRa, which is the main transcription factor controlling ABCA1 (Table SI of Supporting Information). These changes in T-cell- and G1 expression (Fig S8C of Supporting Information). dependent IgG levels resulted in a signiﬁcantly decreased To correlate these in vitro ﬁndings with effects of IL-13 on IgG1:IgG2c ratio indicating an overall Th1-biased response macrophage function in vivo, lesions of cholesterol-fed LDLR (Fig 7C). Similar results were obtained for MDA-LDL-speciﬁc mice that received either IL-13 or PBS (see Fig 1) were analyzed IgG1 and IgG2c titers, respectively (Fig S10A of Supporting for ABCA1 expression by immunohistochemistry. Remarkably, Information). Importantly, the numbers of splenic T cells and atherosclerotic lesions of IL-13-treated mice showed a sig- B cells (including B1 and B2 cells) were not different between niﬁcantly higher number of ABCA1 expressing cells compared the two groups (Table SI of Supporting Information). Consistent to lesions from control mice (Fig 5F). with an inherent Th1 bias, non-atherosclerotic IL-13-deﬁcient Taken together, our data demonstrate that macrophages mice that were used as bone marrow donors were also found to alternatively activated by IL-13 have an overall increased display decreased IgG1 and increased IgG2c levels compared capacity of OxLDL clearance, as they display increased uptake as to wild type controls (Fig S10B of Supporting Information). well as increased cholesterol efﬂux capacities without enhan- These data indicate that IL-13 deﬁciency results in an overall cing foam-cell formation. This should result in a more efﬁcient pro-inﬂammatory environment that inhibits alternative activa- removal of pro-inﬂammatory OxLDL and consequently a less tion of lesional macrophages and promotes atherosclerotic inﬂammatory environment in atherosclerotic lesions. lesion development. IL-13 deficiency accelerates atherosclerosis Finally, to demonstrate the role of IL-13 in the development of DISCUSSION atherosclerotic lesions, we transplanted lethally irradiated / þ/þ LDLR mice with bone marrow from either IL-13 or In the current study, we demonstrate a previously unrecognized IL-13 mice. Four weeks after bone marrow transplantation atheroprotective role of IL-13 in murine models of atherogen- (BMT) and successful replenishment, mice were switched to an esis. It was assumed that IL-13 would have a similar pro- atherogenic diet for the subsequent 16 weeks to induce atherogenic role in atherosclerosis as IL-4, because both atherosclerosis (Fig S9 of Supporting Information). At time of cytokines share similar functions by engaging the same receptor sacriﬁce, the two groups of mice were not different regarding complexes. Our data now point to a differential role in body weight, TC and TG levels (Table SI of Supporting atherosclerosis, which can be explained by unique functions EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1079 Research Article www.embomolmed.org IL-13 protects from atherosclerosis A B -/- IL-13→LDLR ** 35 ** -/- 45 ** -/- ** +/+ ** ** ** 15 +/+ ** 0 50 100 150 200 250 300 350 400 -/- IL-13→LDLR µµµµm +/+ -/- C D E 200 ** 0 0 -/- -/- -/- IL-13→LDLR +/+ -/- IL-13→LDLR +/+ -/- IL-13→LDLR +/+ -/- Figure 6. Increased atherosclerosis in IL-13-deficient LDLR mice. / þ/þ / A. LDLR mice were reconstituted with bone marrow from either IL-13 mice (n¼ 12) or IL-13 mice (n¼ 14) and fed an atherogenic diet for 16 weeks. Increased extent of atherosclerotic lesion size in cross-sections of the aortic origin in mice reconstituted with IL-13 bone marrow. Values represent mm /section throughout the entire aortic origin, (400 mm). p¼ 0.0023. Images show representative H&E stains. Original magnification 50. B. Increased necrotic core area in lesions of recipients of IL-13 bone marrow. Values represent percentages of necrotic core area/total lesion area. p¼ 0.016. / þ/þ C. Lesional macrophage content between recipients of IL-13 or IL-13 bone marrow. Sections were stained with the macrophage-specific anti-mac-3 antibody and values represent the percentages of mac-3 area/cellular lesion area. D. Decreased lesional M2 macrophage content in lesions of recipients of IL-13 bone marrow. Sections were stained with an antibody against Ym-1, which is þ 2 specifically expressed by M2 macrophages and values represent number of Ym-1 cells/mm of cellular lesion area. p¼ 0.0043, Mann–Whitney test. / þ/þ E. Relative lesional M1 macrophage content between recipients of IL-13 or IL-13 bone marrow. Sections were stained with an antibody against iNOS, which is specifically expressed by M1 macrophages and values represent the percentages of iNOS area/cellular lesion area. All data are shown as mean SEM values of all mice of each group. of IL-4 and IL-13 as a consequence of the exclusive engagement and Th1/Th2-dependent IgG isotype levels in sera of these mice. of the alternative receptors IL-4Ra/gc and IL-13Ra2, respec- Our data are consistent with the known impairment in Th2-cell tively, or by differences in ligand afﬁnity for the same IL-4Ra/ development in IL-13-deﬁcient mice (McKenzie et al, 1998a). IL-13Ra1 receptor complex (Kelly-Welch et al, 2003; LaPorte Likely this overall shift in the immune response contributed in et al, 2008). This is exempliﬁed by the ability of only IL-4 to part to the pro-atherogenic effect observed. The profound effects differentiate naı¨ve CD4 T cells into Th2 cells (Seder & Paul, of IL-13 deﬁciency on atherosclerotic lesion formation discour- 1994) or the non-redundant role of IL-13 in parasite expulsion, aged detailed analyses of plaque morphology, as a meaningful allergic inﬂammation and asthma (Bancroft et al, 1998; Grunig interpretation of potential differences in content of lesions of et al, 1998; Liang et al, 2011). different size and stage is virtually impossible. Speciﬁcally, we found that cholesterol-fed chimeric IL-13 To study the atheroprotective effect of IL-13 directly, we LDLR mice develop nearly twofold larger lesions in the aortic examined the impact of exogenous IL-13 administration on / / origin than LDLR mice that were reconstituted with wild type established atherosclerotic lesions in cholesterol-fed LDLR bone marrow. This pro-atherogenic effect of IL-13 deﬁciency mice. To avoid hepatic ﬁbrosis (Wynn, 2008), we chose a low was accompanied by an overall Th1-biased immune phenotype, dose of IL-13 administration to achieve only three times higher as judged by cytokine release of stimulated splenocyte cultures serum levels than found in atherosclerotic mice. Importantly, 1080 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 4 2 Lesion area (x10 µµµµm ) Macrophage content (%) M2 macrophages + 2 (Ym1 cells/mm ) M1 macrophages Necrotic core (%) (% iNOS area/cellular area) www.embomolmed.org Research Article Larissa Cardilo-Reis et al. A B 45 200 2.5 *** 175 n.s 2.0 1.5 1.0 0.5 0 0 -/- -/- -/- IL-13→LDLR IL-13→LDLR +/+ -/- +/+ -/- +/+ -/- +/+ -/- IL-13→LDLR +/+ -/- +/+ -/- αCD3/CD28 IgG1 IgG2c - αCD3/CD28 - 20 3.5 17.5 3.0 *** *** 2.5 12.5 2.0 1.5 7.5 1.0 0.5 2.5 0 0 -/- -/- -/- IL-13→LDLR IL-13→LDLR IL-13→LDLR +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- αCD3/CD28 αCD3/CD28 - - Figure 7. Effect of IL-13 deficiency on splenic cytokine production and antibody isotype levels. / þ/þ / A. LDLR mice were reconstituted with bone marrow from either IL-13 mice (n¼ 12) or IL-13 mice (n¼ 14) and fed an atherogenic diet for 16 weeks. At time of sacrifice, spleens and blood were collected from all mice. Recipients of IL-13-deficient bone marrow show a decreased production of Th2 cytokines (IL-13, IL-4 and IL-10) but not IFN-g by splenocytes stimulated with anti-CD3/CD28 in vitro. Data are presented as ng/ml cytokine of splenocyte cultures p¼ 0.0001. B. Increased levels of total IgG2c antibodies in sera of IL-13-deficient LDLR mice. Data are presented as mg/ml of indicated serum IgG isotypes. p¼ 0.029. C. Decreased ratio of IgG1:IgG2c antibodies in IL-13-deficient LDLR mice. p¼ 0.04. All data are shown as mean SEM values of all mice of each group. this interventional strategy did not result in an alteration of the In this regard it is important to point out that Fisher and Th1/Th2 phenotype of the immune response. Nevertheless, we colleagues recently reported that regression of atherosclerotic discovered that IL-13 administration resulted in signiﬁcantly lesions is associated with the up-regulation of markers of increased lesional collagen content, which is consistent with the alternative macrophage activation (M2) (Feig et al, 2011a,b; known strong pro-ﬁbrotic role of IL-13 (Wynn, 2008), as well as Rayner et al, 2011). M2 macrophages have been documented in signiﬁcantly decreased lesional macrophage content. These murine and human lesions (Bouhlel et al, 2007; Chinetti- alterations in plaque morphology are strongly reminiscent of Gbaguidi et al, 2011; El Hadri et al, 2012; Feig et al, 2012; changes that were reported to occur as a result of lesion Khallou-Laschet et al, 2010). We now show that IL-13 regression induced by lowering serum cholesterol or increasing administration also induces relative and absolute increases in serum HDL in mice (Reis et al, 2001; Rong et al, 2001; Williams M2 macrophages in cholesterol-fed LDLR mice and that the et al, 2008). Of interest, the changes in our model of IL-13 same lesions that show increased collagen and a decreased administration occurred without changes in serum cholesterol macrophage content have signiﬁcantly increased numbers of levels. It remains to be shown whether IL-13 is also alternatively activated macrophages and furthermore, conco- mechanistically involved in atherosclerotic lesion stabilization mitantly decreased numbers of classically activated macro- during lipid lowering or active lesion regression. phages (M1). Because our quantitative assessment of lesional EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1081 IL-4 (ng/mL) IL-13 (ng/mL) IFN-γγγγ (ng/mL) IL-10 (ng/mL) Ratio IgG1/IgG2c Total IgG (mg/mL) Research Article www.embomolmed.org IL-13 protects from atherosclerosis macrophages suggests the presence of still ‘uncommitted’ ment seems to be largely a consequence of decreased macrophages (i.e. iNOS and Ym-1 ), we believe that IL-13 endothelial VCAM-1 expression, which we observed in athero- primarily acts on this particular population of existing sclerotic mice treated with IL-13. A mechanistic role for VCAM-1 macrophages. In contrast, lesions of cholesterol-fed chimeric is further supported by the fact that only recruitment of / / IL-13 LDLR mice displayed decreased numbers of M2 monocytes, but not neutrophils, was affected by the IL-13 macrophages. Although this dual classiﬁcation pattern of intervention. Furthermore, no difference between the recruit- lo hi macrophages has been considered overly simplistic, it helped ment of nonclassical Ly6C or classical Ly6C monocytes was characterizing macrophage heterogeneity and plasticity within observed, which is typically dependent on the expression of atherosclerotic plaques (Feig et al, 2011a,b; Khallou-Laschet speciﬁc chemokines (Combadiere et al, 2008; Ingersoll et al, et al, 2010; Stoger et al, 2010). Our data show that the 2011; Tacke et al, 2007). In agreement with that, we did not macrophage phenotype in atherosclerotic lesions can be observe an alteration of CCL2 presentation by endothelial cells modulated by IL-13 independent of cholesterol-lowering. in the carotid arteries of atherosclerotic ApoE mice injected We also addressed the functional consequences of increased with IL-13. Finally, in analogy to the parallels with lesion numbers of M2 macrophages with respect to uptake of OxLDL regression discussed above, Potteaux et al recently reported that and cholesterol efﬂux, rate limiting steps in foam-cell formation decreased monocyte recruitment during lesion regression was during atherogenesis (Steinberg & Witztum, 2010). Using also associated with decreased endothelial VCAM-1 expression murine primary macrophages differentially activated with (Potteaux et al, 2011). Endothelial VCAM-1 expression is a key either IL-13 or IFN-g, respectively, to model potential extremes event during atherosclerotic lesion formation (Cybulsky et al, of cytokine exposure inside the plaques, we observed an 2001; Dansky et al, 2001). However, it is unlikely that IL-13 increased capacity of IL-13-stimulated macrophages to take up administration had a direct effect on VCAM-1 expression, OxLDL. This is consistent with previous studies showing that as a previous study demonstrated that IL-13 in fact promoted M2 macrophages exhibit increased expression of scavenger the up-regulation of VCAM-1 on activated-endothelial cells receptor CD36 and possess higher phagocytic activity (Berry in vitro (Bochner et al, 1995; Woltmann et al, 2000). It is et al, 2007; Gordon & Martinez, 2010). Previously, IL-4/IL-13 known that VCAM-1 is strongly induced by OxLDL in stimulation has been shown to activate PPARg, leading to endothelial cells in vitro and at lesion prone-sites even before upregulation of CD36 through the generation of endogenous the appearance of visible lesions (Cybulsky & Gimbrone, 1991; ligands in murine and human macrophages (Huang et al, 1999; Khan et al, 1995). Therefore, considering our data, we would Rey et al, 1998), which might be predicted to lead to enhanced speculate that a more likely explanation for the decreased foam-cell formation. Indeed, we could also demonstrate VCAM-1 expression is a reduced intimal content of OxLDL as a increased expression of CD36, but not SRA-1 or LOX-1, in consequence of enhanced IL-13-induced M2 macrophage- IL-13-activated macrophage foam cells. We also found that mediated clearance. OxLDL-loaded IL-13-activated macrophages exhibited a higher In conclusion, our data indicate a key role for IL-13 in halting cholesterol-efﬂux capacity and had increased expression of the progression of atherogenesis and promoting plaque ABCA1 and ABCG1 compared to IFN-g-activated macrophages, stabilization. We provide evidence that IL-13 leads to decreased resulting in no net increase in cholesterol accumulation. Our VCAM-1 mediated monocyte recruitment to atherosclerotic data is supported by a previous report demonstrating decreased lesions, enhanced phenotypic modulation towards the repara- ABCA1 expression and cholesterol-efﬂux of IFN-g-treated tive and atheroprotective M2 phenotype, and enhanced collagen murine foam cells compared to unstimulated foam cells deposition. Even in the absence of decreased plasma cholesterol (Panousis & Zuckerman, 2000). If translatable to the in vivo levels, the changes induced by IL-13 are strongly reminiscent situation, our data suggest an enhanced ability of IL-13- of effects seen during plaque regression in response to stimulated M2 type macrophages to clear OxLDL from the cholesterol lowering. Thus, our ﬁndings identify a potential extracellular environment and efﬁciently promote efﬂux of free new target for the prevention and treatment of atherosclerosis. cholesterol via ABCA1/G1 pathways without enhancing foam- cell formation—a protective response that would be desirable in a lesional macrophage. In fact, we did ﬁnd increased expression MATERIALS AND METHODS of ABCA1 in lesions of IL-13-treated mice. It is noteworthy that Chinetti-Gbaguidi et al recently reported that IL-4-activated An expanded Materials and Methods Section is available in the human monocyte-derived macrophages are less prone to foam- Supporting Information. cell formation, although they found lower efﬂux capacity and increased cholesterol esteriﬁcation in these cells compared to Animal and intervention studies untreated cells. This comparison to ‘neutral’ monocytes and LDL receptor-deficient mice (LDLR ) and C57BL/6J were from The differences between IL-4 and IL-13 may explain the discrepan- Jackson Laboratories (Bar Harbor, Maine, USA); IL-13 mice were a cies (Chinetti-Gbaguidi et al, 2011). kind gift of Dr. Thomas Wynn (NIAID/NIH, Bethesda, USA). All mice A prominent consequence of IL-13 administration was were on a C57BL/6J background (tenth generation) and were bred decreased recruitment of monocytes to carotid arteries of in-house. All experimental protocols were approved by the institu- atherosclerotic ApoE mice, whereas no effect on lesional tional animal experimentation committee and the Austrian Ministry of macrophage egression was observed. The diminished recruit- Science. 1082 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. The paper explained PROBLEM: Atherosclerosis is a chronic inflammatory disease of the vessel activation states of plaque macrophages, and we could show that wall and the underlying cause of heart attacks and a majority of IL-13-activated macrophages (M2) possess more efficient, strokes. Atherosclerotic lesions are characterized by infiltrating beneficial lipid handling capacities compared to IFN-g-activated macrophages that take up accumulating lipids resulting in the macrophages (M1) in vitro. Importantly, atherosclerosis-prone formation of pro-inflammatory foam cells. Innate and adaptive mice that were incapable of secreting IL-13 by hematopoietic immunity modulate the development and progression of cells developed significantly bigger and more advanced ather- atherosclerotic lesions, which provides potential therapeutic osclerotic plaques. targets in addition to existing cholesterol lowering strategies. The functions of a number of cytokines, including the pro- IMPACT: inflammatory effects of IFN-g, are well established. However, the Our findings indicate a protective role for IL-13 in athero- role of interleukin-13 (IL-13) is still unknown. sclerosis. Remarkably, IL-13 has the capacity to alter plaque morphology in the presence of high serum cholesterol levels RESULTS: towards more stable, less vulnerable plaques. Because IL-13 In our study, we show that IL-13 administration limits the leads to alternative macrophage activation in atherosclerotic recruitment of macrophages to and promotes collagen produc- lesions, and these macrophages have anti-atherogenic proper- tion in established lesions of atherosclerosis-prone mice. These ties, our data identify macrophage polarization by IL-13 as novel effects are paralleled by IL-13-dependent changes in the point for therapeutic intervention. Bone marrow transplantation studies were performed as previously California, USA), smooth-muscle cell actin (Sigma-Aldrich), CD3 (DAKO, described (Binder et al, 2004). Thirty 8-week-old male LDLR mice Glostrup, Denmark), iNOS (ABCAM, Cambridge, UK), CD206 (BioLegend, were given a single dose of 9-Gy lethal irradiation and irradiated mice San Diego, California, USA), Ym1/2 (a kind gift of Dr. Shioko Kimura, were injected intravenously with 2 10 bone marrow cells harvested NIH/NCI, Bethesda, USA) and ABCA1 (Novus Biological, Littleton, / þ/þ from either IL-13 (n¼ 15) or IL-13 (n¼ 15) mice. Mice were fed Colorado, USA). The photographed images were analyzed using ImageJ regular chow diet for 4 weeks after BMT to allow for bone marrow 1.41 software. reconstitution and then switched to an atherogenic diet containing 21% fat and 0.2% cholesterol (TD88137, Ssniff Spezialdia ¨ten GmbH, Macrophage foam-cell assays Soest, Germany) for an additional 16 weeks to induce lesion For all in vitro experiments, C57BL/6J mice, 12–16 weeks of age, were formation. injected intraperitoneally with 2 ml of 3% thioglycollate (Difco, For the atherosclerosis intervention study, twenty-four 12-week-old Thermo Fischer Scientific). After 3 days, thioglycollate-elicited macro- female LDLR mice were fed an atherogenic diet (Ssniff) for a total of phages were harvested and differentiated into classically activated 16 weeks to induce lesion formation. At week 11, mice were divided macrophages (M1) with 100 ng/ml IFN-g (R&D systems) or alterna- randomly into two groups and injected intraperitoneally with PBS tively activated macrophages (M2) with 5 ng/ml IL-13 (R&D systems) (n¼ 11) or IL-13 (50 ng/mouse R&D systems, Minneapolis, Minnesota, for 16 h. Following differentiation, cells were stimulated with USA; n¼ 13) twice per week for the following remaining 5 weeks. 50 mg/ml CuOx-LDL in the presence or absence of 10 mg/ml of HDL in full culture medium containing 1% mouse serum for 24 h to induce Evaluation and phenotypic analysis of atherosclerotic lesions foam-cell formation. Subsequently, cholesterol efflux, RNA/protein The extent of atherosclerosis was determined in a blinded fashion isolation and cellular cholesterol quantification experiments were in en face preparations of the entire aorta, as well as in cross performed as described in the Supporting Information. For primer sections through the aortic origin, by computer-assisted image sequences see Table SII of Supporting Information. analysis as previously described (Binder et al, 2004). Lesion phenotype was determined by the content of collagen, size of necrotic core area, Monocyte labeling and macrophage egression assessment and the presence of macrophages, smooth-muscle cells, T cells, Twenty-two male, 8-week-old, ApoE mice (C57BL/6J background) classically activated (M1) macrophages, alternatively activated (M2) were fed a standard western diet containing 21% fat and 0.2% macrophages, and ABCA1 expressing cells in lesions of equal size. For cholesterol (Altromin Spezialfutter GmbH & Co.KG, Lage, Germany) for the collagen content, sections were stained with Sirius Red, and for the 6 weeks to induce lesion formation. At week 3, circulating classical hi assessment of necrotic cores, sections were stained with a modified Ly6C monocytes were labelled by intravenous (i.v) injection of 1 mm elastic-trichrome stain. For the presence of macrophages, smooth- Fluoresbrite green fluorescent (YG) plain microspheres (Polysciences muscle cells, T cells, M1 macrophages, M2 macrophages and ABCA1 Inc., Warrington, Pennsylvania, USA), 24 h after clodronate-liposome expressing cells immunohistochemistry was performed using anti- depletion of monocytes. Latex-beads monocytes were allowed to accumulate within atherosclerotic plaques for 1 week. At week 4, mice bodies against mouse Mac-3 (BD-Biosciences Pharmingen, San Diego, EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1083 Research Article www.embomolmed.org IL-13 protects from atherosclerosis were divided into three groups. One group was sacrificed for the Academy of Sciences (CJB), the Fondation Leducq (CJB), the quantification of bead and macrophage (mac-2 cells) content Austrian Science Fund (FWF) Project P22838 (HS) and the (baseline, n¼ 7); the remaining two groups received biweekly Deutsche Forschungsgemeinschaft (FOR809 and SFB914 TP intraperitoneal injections of either PBS (n¼ 7) or IL-13 (50 ng/mouse, B08 to CW and OS). R&D systems n¼ 8) for the remaining 2 weeks. All animal experiments were approved by the local ethical committee (Regierung von Supporting Information is available at EMBO Molecular Oberbayern). Macrophage emigration from atherosclerotic lesions Medicine Online. was analyzed as described previously (Potteaux et al, 2011). The authors declare that they have no conflict of interest. Intravital microscopy gfp/wt / Sixteen male, 8-week-old, Cx3cr1 ApoE mice (C57BL/6J background, for leukocyte endothelial interactions) and 16 male, References 8-week-old, ApoE mice (C57BL/6J background, for endothelial Ait-Oufella H, Taleb S, Mallat Z, Tedgui A (2011) Recent advances on the adhesion molecules expression) were fed an atherogenic diet contain- role of cytokines in atherosclerosis. Arterioscler Thromb Vasc Biol 31: 969- ing 21% fat and 0.2% cholesterol (Altromin) for 6 weeks to induce lesion formation. At week 4, they were randomly divided into two Bancroft AJ, McKenzie AN, Grencis RK (1998) A critical role for IL-13 in groups and injected intraperitoneally with PBS (n¼ 8) or IL-13 resistance to intestinal nematode infection. J Immunol 160: 3453-3461 Barlow JL, McKenzie AN (2011) Nuocytes: expanding the innate cell repertoire (50 ng/mouse, R&D systems, n¼ 8) twice per week for the remaining in type-2 immunity. J Leukoc Biol 90: 867-874 2 weeks. At the end of this period, mice were anaesthetized with Berry A, Balard P, Coste A, Olagnier D, Lagane C, Authier H, Benoit-Vical F, ketamine/xylazine and leukocyte endothelial interactions (Drechsler ´ ´ Lepert JC, Seguela JP, Magnaval JF et al (2007) IL-13 induces expression of et al, 2010) and expression of endothelial adhesion molecules (Engel CD36 in human monocytes through PPArg activation. Eur J Immunol 37: et al, 2011) were analyzed by intravital microscopy of the left carotid 1642-1652 artery as described previously. To permit discrimination of phagocyte Binder CJ, Chang MK, Shaw PX, Miller YI, Hartvigsen K, Dewan A, Witztum JL (2002) Innate and acquired immunity in atherogenesis. Nat Med 8: 1218- subsets a PE-conjugated antibody to Gr1 (eBioscience) was introduced via an intravenous catheder 5 min prior to recording. All animal Binder CJ, Hartvigsen K, Chang MK, Miller M, Broide D, Palinski W, Curtiss LK, experiments were approved by the local ethical committee (Regierung Corr M, Witztum JL (2004) IL-5 links adaptive and natural immunity speciﬁc von Oberbayern). for epitopes of oxidized LDL and protects from atherosclerosis. J Clin Invest 114: 427-437 Statistical analysis Bochner BS, Klunk DA, Sterbinsky SA, Coffman RL, Schleimer RP (1995) IL-13 Statistical analyses were performed by unpaired Student t-test for all selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells. J Immunol 154: 799-803 results of in vivo studies and by one-way analysis of variance (with Bouhlel MA, Derudas B, Rigamonti E, Dievart R, Brozek J, Haulon S, Zawadzki C, Bonferoni post-test analysis) for all in vitro data (unless indicated Jude B, Torpier G, Marx N et al (2007) PPARgamma activation primes human differentially) to determine statistical significance between the groups. monocytes into alternative M2 macrophages with anti-inﬂammatory Data are presented as mean SEM and p< 0.05 was considered properties. Cell Metab 6: 137-143 significant. Buono C, Come CE, Stavrakis G, Maguire GF, Connelly PW, Lichtman AH (2003) Inﬂuence of interferon-gamma on the extent and phenotype of diet- induced atherosclerosis in the LDLR-deﬁcient mouse. Arterioscler Thromb Vasc Biol 23: 454-460 Author contributions Buono C, Binder CJ, Stavrakis G, Witztum JL, Glimcher LH, Lichtman AH (2005) The study was conceived by CJB. LCR and CJB designed the T-bet deﬁciency reduces atherosclerosis and alters plaque antigen-speciﬁc study and analyzed the data with contributions of OW. LCR immune responses. Proc Natl Acad Sci USA 102: 1596-1601 and SG performed most of the experiments with contributions Chiaramonte MG, Schopf LR, Neben TY, Cheever AW, Donaldson DD, Wynn TA of NPM. SMS and HS contributed to the cholesterol efﬂux (1999) IL-13 is a key regulatory cytokine for Th2 cell-mediated pulmonary experiments. MD and OS performed and analyzed carotid granuloma formation and IgE responses induced by Schistosoma mansoni eggs. J Immunol 162: 920-930 intravital microscopy and macrophage egression experiments Chinetti-Gbaguidi G, Baron M, Bouhlel MA, Vanhoutte J, Copin C, Sebti Y, with contributions of CW. LCR and CJB wrote the manuscript Derudas B, Mayi T, Bories G, Tailleux A et al (2011) Human atherosclerotic with contributions of HS and OS. All authors read and approved plaque alternative macrophages display low cholesterol handling but high the manuscript. phagocytosis because of distinct activities of the PPARgamma and LXRalpha pathways. Circ Res 108: 985-995 Chomarat P, Banchereau J (1998) Interleukin-4 and interleukin-13: their Acknowledgements similarities and discrepancies. Int Rev Immunol 17: 1-52 Combadiere C, Potteaux S, Rodero M, Simon T, Pezard A, Esposito B, Merval R, We are grateful to Maria Ozsvar-Kozma and Laura Go¨derle for Proudfoot A, Tedgui A, Mallat Z (2008) Combined inhibition of CCL2, precise and immeasurable technical support, Gernot Schab- CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost bauer and Pavel Uhrin for assistance with animal experiments, abolishes atherosclerosis in hypercholesterolemic mice. Circulation 117: and the staff of the Department of Laboratory Medicine. We 1649-1657 thank Joseph L. Witztum and Esther Lutgens for critical Cybulsky MI, Gimbrone MA Jr., (1991) Endothelial expression of a reading of the manuscript. This work was supported by grants mononuclear leukocyte adhesion molecule during atherogenesis. Science from the GenAU program (DRAGON) (CJB), the Austrian 251: 788-791 1084 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos vascular cell adhesion molecule-1 gene expression in human vascular JC, Connelly PW, Milstone DS (2001) A major role for VCAM-1, but not ICAM- endothelial cells. J Clin Invest 95: 1262-1270 1, in early atherosclerosis. J Clin Invest 107: 1255-1262 King VL, Szilvassy SJ, Daugherty A (2002) Interleukin-4 deﬁciency decreases Dansky HM, Barlow CB, Lominska C, Sikes JL, Kao C, Weinsaft J, Cybulsky MI, atherosclerotic lesion formation in a site-speciﬁc manner in female LDL Smith JD (2001) Adhesion of monocytes to arterial endothelium and initiation receptor/ mice. Arterioscler Thromb Vasc Biol 22: 456-461 of atherosclerosis are critically dependent on vascular cell adhesion King VL, Cassis LA, Daugherty A (2007) Interleukin-4 does not inﬂuence molecule-1 gene dosage. Arterioscler Thromb Vasc Biol 21: 1662-1667 development of hypercholesterolemia or angiotensin II-induced Davenport P, Tipping PG (2003) The role of interleukin-4 and interleukin-12 in atherosclerotic lesions in mice. Am J Pathol 171: 2040-2047 the progression of atherosclerosis in apolipoprotein E-deﬁcient mice. Am J Kumar RK, Herbert C, Yang M, Koskinen AM, McKenzie AN, Foster PS (2002) Pathol 163: 1117-1125 Role of interleukin-13 in eosinophil accumulation and airway remodelling Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O (2010) in a mouse model of chronic asthma. Clin Exp Allergy 32: 1104-1111 Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Kuperman DA, Schleimer RP (2008) Interleukin-4, interleukin-13, signal Circulation 122: 1837-1845 transducer and activator of transcription factor 6, and allergic asthma. Curr El Hadri K, Mahmood DF, Couchie D, Jguirim-Souissi I, Genze F, Diderot V, Mol Med 8: 384-392 Syrovets T, Lunov O, Simmet T, Rouis M (2012) Thioredoxin-1 promotes LaPorte SL, Juo ZS, Vaclavikova J, Colf LA, Qi X, Heller NM, Keegan AD, Garcia KC anti-inﬂammatory macrophages of the M2 phenotype and antagonizes (2008) Molecular and structural basis of cytokine receptor pleiotropy in the atherosclerosis. Arterioscler Thromb Vasc Biol 32: 1445-1452 interleukin-4/13 system. Cell 132: 259-272 Engel D, Beckers L, Wijnands E, Seijkens T, Lievens D, Drechsler M, Gerdes N, Liang HE, Reinhardt RL, Bando JK, Sullivan BM, Ho IC, Locksley RM (2011) Soehnlein O, Daemen MJ, Stan RV et al (2011) Caveolin-1 deﬁciency Divergent expression patterns of IL-4 and IL-13 deﬁne unique functions in decreases atherosclerosis by hampering leukocyte inﬂux into the arterial allergic immunity. Nat Immunol 13: 58-66 wall and generating a regulatory T-cell response. FASEB J 25: 3838-3848 Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF, Soubrier F, Feig JE, Parathath S, Rong JX, Mick SL, Vengrenyuk Y, Grauer L, Young SG, Fisher Esposito B, Duez H, Fievet C et al (1999) Protective role of interleukin-10 in EA (2011a) Reversal of hyperlipidemia with a genetic switch favorably atherosclerosis. Circ Res 85: e17-24 affects the content and inﬂammatory state of macrophages in McKenzie G, Emson C, Bell S, Anderson S, Fallon P, Zurawski G, Murray R, atherosclerotic plaques. Circulation 123: 989-998 Grencis R, McKenzie AN (1998a) Impaired development of Th2 cells in IL- Feig JE, Rong JX, Shamir R, Sanson M, Vengrenyuk Y, Liu J, Rayner K, Moore K, 13-deﬁcient mice. Immunity 9: 423-432 Garabedian M, Fisher EA (2011b) HDL promotes rapid atherosclerosis McKenzie GJ, Bancroft A, Grencis RK, McKenzie AN (1998b) A distinct role for regression in mice and alters inﬂammatory properties of plaque monocyte- interleukin-13 in Th2-cell-mediated immune responses. Curr Biol 8: 339- derived cells. Proc Natl Acad Sci USA 108: 7166-7171 342 Feig JE, Vengrenyuk Y, Reiser V, Wu C, Statnikov A, Aliferis CF, Garabedian MJ, Oliphant CJ, Barlow JL, McKenzie AN (2011) Insights into the initiation of type Fisher EA, Puig O (2012) Regression of atherosclerosis is characterized by 2 immune responses. Immunology 134: 378-385 broad changes in the plaque macrophage transcriptome. PLoS One 7: Oriente A, Fedarko NS, Pacocha SE, Huang SK, Lichtenstein LM, Essayan DM e39790 (2000) Interleukin-13 modulates collagen homeostasis in human skin and Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A (2006) IL-13 keloid ﬁbroblasts. J Pharmacol Exp Ther 292: 988-994 signaling through the IL-13alpha2 receptor is involved in induction of Panousis CG, Zuckerman SH (2000) Interferon-gamma induces TGF-beta1 production and ﬁbrosis. Nat Med 12: 99-106 downregulation of Tangier disease gene (ATP-binding-cassette transporter Gautier EL, Jakubzick C, Randolph GJ (2009) Regulation of the migration and 1) in macrophage-derived foam cells. Arterioscler Thromb Vasc Biol 20: survival of monocyte subsets by chemokine receptors and its relevance to 1565-1571 atherosclerosis. Arterioscler Thromb Vasc Biol 29: 1412-1418 Peters W, Charo IF (2001) Involvement of chemokine receptor 2 and its ligand, Gordon S, Martinez FO (2010) Alternative activation of macrophages: monocyte chemoattractant protein-1, in the development of mechanism and functions. Immunity 32: 593-604 atherosclerosis: lessons from knockout mice. Curr Opin Lipidol 12: 175-180 Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, Pinderski Oslund LJ, Hedrick CC, Olvera T, Hagenbaugh A, Territo M, Berliner JA, Sheppard D, Mohrs M, Donaldson DD, Locksley RM et al (1998) Requirement Fyfe AI (1999) Interleukin-10 blocks atherosclerotic events in vitro and for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261- in vivo. Arterioscler Thromb Vasc Biol 19: 2847-2853 2263 Potteaux S, Gautier EL, Hutchison SB, van Rooijen N, Rader DJ, Thomas MJ, Gupta S, Pablo AM, Jiang X, Wang N, Tall AR, Schindler C (1997) IFN-gamma Sorci-Thomas MG, Randolph GJ (2011) Suppressed monocyte recruitment potentiates atherosclerosis in ApoE knock-out mice. J Clin Invest 99: 2752- drives macrophage removal from atherosclerotic plaques of Apoe/ mice 2761 during disease regression. J Clin Invest 121: 2025-2036 Hansson GK (2005) Inﬂammation, atherosclerosis, and coronary artery Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van Gils JM, disease. N Engl J Med 352: 1685-1695 Rayner AJ, Chang AN, Suarez Y et al (2011) Antagonism of miR-33 in mice Hansson GK, Hermansson A (2011) The immune system in atherosclerosis. Nat promotes reverse cholesterol transport and regression of atherosclerosis. J Immunol 12: 204-212 Clin Invest 121: 2921-2931 Huang JT, Welch JS, Ricote M, Binder CJ, Willson TM, Kelly C, Witztum JL, Reis ED, Li J, Fayad ZA, Rong JX, Hansoty D, Aguinaldo JG, Fallon JT, Fisher EA Funk CD, Conrad D, Glass CK (1999) Interleukin-4-dependent production of (2001) Dramatic remodeling of advanced atherosclerotic plaques of the PPAR-gamma ligands in macrophages by 12/15-lipoxygenase. Nature 400: apolipoprotein E-deﬁcient mouse in a novel transplantation model. J Vasc 378-382 Surg 34: 541-547 Ingersoll MA, Platt AM, Potteaux S, Randolph GJ (2011) Monocyte trafﬁcking in Rey A, M’Rini C, Sozzani P, Lamboeuf Y, Beraud M, Caput D, Ferrara P, Pipy B acute and chronic inﬂammation. Trends Immunol 32: 470-477 (1998) IL-13 increases the cPLA2 gene and protein expression and the Kelly-Welch AE, Hanson EM, Boothby MR, Keegan AD (2003) Interleukin-4 and mobilization of arachidonic acid during inﬂammatory process in mouse interleukin-13 signaling connections maps. Science 300: 1527-1528 peritoneal macrophages. Biochem Biophys Acta 1393: 244-252 Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Clement M, Rong JX, Li J, Reis ED, Choudhury RP, Dansky HM, Elmalem VI, Fallon JT, Breslow Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A et al (2010) Macrophage JL, Fisher EA (2001) Elevating high-density lipoprotein cholesterol in plasticity in experimental atherosclerosis. PLoS One 5: e8852 apolipoprotein E-deﬁcient mice remodels advanced atherosclerotic lesions Khan BV, Parthasarathy SS, Alexander RW, Medford RM (1995) Modiﬁed low by decreasing macrophage and increasing smooth muscle cell content. density lipoprotein and its constituents augment cytokine-activated Circulation 104: 2447-2452 EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1085 Research Article www.embomolmed.org IL-13 protects from atherosclerosis Seder R, Paul W (1994) Acquisition of lymphokine-producing phenotype by Tedgui A, Mallat Z (2006) Cytokines in atherosclerosis: pathogenic and CD4þ T cells. Annu Rev Immunol 12: 635-673 regulatory pathways. Physiol Rev 86: 515-581 Steinberg D, Witztum JL (2010) Oxidized low-density lipoprotein and Tellides G, Tereb DA, Kirkiles-Smith NC, Kim RW, Wilson JH, Schechner JS, atherosclerosis. Arterioscler Thromb Vasc Biol 30: 2311-2316 Lorber MI, Pober JS (2000) Interferon-gamma elicits arteriosclerosis in the Stoger JL, Goossens P, de Winther MP (2010) Macrophage heterogeneity: absence of leukocytes. Nature 403: 207-211 relevance and functional implications in atherosclerosis. Curr Vasc Williams KJ, Feig JE, Fisher EA (2008) Rapid regression of atherosclerosis: Pharmacol 8: 233-248 insights from the clinical and experimental literature. Nat Clin Pract Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jaffer FA, Libby P, Weissleder R Cardiovasc Med 5: 91-102 (2006) Monocyte accumulation in mouse atherogenesis is progressive and Woltmann G, McNulty CA, Dewson G, Symon FA, Wardlaw AJ (2000) proportional to extent of disease. Proc Natl Acad Sci USA 103: 10340-10345 Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek R, Llodra J, Garin A, Liu J, eosinophils, but not neutrophils, to human umbilical vein endothelial cells Mack M, van Rooijen N et al (2007) Monocyte subsets differentially employ under ﬂow. Blood 95: 3146-3152 CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques. J Wynn TA (2008) Cellular and molecular mechanisms of ﬁbrosis. J Pathol 214: Clin Invest 117: 185-194 199-210 1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EMBO Molecular Medicine Pubmed Central http://www.deepdyve.com/lp/pubmed-central/interleukin-13-protects-from-atherosclerosis-and-modulates-plaque-9ahLUIaOmM

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References (82)

Gene expression of alternatively activated M2 macrophages in vitro
Jonathan Feig, Saj Parathath, J. Rong, S. Mick, Y. Vengrenyuk, Lisa Grauer, S. Young, E. Fisher (2011)
Reversal of Hyperlipidemia With a Genetic Switch Favorably Affects the Content and Inflammatory State of Macrophages in Atherosclerotic Plaques
Circulation, 123
TA Wynn (2008)
Cellular and molecular mechanisms of fibrosis
J Pathol, 214
G. Tellides, D. Tereb, N. Kirkiles-Smith, R. Kim, J. Wilson, J. Schechner, M. Lorber, J. Pober (2000)
Interferon-gamma elicits arteriosclerosis in the absence of leukocytes.
Nature, 403 6766
B. Bochner, David Klunk, S. Sterbinsky, R. Coffman, R. Schleimer (1995)
IL-13 selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells.
Journal of immunology, 154 2
W. Peters, I. Charo (2001)
Involvement of chemokine receptor 2 and its ligand, monocyte chemoattractant protein-1, in the development of atherosclerosis: lessons from knockout mice
Current Opinion in Lipidology, 12
G. Chinetti-Gbaguidi, Morgane Baron, M. Bouhlel, Jonathan Vanhoutte, C. Copin, Y. Sebti, B. Derudas, T. Mayi, Gael Bories, A. Tailleux, S. Haulon, C. Zawadzki, B. Jude, B. Staels (2011)
Human Atherosclerotic Plaque Alternative Macrophages Display Low Cholesterol Handling but High Phagocytosis Because of Distinct Activities of the PPAR&ggr; and LXR&agr; Pathways
Circulation Research, 108
D. Kuperman, R. Schleimer (2008)
Interleukin-4, interleukin-13, signal transducer and activator of transcription factor 6, and allergic asthma.
Current molecular medicine, 8 5
E. Reis, J. Li, Z. Fayad, J. Rong, Dipal Hansoty, J. Aguinaldo, J. Fallon, E. Fisher (2001)
Dramatic remodeling of advanced atherosclerotic plaques of the apolipoprotein E-deficient mouse in a novel transplantation model.
Journal of vascular surgery, 34 3
Hong-Erh Liang, R. Reinhardt, Jennifer Bando, B. Sullivan, I. Ho, R. Locksley (2011)
Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity
Nature immunology, 13
A. Berry, Patricia Balard, Agnès Coste, D. Olagnier, Céline Lagane, H. Authier, F. Benoit‐Vical, J. Lepert, J. Séguéla, J. Magnaval, P. Chambon, D. Metzger, B. Desvergne, W. Wahli, J. Auwerx, B. Pipy (2007)
IL‐13 induces expression of CD36 in human monocytes through PPARγ activation
European Journal of Immunology, 37
J. Rong, Jie Li, E. Reis, R. Choudhury, H. Dansky, Valerie Elmalem, J. Fallon, J. Breslow, E. Fisher (2001)
Elevating High-Density Lipoprotein Cholesterol in Apolipoprotein E—Deficient Mice Remodels Advanced Atherosclerotic Lesions by Decreasing Macrophage and Increasing Smooth Muscle Cell Content
Circulation: Journal of the American Heart Association, 104
Laura Oslund, C. Hedrick, Tristana Olvera, A. Hagenbaugh, M. Territo, J. Berliner, A. Fyfe (1999)
Interleukin-10 blocks atherosclerotic events in vitro and in vivo.
Arteriosclerosis, thrombosis, and vascular biology, 19 12
Jonathan Feig, Y. Vengrenyuk, V. Reiser, Chaowei Wu, A. Statnikov, C. Aliferis, M. Garabedian, E. Fisher, O. Puig (2012)
Regression of Atherosclerosis Is Characterized by Broad Changes in the Plaque Macrophage Transcriptome
PLoS ONE, 7
Increased HDL-dependent cholesterol efflux by foam cells stimulated with IL-13
J. Barlow, A. McKenzie (2011)
Nuocytes: expanding the innate cell repertoire in type‐2 immunity
Journal of Leukocyte Biology, 90
K. Hadri, Mahmood Darweesh, D. Couchie, I. Jguirim-Souissi, F. Genze, V. Diderot, T. Syrovets, O. Lunov, T. Simmet, M. Rouis (2012)
Thioredoxin-1 Promotes Anti-Inflammatory Macrophages of the M2 Phenotype and Antagonizes Atherosclerosis
Arteriosclerosis, Thrombosis, and Vascular Biology, 32
C. Oliphant, J. Barlow, A. McKenzie (2011)
Insights into the initiation of type 2 immune responses
Immunology, 134
G. Woltmann, C. McNulty, Grant Dewson, F. Symon, Andrew Wardlaw (2000)
Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow.
Blood, 95 10
C. Buono, C. Come, G. Stavrakis, G. Maguire, P. Connelly, A. Lichtman (2003)
Influence of Interferon-&ggr; on the Extent and Phenotype of Diet-Induced Atherosclerosis in the LDLR-Deficient Mouse
Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association, 23
F. Tacke, David Alvarez, Theodore Kaplan, C. Jakubzick, R. Spanbroek, Jaime Llodrá, Alexandre Garin, Jianhua Liu, M. Mack, N. Rooijen, S. Lira, A. Habenicht, G. Randolph (2007)
Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques.
The Journal of clinical investigation, 117 1
R. Seder (1994)
Acquisition of lymphokine-producing phenotype by CD4+ T cells.
The Journal of allergy and clinical immunology, 94 6 Pt 2
P. Chomarat, J. Banchereau (1998)
Interleukin-4 and interleukin-13: their similarities and discrepancies.
International reviews of immunology, 17 1-4
GK Hansson (2005)
Inflammation, atherosclerosis, and coronary artery disease
N Engl J Med, 352
J. Khallou-Laschet, Aditi Varthaman, G. Fornasa, C. Compain, A. Gaston, M. Clément, M. Dussiot, O. Levillain, S. Graff-Dubois, A. Nicoletti, G. Caligiuri (2010)
Macrophage Plasticity in Experimental Atherosclerosis
PLoS ONE, 5
E. Gautier, C. Jakubzick, G. Randolph (2009)
Regulation of the migration and survival of monocyte subsets by chemokine receptors and its relevance to atherosclerosis.
Arteriosclerosis, thrombosis, and vascular biology, 29 10
S. Gordon, F. Martínez (2010)
Alternative activation of macrophages: mechanism and functions.
Immunity, 32 5
Z. Mallat, C. Heymes, J. Ohan, E. Faggin, G. Lesèche, A. Tedgui (1999)
Expression of interleukin-10 in advanced human atherosclerotic plaques: relation to inducible nitric oxide synthase expression and cell death.
Arteriosclerosis, thrombosis, and vascular biology, 19 3
S. Fichtner-Feigl, W. Strober, K. Kawakami, R. Puri, A. Kitani (2006)
IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis.
Nature medicine, 12 1
A. Rey, C. M'rini, P. Sozzani, Yves Lamboeuf, Maryse Beraud, Daniel Caput, Pascual Ferrara, B. Pipy (1998)
IL-13 increases the cPLA2 gene and protein expression and the mobilization of arachidonic acid during an inflammatory process in mouse peritoneal macrophages.
Biochimica et biophysica acta, 1393 2-3
J. Stöger, P. Goossens, M. Winther (2010)
Macrophage heterogeneity: relevance and functional implications in atherosclerosis.
Current vascular pharmacology, 8 2
R. Seder, W. Paul (1994)
Acquisition of lymphokine-producing phenotype by CD4+ T cells.
Annual review of immunology, 12
Z. Mallat, Sandrine Besnard, M. Duriez, V. Deleuze, F. Emmanuel, M. Bureau, F. Soubrier, B. Esposito, H. Duez, C. Fiévet, B. Staels, N. Duverger, D. Scherman, A. Tedgui (1999)
Protective role of interleukin-10 in atherosclerosis.
Circulation research, 85 8
C. Combadière, Stephane Potteaux, M. Rodero, T. Simon, A. Pézard, B. Esposito, R. Merval, A. Proudfoot, A. Tedgui, Z. Mallat (2008)
Combined Inhibition of CCL2, CX3CR1, and CCR5 Abrogates Ly6Chi and Ly6Clo Monocytosis and Almost Abolishes Atherosclerosis in Hypercholesterolemic Mice
Circulation, 117
D. Engel, Linda Beckers, Erwin Wijnands, T. Seijkens, D. Lievens, Maik Drechsler, N. Gerdes, O. Soehnlein, M. Daemen, R. Stan, E. Biessen, E. Lutgens (2011)
Caveolin‐1 deficiency decreases atherosclerosis by hampering leukocyte influx into the arterial wall and generating a regulatory T‐cell response
The FASEB Journal, 25
Rakesh Kumar, Cristan Herbert, Ming Yang, Aulikki Koskinen, A. McKenzie, Paul Foster (2002)
Role of interleukin‐13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma
Clinical & Experimental Allergy, 32
A. Bancroft, A. McKenzie, R. Grencis (1998)
A critical role for IL-13 in resistance to intestinal nematode infection.
Journal of immunology, 160 7
H. Ait-Oufella, S. Taleb, Z. Mallat, A. Tedgui (2011)
Recent Advances on the Role of Cytokines in Atherosclerosis
Arteriosclerosis, Thrombosis, and Vascular Biology, 31
G. McKenzie, A. Bancroft, R. Grencis, A. McKenzie (1998)
A distinct role for interleukin-13 in Th2-cell-mediated immune responses
Current Biology, 8
M. Cybulsky, M. Gimbrone (1991)
Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis.
Science, 251 4995
G. Grünig (1998)
Requirement for IL-13 independently of IL-4 in experimental asthma
Nature, 282
Alfonso Oriente, Neal Fedarko, S. Pacocha, S. Huang, L. Lichtenstein, D. Essayan (2000)
Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts.
The Journal of pharmacology and experimental therapeutics, 292 3
M. Chiaramonte, Lisa Schopf, T. Neben, Allen Cheever, Debra Donaldson, Thomas Wynn (1999)
IL-13 is a key regulatory cytokine for Th2 cell-mediated pulmonary granuloma formation and IgE responses induced by Schistosoma mansoni eggs.
Journal of immunology, 162 2
A. Basbaum, D. Bautista, G. Scherrer, D. Julius (2009)
Cellular and Molecular Mechanisms of Pain
Cell, 139
M. Bouhlel, B. Derudas, E. Rigamonti, R. Dièvart, J. Brozek, S. Haulon, C. Zawadzki, B. Jude, G. Torpier, N. Marx, B. Staels, G. Chinetti-Gbaguidi (2007)
PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties.
Cell metabolism, 6 2
Maik Drechsler, R. Megens, M. Zandvoort, C. Weber, O. Soehnlein (2010)
Hyperlipidemia-Triggered Neutrophilia Promotes Early Atherosclerosis
Circulation, 122
Stephane Potteaux, E. Gautier, Susan Hutchison, N. Rooijen, D. Rader, Michael Thomas, M. Sorci-Thomas, G. Randolph (2011)
Suppressed monocyte recruitment drives macrophage removal from atherosclerotic plaques of Apoe-/- mice during disease regression.
The Journal of clinical investigation, 121 5
Ann Kelly-Welch, E. Hanson, M. Boothby, A. Keegan (2003)
Interleukin-4 and Interleukin-13 Signaling Connections Maps
Science, 300
C. Buono, C. Binder, G. Stavrakis, J. Witztum, L. Glimcher, A. Lichtman (2005)
T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses.
Proceedings of the National Academy of Sciences of the United States of America, 102 5
H. Dansky, Courtenay Barlow, Chris Lominska, J. Sikes, Catherine Kao, Jonathan Weinsaft, M. Cybulsky, Jonathan Smith (2001)
Adhesion of Monocytes to Arterial Endothelium and Initiation of Atherosclerosis Are Critically Dependent on Vascular Cell Adhesion Molecule-1 Gene Dosage
Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association, 21
G. Tellides, D. Tereb, N. Kirkiles-Smith, R. Kim, Jean Wilson, J. Schechner, M. Lorber, J. Pober (2000)
Interferon-γ elicits arteriosclerosis in the absence of leukocytes
Nature, 403
D. Steinberg, J. Witztum (2010)
Oxidized low-density lipoprotein and atherosclerosis.
Arteriosclerosis, thrombosis, and vascular biology, 30 12
F. Swirski, M. Pittet, M. Kircher, E. Aikawa, F. Jaffer, P. Libby, R. Weissleder (2006)
Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease
Proceedings of the National Academy of Sciences, 103
Sanjay Gupta, A. Pablo, X. Jiang, N. Wang, A. Tall, C. Schindler (1997)
IFN-gamma potentiates atherosclerosis in ApoE knock-out mice.
The Journal of clinical investigation, 99 11
S. LaPorte, Z. Juo, J. Václavíková, L. Colf, Xiulan Qi, N. Heller, A. Keegan, K. Garcia (2008)
Molecular and Structural Basis of Cytokine Receptor Pleiotropy in the Interleukin-4/13 System
Cell, 132
(2012)
EMBO Mol Med
M. Eaman (2000)
Immune system.
Nursing standard (Royal College of Nursing (Great Britain) : 1987), 15 3
V. King, L. Cassis, A. Daugherty (2007)
Interleukin-4 does not influence development of hypercholesterolemia or angiotensin II-induced atherosclerotic lesions in mice.
The American journal of pathology, 171 6
C. Binder, K. Hartvigsen, Mi-Kyung Chang, Marina Miller, D. Broide, W. Palinski, L. Curtiss, M. Corr, J. Witztum (2004)
IL-5 links adaptive and natural immunity specific for epitopes of oxidized LDL and protects from atherosclerosis.
The Journal of clinical investigation, 114 3
Jonathan Feig, J. Rong, R. Shamir, M. Sanson, Y. Vengrenyuk, Jianhua Liu, K. Rayner, K. Moore, M. Garabedian, E. Fisher (2011)
HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells
Proceedings of the National Academy of Sciences, 108
P. Davenport, P. Tipping (2003)
The role of interleukin-4 and interleukin-12 in the progression of atherosclerosis in apolipoprotein E-deficient mice.
The American journal of pathology, 163 3
A. Tedgui, Z. Mallat (2006)
Cytokines in atherosclerosis: pathogenic and regulatory pathways.
Physiological reviews, 86 2
V. King, S. Szilvassy, A. Daugherty (2002)
Interleukin-4 Deficiency Decreases Atherosclerotic Lesion Formation in a Site-Specific Manner in Female LDL Receptor−/− Mice
Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association, 22
M. Ingersoll, A. Platt, Stephane Potteaux, G. Randolph (2011)
Monocyte trafficking in acute and chronic inflammation.
Trends in immunology, 32 10
Grahame McKenzie, Claire Emson, Sarah Bell, Shannon Anderson, P. Fallon, G. Zurawski, Richard Murray, R. Grencis, A. McKenzie (1998)
Impaired development of Th2 cells in IL-13-deficient mice.
Immunity, 9 3
C. Binder, S. Hörkkö, A. Dewan, Mi-Kyung Chang, E. Kieu, C. Goodyear, P. Shaw, W. Palinski, J. Witztum, G. Silverman (2003)
Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL
Nature Medicine, 9
J. Huang, J. Welch, M. Ricote, C. Binder, T. Willson, C. Kelly, J. Witztum, C. Funk, D. Conrad, C. Glass (1999)
Interleukin-4-dependent production of PPAR-gamma ligands in macrophages by 12/15-lipoxygenase.
Nature, 400 6742
Jannet Huang, J. Welch, M. Ricote, C. Binder, T. Willson, C. Kelly, J. Witztum, C. Funk, D. Conrad, C. Glass (1999)
Interleukin-4-dependent production of PPAR-γ ligands in macrophages by 12/15-lipoxygenase
Nature, 400
Zoher Kapasi, Catherine Goodman (2020)
The immune system
Anaesthesia & Intensive Care Medicine
A. Sica, A. Mantovani (2012)
Macrophage plasticity and polarization: in vivo veritas.
The Journal of clinical investigation, 122 3
K. Rayner, Frederick Sheedy, Christine Esau, F. Hussain, R. Temel, Saj Parathath, J. Gils, Alistair Rayner, Aaron Chang, Y. Suárez, C. Fernández-Hernando, E. Fisher, K. Moore (2011)
Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis.
The Journal of clinical investigation, 121 7
M. Cybulsky, K. Iiyama, Hongmei Li, Su-Ning Zhu, Mian Chen, M. Iiyama, V. Davis, J. Gutiérrez-Ramos, P. Connelly, D. Milstone (2001)
A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.
The Journal of clinical investigation, 107 10
Bobby Khan, Sampath Parthasarathy, R. Alexander, Russell, M. Medford (1995)
Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells.
The Journal of clinical investigation, 95 3
Research Article www.embomolmed.org IL-13 protects from atherosclerosis
G. Hansson, A. Hermansson (2011)
The immune system in atherosclerosis
Nature Immunology, 12
Y. Momiyama, H. Adachi, D. Fairweather, N. Ishizaka, Emi Saita (2014)
Inflammation, Atherosclerosis and Coronary Artery Disease
Clinical Medicine Insights. Cardiology, 8
J. Scoazec, F. Verrecchia, M. Jacob, P. Bruneval (2006)
[Cellular and molecular mechanisms of fibrosis].
Annales de pathologie, 26 Spec No 1
A Rey, C M'Rini, P Sozzani, Y Lamboeuf, M Beraud, D Caput, P Ferrara, B Pipy (1998)
IL‐13 increases the cPLA2 gene and protein expression and the mobilization of arachidonic acid during inflammatory process in mouse peritoneal macrophages
Biochem Biophys Acta, 1393
C. Panousis, S. Zuckerman (2000)
Interferon-gamma induces downregulation of Tangier disease gene (ATP-binding-cassette transporter 1) in macrophage-derived foam cells.
Arteriosclerosis, thrombosis, and vascular biology, 20 6
K. Williams, Jonathan Feig, E. Fisher (2008)
Rapid regression of atherosclerosis: insights from the clinical and experimental literature
Nature Clinical Practice Cardiovascular Medicine, 5
C. Binder, Mi-Kyung Chang, P. Shaw, Yury Miller, K. Hartvigsen, A. Dewan, J. Witztum (2002)
Innate and acquired immunity in atherogenesis
Nature Medicine, 8
(1998)
13-deficient mice. Immunity

Publisher: Pubmed Central
Copyright: Copyrights © 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO
ISSN: 1757-4676
eISSN: 1757-4684
DOI: 10.1002/emmm.201201374
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Abstract

OPEN TRANSPARENT Research Article ACCESS PROCESS IL-13 protects from atherosclerosis Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype 1,2 1,2 3 4 Larissa Cardilo-Reis , Sabrina Gruber , Sabine M. Schreier , Maik Drechsler , 1,2 4 1 3 Nikolina Papac-Milicevic , Christian Weber , Oswald Wagner , Herbert Stangl , 4 1,2 Oliver Soehnlein , Christoph J. Binder Keywords: alternatively activated Atherosclerotic lesions are characterized by the accumulation of oxidized LDL macrophages (M2); atherosclerosis; (OxLDL) and the inﬁltration of macrophages and T cells. Cytokine expression in cytokines; interleukin-13; oxidized LDL the microenvironment of evolving lesions can profoundly contribute to plaque development. While the pro-atherogenic effect of T helper (Th) 1 cytokines, such as IFN-g, is well established, the role of Th2 cytokines is less clear. Therefore, we DOI 10.1002/emmm.201201374 characterized the role of the Th2 cytokine interleukin (IL)-13 in murine athero- sclerosis. Here, we report that IL-13 administration favourably modulated the Received March 14, 2012 Revised July 16, 2012 morphology of already established atherosclerotic lesions by increasing lesional Accepted August 03, 2012 collagen content and reducing vascular cell adhesion molecule-1 (VCAM-1)- dependent monocyte recruitment, resulting in decreased plaque macrophage content. This was accompanied by the induction of alternatively activated (M2) macrophages, which exhibited increased clearance of OxLDL compared to IFN-g- activated (M1) macrophages in vitro. Importantly, deﬁciency of IL-13 results in accelerated atherosclerosis in LDLR mice without affecting plasma cholesterol levels. Thus, IL-13 protects from atherosclerosis and promotes a favourable plaque morphology, in part through the induction of alternatively activated macrophages. INTRODUCTION ﬁbrosis in the vessel wall of arteries (Binder et al, 2002; Hansson & Hermansson, 2011). T cells are a prominent component of the Atherosclerosis is a chronic inﬂammatory disease of the artery atherosclerotic plaque and exhibit signs of activation, including wall, whose pathogenesis is inﬂuenced by dyslipidemia (high the expression of cytokines (Ait-Oufella et al, 2011; Hansson & LDL/low high density lipoprotein, HDL) and inﬂammation Hermansson, 2011). Substantial evidence supports that T helper (Hansson, 2005). Atherosclerotic lesions are characterized by (Th) 1-driven responses, mainly characterized by IFN-g the accumulation of oxidized LDL (OxLDL), the inﬁltration of production, are detrimental and correlate with progression of activated macrophages and T cells, as well as cell death and atherosclerosis (Buono et al, 2003, 2005; Gupta et al, 1997; Tellides et al, 2000). These pro-atherogenic responses have been shown to be dampened by the presence of speciﬁc regulatory (1) Department of Laboratory Medicine, Medical University of Vienna, T cells, which secrete the anti-atherogenic cytokines transform- Vienna, Austria (2) Center for Molecular Medicine (CeMM) of the Austrian Academy of ing growth factor (TGF)-b and interleukin (IL)-10 (Ait-Oufella Sciences, Vienna, Austria et al, 2011). Indeed, IL-10 secreted by T cells decreases (3) Department of Medical Chemistry, Center for Pathobiochemistry and atherogenesis in mice (Mallat et al, 1999; Pinderski Oslund Genetics, Medical University of Vienna, Vienna, Austria et al, 1999). On the other hand, the role of Th2 responses in (4) Institute for Cardiovascular Prevention, Ludwig-Maximilians University lesion formation is more complex. For example, previous Munich, Munich, Germany studies reported a pro-atherogenic role for IL-4 (Davenport & *Corresponding author: Tel: þ43 1 4040073755; Fax: þ43 1 4040073588; Tipping, 2003; King et al, 2002), while a later report found no E-mail: [email protected] 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License (CC BY-NC 3.0), which permits use, distribution 1072 and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. effect (King et al, 2007). We have shown previously that the and reduces macrophage content of existing lesions. The latter / / atheroprotective immunization of LDLR mice with malon- was conﬁrmed in cholesterol-fed ApoE mice, and found to be dialdehyde-modiﬁed LDL (MDA-LDL) induced a Th2-biased a consequence of decreased vascular cell adhesion molecule-1 immune response that was characterized by antigen-speciﬁc (VCAM-1)-dependent monocyte recruitment. In addition, IL-13 production of IL-5 and IL-13 but only small amounts of IL-4 and administration induces a switch of the lesional macrophage IFN-g. In the same study, we demonstrated the capacity of IL-5 phenotype in vivo towards alternative activation. Macrophages, to stimulate natural atheroprotective IgM speciﬁc for OxLDL and which were alternatively activated with IL-13 in vitro, have an its ability to protect from atherosclerosis (Binder et al, 2004). increased capacity of clearing OxLDL by promoting its uptake as In addition to their role in providing help for B cells to secrete well as cholesterol efﬂux without increasing net foam-cell antibodies, another potentially important function of cytokines formation. Moreover, we show that LDLR mice that were in atherogenesis is their capacity to modulate the activation state reconstituted with bone marrow of IL-13-deﬁcient mice develop of macrophages. Continuous recruitment of monocyte/macro- accelerated atherosclerosis. phages into lesions has been shown to be related to plaque progression (Gautier et al, 2009; Peters & Charo, 2001; Potteaux et al, 2011; Swirski et al, 2006). IFN-g (and LPS) can promote RESULTS classical activation of macrophages (M1), which is known to be pro-inﬂammatory and may promote atherogenesis (Khallou- IL-13 administration modulates established atherosclerosis Laschet et al, 2010). On the other hand, IL-4 and/or IL-13 induce To study the capacity of IL-13 to inﬂuence existing athero- alternative macrophage activation (M2), which results in potent sclerosis, we performed an intervention study in which IL-13 anti-inﬂammatory and tissue repair capacities (Gordon & was administered exogenously to mice with established Martinez, 2010). Thus, the cytokine proﬁle of the microenvi- atherosclerotic lesions. LDLR mice were fed an atherogenic ronment can profoundly affect the activation state of macro- diet for 16 weeks and received biweekly intraperitoneal phages and their function, e.g. in foam-cell formation. Recently, injections of IL-13 or phosphate-buffered saline (PBS) during Khallou-Laschet et al (2010) showed that lesion-inﬁltrated the last 5 weeks of diet. Based on our observation that / / macrophages of young ApoE mice exhibit predominantly the atherosclerotic LDLR mice have IL-13 serum levels of M2 phenotype, while M1 macrophages were dominant in more approximately 2.5 ng/ml, we decided to administer 50 ng of advanced lesions of aged mice and their presence correlated IL-13 per LDLR mouse twice per week, which corresponds to with lesion progression. These data suggest a potentially only threefold higher amounts of systemic IL-13 per day. At time atheroprotective role of anti-inﬂammatory M2 macrophages of sacriﬁce, mice were not different with respect to body weight, and the cytokines involved in alternative macrophage activa- total plasma cholesterol (TC), triglycerides (TG), or levels of tion. Importantly, the presence of anti-inﬂammatory M2 total IgG1 or IgG2c antibodies (Table 1). Furthermore, there macrophages has also been documented in human carotid were no differences in the frequencies of splenic T or B cells atherosclerotic lesions (Bouhlel et al, 2007). (Table 1). Stimulated splenocytes from atherosclerotic LDLR IL-13 is exclusively produced by hematopoietic cells, mice treated with PBS or IL-13 produced similar amounts of Th2 including activated Th2 cells and the recently discovered (IL-4, IL-5, IL-10 and IL-13) and Th1 (IFN-g) cytokines, nuocytes (Barlow & McKenzie, 2011; Oliphant et al, 2011). indicating that IL-13 administration at this dose did not alter Because IL-4 and IL-13 share a common receptor signalling the overall Th1/Th2 balance (Fig S1 of Supporting Information). pathway (IL-4Ra/IL-13Ra1/STAT6) similar functions for both In addition, no induction of liver or lung ﬁbrosis due to cytokines have been described (Chomarat & Banchereau, 1998; the continuous IL-13 injection was observed (unpublished Kuperman & Schleimer, 2008). However, IL-13 also possesses observations). As expected with the relatively short time of functions that differ from those of IL-4 (Chiaramonte et al, 1999; administration only during the last 5 weeks of a 16-week feeding Kumar et al, 2002; Liang et al, 2011; McKenzie et al, 1998b; period, there were no differences in the extent of atherosclerosis Oriente et al, 2000). For example, IL-13 can initiate signalling via in the cross-sectional analyses of the aortic origin or in the the alternative IL-13 receptor IL-13Ra2, which exclusively binds entire aorta by en face analyses between atherosclerotic IL-13 and which has been shown to induce TGF-b1 production LDLR mice treated with PBS or IL-13, respectively (Fig 1A in macrophages via a STAT6-independent pathway, leading to and Table 1). collagen deposition in vivo (Fichtner-Feigl et al, 2006). Although As plaque size was similar between the two groups, we were it has been assumed that IL-13 affects atherosclerosis in able to directly compare lesion composition. Morphological the same way as IL-4, no studies are currently available to analyses of the cross-sectional lesions showed no signiﬁcant support this notion (Tedgui & Mallat, 2006). Therefore, we differences in the necrotic core area, suggesting that both groups tested the role of IL-13 in atherosclerotic lesion formation. Based of mice had the same stage of lesion development (Fig S2A of on the prominent functions of IL-13 in inducing ﬁbrosis and Supporting Information). In contrast, the collagen content was alternative macrophage activation, we hypothesized that IL-13 signiﬁcantly higher in atherosclerotic LDLR mice treated might have an atheroprotective role by modulating plaque with IL-13 compared to PBS-treated mice (Fig 1B), while the composition. smooth-muscle cell content remained unchanged between Here, we demonstrate that exogenous administration of IL-13 the groups (Fig S2B of Supporting Information). These results to cholesterol-fed LDLR mice promotes collagen formation suggest that IL-13 injections stimulated the production of EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1073 Research Article www.embomolmed.org IL-13 protects from atherosclerosis Table 1. Overview of experimental data of the IL-13 administration study / / LDLR inj! PBS (n¼ 11) LDLR inj! IL-13 (n¼ 13) Atherosclerosis 4 2 Aortic origin (10 mm /section) 48.16 3.56 47.15 1.22 En face (% of aorta) 6.63 0.94 6.22 0.66 Metabolic parameters Weight (g) 23.6 0.7 22.5 0.5 TC (mg/dl) 1493 49 1362 93 TG (mg/dl) 863 85 777 81 Serum antibody titers Total IgM (mg/ml) 0.97 0.11 0.85 0.11 Total IgG1 (mg/ml) 1.11 0.10 0.97 0.11 Total IgG2c (mg/ml) 1.84 0.16 1.58 0.16 Serum chemokines CCL2/MCP-1 (pg/ml) 59.1 8.3 69.3 8.1 CXCL1/KC (pg/ml) 1709 247 1535 181 Characterization of PEC Total peritoneal cells (10 ) 3.38 0.52 2.22 0.29 CD5 T cells (% of total) 20.40 3.74 15.25 2.71 CD11b Mac cells (% of total) 13.96 2.16 18.78 2.32 CD19 B cells (% of total) 53.25 3.43 51.84 3.29 þ þ CD11b CD5 B1a cells (% of B cells) 27.24 2.71 24.56 3.29 CD11b CD5 B1b cells (% of B cells) 16.95 1.35 19.37 1.47 þ þ CD19 CD23 B2 cells (% of B cells) 28.43 3.39 32.69 3.68 Characterization of PBC Total white blood cells/ml (10 ) 1.40 0.18 1.59 0.25 þ lo CD11b Ly6C monocytes (% of total) 4.11 0.34 4.96 0.43 þ hl CD11b Ly6C monocytes (% of total) 2.59 0.36 2.15 0.36 þ þ CD11b Ly6G neutrophils (% of total) 3.97 0.46 4.51 0.65 Characterization of splenocytes Total spleen cells/ml (10 ) 67.3 11.3 62.3 9.4 CD43 T cells/ml (% of total) 24.83 2.26 23.39 1.42 B220 B cells/ml (% of total) 45.35 3.32 46.67 1.92 þ þ CD43 IgM B1 cells/ml (% of B cells) 16.29 1.27 17.16 1.22 CD43 IgM B2 cells/ml (% of B cells) 81.76 1.21 80.97 1.19 Atherosclerosis in the aortic origin was analyzed by cross-sections through the aortic origin and values represent the average mm /section. En face measurements are given in percent lesion area of the entire aorta. TC, total serum cholesterol; TG, serum triglycerides; PEC, peritoneal exudate cells; PBC, peripheral blood cells. Data are shown as mean SEM. PEC cellular populations as measured by ﬂow cytometry. PBC cellular populations as measured by ﬂow cytometry. Spleen cellular populations were analyzed by ﬂow cytometry. collagen by either macrophages or smooth-muscle cells rather IL-13 administration reduces macrophage content in lesions than the proliferation of these cells, consistent with the pro- of atherosclerotic ApoE mice, independent of macro- ﬁbrotic function of IL-13. In addition, the numbers of T cells were phage egress not different between the two groups (Fig S2C of Supporting The observed morphological changes of atherosclerotic lesions Information). Remarkably, immunohistological analyses of following IL-13 administration suggested the possibility of mac-3 macrophages uncovered a signiﬁcant reduction in macrophage egression. Because CCR7 has been implicated as a lesional macrophages in the IL-13-treated mice (Fig 1C). These key chemokine receptor in this, we ﬁrst evaluated CCR7 differences in macrophage numbers were not due to changes in expression in lesions of the cholesterol-fed LDLR mice that circulating blood cell counts, as total numbers of white blood were treated with either IL-13 or PBS, respectively. There was no hi lo þ cells, monocytes (both classical Ly6C and nonclassical Ly6C ) signiﬁcant difference in the percentage of CCR7 lesion area or neutrophils in the peripheral blood of mice were similar between the two groups, suggesting no major contribution of (Table 1). Moreover, the plasma levels of CCL2/MCP-1 and CCR7-mediated emigration (Fig S3 of Supporting Information). CXCL1/KC, the two major chemokines involved in monocyte We then directly evaluated the ability of IL-13 to induce recruitment, were not reduced in IL-13-injected mice (Table 1). macrophage egress from existing lesions using a method that is Taken together, our data showed that IL-13 modulates based on the tracking of ﬂuorescent bead-labelled monocyte/ established atherosclerotic lesions by inducing a more stable macrophages in ApoE mice (Potteaux et al, 2011). To achieve plaque composition with higher collagen content and fewer comparable lesion development in these mice, they were fed an macrophages. atherogenic diet for a total of 6 weeks and received PBS or IL-13 1074 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. A B C PBS -/- LDLR inj IL-13 *** 70 1500 0 0 -/- -/- 0 50 100 150 200 250 300 LDLR inj PBS IL-13 LDLR inj PBS IL-13 µm PBS IL-13 PBS IL-13 PBS IL-13 Figure 1. IL-13 administration alters plaque morphology of established atherosclerotic lesions. A. LDLR mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n¼ 11) or IL-13 (n¼ 13) during the last / 2 5 weeks. Equal extent of atherosclerotic lesion size in cross-sections of the aortic origin in injected LDLR mice. Values represent mm /section throughout the entire aortic origin (300 mm). Original magnification 50. B. Increased collagen content in lesions of LDLR mice injected with IL-13. Sections were stained with Sirius Red for the presence of collagen, and values represent the percentages of Sirius Red area/total lesion area. p¼ 0.035. Original magnification 100. C. Decreased macrophage content in lesions of LDLR mice injected with IL-13. Sections were stained with the macrophage-specific anti-mac-3 antibody and þ 2 values represent the numbers of mac-3 cells/mm of total lesion area. p¼ 0.0007. Original magnification 100. All data are mean SEM values of all mice of each group. Images show representative examples of the respective stainings. during the last 2 weeks of diet. Following depletion of counts, similar plasma levels of important chemokines, and no monocyte/macrophages by clodronate-liposome injection effect on macrophage egression, we hypothesized that IL-13 20 days after initiation of the atherogenic diet, circulating reduced the recruitment and adhesion of monocytes to the monocytes were labelled with latex-beads to monitor lesional atherosclerotic wall. To elucidate this directly, we assessed the macrophage migration. At day 28, one-third of mice were adhesion of different leukocyte populations by intravital gfp/wt sacriﬁced to obtain a baseline number of beads per plaque microscopy of carotid arteries of Cx cr1 mice that were area in the aortic root. The other mice were divided into available on an ApoE background. Following injection of two groups and received biweekly injections of either IL-13 a ﬂuorescent antibody to GR1 (Ly6C/G), this animal model or PBS, respectively, for the remaining 2 weeks (Fig 2A). At enabled us to differentiate, at the same time, between the time of sacriﬁce, cross-sections of the aortic origin were adhesion of two subtypes of circulating monocytes (nonclassical hiGFP þ loGFP analyzed for the presence of ﬂuorescent beads as well as Gr1 Cx cr1 and classical Gr1 Cx cr1 ) and neutro- 3 3 þ þ mac-2 cells. The numbers of beads per plaque area at phils (Gr1 gfp ). Indeed, the adhesion of circulating monocytes week 6 were not different between PBS- and IL-13-injected to carotid arteries was signiﬁcantly decreased in atherosclerotic animals and comparable to the numbers at baseline mice injected with IL-13 compared to PBS-treated mice (week 4), indicating that IL-13 had no effect on macrophage (Fig 3A), whereas adhesion of circulating neutrophils was egression (Fig 2B). Nevertheless, macrophage content of the similar (Fig 3A). In addition, the adhesion of the two monocyte same lesions was signiﬁcantly lower in IL-13-injected mice subsets was equally reduced (unpublished observation), compared to PBS-injected mice, thereby conﬁrming our initial suggesting that IL-13 administration reduces monocyte observation in a different atherosclerosis-prone mouse strain recruitment through a common pathway. Thus, we hypothe- (Fig 2C). sized that IL-13-induced changes may result in decreased Thus, IL-13 limits lesional macrophage content by a endothelial cell activation. To test this, we performed intravital mechanism other than macrophage emigration. microscopy of carotid arteries of ApoE mice that were fed an atherogenic diet for a total of 6 weeks and received PBS IL-13 administration results in decreased VCAM-1 expression or IL-13 during the last 2 weeks of diet, followed by the and monocyte adhesion in lesions of atherosclerotic ApoE injection of ﬂuorescently labelled beads coupled with anti- mice in vivo VCAM-1 or anti-CCL2 antibodies to analyze the carotid To explain the decreased numbers of macrophages in athero- endothelial activation state in these mice. VCAM-1 expression sclerotic lesions despite unchanged peripheral blood monocyte was signiﬁcantly decreased in IL-13-treated mice (Fig 3B), EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1075 4 2 Lesion area (x10 µµµµm ) Collagen content (%) Macrophage content + 2 (mac-3 cells/mm ) Research Article www.embomolmed.org IL-13 protects from atherosclerosis Figure 2. IL-13 administration has no effect Monocyte Baseline or on macrophage egress from established Clodronate Labeling PBS vs. IL-13 atherosclerotic lesions. ApoE mice were fed an injections (beads) treatment Analysis atherogenic diet for 4 weeks (baseline) or 6 weeks (PBS and IL-13, respectively). After 3 weeks, circulating monocytes of all mice were labelled with fluorescent latex beads following clodronate- 24h liposome depletion 24 h before. One week later, one 0 3 4 6 group of mice was sacrificed for baseline (n¼ 7) Weeks on atherogenic diet measurements of bead and macrophage content and the remaining mice received biweekly B C intraperitoneal injections with either PBS (n¼ 7) or PBS -/- ApoE inj IL-13 (n¼ 8) until sacrifice. 250 IL-13 *** A. Diagram of experimental design. n.s B. Similar content of fluorescent beads per plaque area in the aortic root of PBS and IL-13 admin- istration, respectively, compared to baseline. C. Decreased macrophage content in lesions of ApoE mice injected with IL-13. Sections were 100 stained with the macrophage-specific anti-mac-2 antibody and values represent the percentages of þ þ mac-2 cells/total DAPI cells. p¼ 0.0001. All data are shown as mean SEM values of all mice of each group. -/- ApoE inj PBS IL-13 Weeks on atherogenic diet whereas the endothelial presentation of CCL2 was similar in IL-13 administration skews macrophage phenotype towards both groups (Fig S4 of Supporting Information). alternatively activated macrophages (M2) in vivo These data strongly suggest that the decrease in lesional Because IL-13 is known to induce alternative macrophage macrophage content observed in IL-13-treated mice is activation, we investigated whether IL-13 administration had caused by a reduction in the VCAM-1-dependent monocyte the capacity to generate M2 macrophages in vivo. Indeed, we recruitment. observed a shift within the macrophage population towards A B * *** PBS IL-13 12 35 PBS -/- ApoE inj IL-13 30 2 5 Monocytes Neutrophils PBS IL13 -/- ApoE inj Figure 3. IL-13 administration reduces VCAM-1-dependent monocyte recruitment in established atherosclerotic lesions. gfp/wt / A. Cx3cr1 ApoE mice were fed an atherogenic diet for 6 weeks and received biweekly intraperitoneal injections with PBS (n¼ 8) or IL-13 (n¼ 8) during the last 2 weeks. At the time of sacrifice, PE-conjugated anti-GR1 antibodies were injected intravenously and the number of monocytes (GFP cells) and neutrophils (PE cells) adhering to the carotid bifurcation was assessed by intravital microscopy. Values represent the numbers of cells/optical field. p¼ 0.0185, Mann–Whitney test. B. ApoE mice were fed an atherogenic diet for 6 weeks and received biweekly intraperitoneal injections with PBS (n¼ 8) or IL-13 (n¼ 8) during the last 2 weeks. At time of sacrifice, fluorescent anti-VCAM-1 beads were injected intravenously and the number of beads adhering to the carotid bifurcation was assessed by intravital microscopy. Values represent the numbers of beads/optical field. p¼ 0.0001. Representative microscopy images are shown. Bar: 100 mm. All data are shown as mean SEM values of all mice of each group. 1076 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 Leukocytes adhering to beads/plaque area the carotid bifurcation (cells/field) α α α αVCAM1 beads adhering to Macrophage content + + (%mac-2 cells/DAPI cells) the carotid bifurcation (beads/field) www.embomolmed.org Research Article Larissa Cardilo-Reis et al. þ þ more CD206 macrophages (M2) and less CD80 macrophages fewer M1 macrophages in lesions of IL-13-injected mice (M1) in the peritoneal cavities of IL-13-treated mice as assessed (Fig 4B). Moreover, immunostaining for the expression of by ﬂow cytometry (Fig 4A) and further conﬁrmed by two different M2 markers, Ym-1 and mannose receptor (MR, quantitative polymerase chain reaction (PCR), demonstrating CD206), demonstrated signiﬁcantly higher numbers of M2 a signiﬁcant up-regulation of common M2-related genes, macrophages in lesions of IL-13-injected mice, despite an overall including arginase-1 (Arg-1), chitinase 3-like 3 protein decreased macrophage content (Fig 4C and Fig S5B of (Chi3l3/Ym-1) as well as CCL9, and a concomitant down- Supporting Information, respectively). These changes in regulation of M1-related genes such as inducible nitric oxide macrophage activation states resulted in a signiﬁcantly synthase (iNOS), CD86 and CXCL10 (Fig S5A of Supporting increased M2:M1 ratio in lesions of IL-13-treated mice (Fig 4D). Information). The total numbers of peritoneal macrophages, In summary, we could demonstrate that the decreased T and B cells (including B1 and B2 cells) were not different lesional macrophage content was paralleled by the induction between the two groups (Table 1). Importantly, in analogy to the of M2 macrophages and concomitantly the reduction of M1 activation state of peritoneal macrophages, immunostaining of macrophages in atherosclerotic lesions by exogenous adminis- lesional macrophages for the expression of iNOS demonstrated tration of IL-13. A B * PBS IL-13 -/- LDLR inj PBS IL-13 C D PBS 0.8 *** 0.6 0.4 IL-13 0.2 0.0 -/- -/- LDLR inj PBS IL-13 LDLR inj PBS IL-13 Figure 4. IL-13 administration skews macrophage phenotype towards alternatively activated (M2) macrophages in vivo. A. LDLR mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n¼ 11) or IL-13 (n¼ 13) during the last 5 weeks. Increased frequencies of M2 macrophages and decreased frequencies of M1 macrophages in the peritoneal cavities of LDLR mice injected with IL-13. Peritoneal cells were stained with antibodies against CD80 (M1) and CD206 (M2) and identified by flow cytometry. CD80/CD206 double-positive cells were classified as M1/M2 macrophages. Values represent the percentages of individual macrophage subtypes/total macrophages. p¼ 0.04. B. Decreased numbers of M1 macrophages in lesions of LDLR mice injected with IL-13. Sections were stained with an antibody against iNOS, which is þ 2 specifically expressed by M1 macrophages and values represent the numbers of iNOS cells/mm of total lesion area. p¼ 0.012. C. Increased numbers of M2 macrophages in lesions of LDLR mice injected with IL-13. Sections were stained with an antibody against Ym-1, which is þ 2 specifically expressed by M2 macrophages. Values represent the numbers of Ym-1 cells/mm of total lesion area. p¼ 0.024. D. Increased ratio of M2:M1 macrophages in lesions of LDLR mice injected with IL-13. p¼ 0.0001. All data are shown as mean SEM values of all mice of each group. Images show representative examples of the respective stainings. Original magnification: 400. EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1077 M2 macrophages + 2 (Ym1 cells/mm ) M1 macrophages + 2 (iNOS cells/mm ) M2/M1 ratio Research Article www.embomolmed.org IL-13 protects from atherosclerosis Alternatively activated macrophages by IL-13 show higher stimulated macrophages and to a signiﬁcantly greater extent in clearance of OxLDL in vitro macrophages stimulated with IL-13 (Fig 5A). An increased To study potential effects of differentially activated macro- uptake of CuOx-LDL was further conﬁrmed by Oil Red O (ORO) phages in atherogenesis, we evaluated the ability of IFN-g staining, which revealed a higher percentage of ORO cells in and IL-13 to modulate macrophage foam-cell formation by IL-13-activated macrophages compared to IFN-g-activated promoting either classical (M1) or alternative (M2) macrophage macrophages (Fig S6B of Supporting Information). Consistent activation, respectively. Thioglycollate-elicited macrophages with that, IL-13 stimulation resulted in increased expression of were stimulated with either IFN-g or IL-13, and successful the scavenger receptor CD36, which was further induced after differentiation into M1 or M2 macrophages was conﬁrmed by CuOx-LDL loading (Fig S6C of Supporting Information). the expression of iNOS (M1) and Arg-1 (M2), respectively The expression of scavenger receptor A-1 (SRA-1) and LOX-1 (Fig S6A of Supporting Information). Differentially activated was not different between the two differentially activated macrophages were then incubated with copper-oxidized macrophage foam cells (Fig S6D and S6E of Supporting LDL (CuOx-LDL) to induce foam-cell formation. Subsequent Information). Addition of HDL to the foam-cell cultures analyses of these cells revealed an increase of total cellular signiﬁcantly reduced the increased cellular cholesterol only in cholesterol content in foam-cell cultures derived from IFN-g- IL-13-activated macrophage cultures, thereby abrogating the A B C *** 6 *** *** 50 5 ABCA-1 40 4 n.s 30 3 ABCG-1 ββββ-Actin 0 0 CuOx-LDL 50 µµµµg/mL -+ + - + + CuOx-LDL 50 µµµµg/mL ++ CuOx-LDL 50 µµg/mL µµ -+ - + IFN-γγγγ 100 ng/mL ++ + - - - IFN-γγγγ 100 ng/mL +- IFN-γγγγ 100 ng/mL++ - - IL-13 5 ng/mL --- + + + IL-13 5 ng/mL -+ IL-13 5 ng/mL-- + + HDL 10 µµµµg/mL -- + - - + HDL 10 µµµµg/mL ++ D E F *** 1500 *** 1.75 0.5 *** 1.50 0.4 1000 PBS ** 1.25 0.3 n.s 1.00 IL13 0.75 0.2 0.50 0.1 0.25 0.0 0.00 CuOx-LDL 50 µµµµg/mL -+ - + CuOx-LDL 50 µµµµg/mL -+ - + -/- LDLR inj PBS IL-13 IFN-γγγγ 100 ng/mL ++ - - IFN-γγγγ 100 ng/mL ++ - - IL-13 5 ng/mL IL-13 5 ng/mL -- + + -- + + Figure 5. Alternatively activated macrophages (M2) exhibit increased clearance of OxLDL in vitro. A. Thioglycollate-elicited macrophages were stimulated with IFN-g or IL-13 into classically (M1) or alternatively (M2) activated macrophages, respectively, and then incubated with CuOx-LDL for 24 h to generate foam cells. Increased cellular cholesterol levels in M2-derived foam cells are reduced in the presence of HDL. M1 and M2 macrophages were incubated with CuOx-LDL in the absence or presence of HDL 10 mg/ml. Lipids were extracted from cell lysates and total cholesterol and protein were measured. Data are shown as mean SEM values of two independent experiments performed in quadruplicates and represent mg cholesterol/mg protein. p¼ 0.04, p¼ 0.0001. B. Increased HDL-dependent cholesterol efflux by M2-derived foam cells. M1 and M2 macrophages were incubated with CuOx-LDL plus 1 mMof [ H]-cholesterol and HDL-dependent efflux was assayed as described in Materials and Methods Section. Data represent percentages of HDL-dependent efflux/total efflux. p¼ 0.010, t-test. C. Increased ABCA1 and ABCG1 expression in M2-derived foam cells. Shown is a representative Western blot for the presence of ABCA1, ABCG1 and b-actin in lysates of cells that were treated as indicated. D,E. The quantification of the band intensity of ABCA1 (D) and ABCG1 (E) related to b-actin. p¼ 0.04, p¼ 0.0075, p¼ 0.0001. All data in (B–E) are shown as mean SEM values of three independent experiments performed in triplicates. / / F. Increased ABCA1 expression in lesions of LDLR mice injected with IL-13. LDLR mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n¼ 11) or IL-13 (n¼ 13) during the last 5 weeks. Sections were stained with an antibody against ABCA1 and values þ 2 represent the numbers of ABCA1 cells/mm of total lesion area, p¼ 0.031. Data are shown as mean SEM values of all mice of each group. Images show representative ABCA1 staining. Original magnification 400. 1078 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 ABCA-1 (band intensity) Cholesterol/protein (mg) ABCG-1 (band intensity) HDL-dependent efflux (%) ABCA1 expression + 2 (ABCA1 cells/mm ) www.embomolmed.org Research Article Larissa Cardilo-Reis et al. increased foam-cell formation (Fig 5A). These data suggested a Information). Importantly, cross-sectional analyses of lesions higher cholesterol efﬂux capacity of IL-13-activated compared to in the aortic origin revealed signiﬁcantly accelerated athero- IFN-g-activated foam cells. sclerosis with almost two times larger lesions in IL-13 bone To further test this hypothesis, we performed in vitro marrow recipients, indicating a protective role of IL-13 in cholesterol efﬂux assays using equal amounts of [ H]-choles- atherogenesis (Fig 6A and Table SI of Supporting Information). terol, which demonstrated a signiﬁcantly higher HDL-dependent In addition, en face analyses also showed a trend towards efﬂux of foam-cell cultures derived from IL-13-stimulated increased lesion formation in recipients of IL-13 bone macrophages compared to IFN-g-stimulated macrophages marrow (Table SI of Supporting Information). (Fig 5B). Moreover, to evaluate the ability of IL-13 to directly Lesions of IL-13 bone marrow chimeras displayed increase cholesterol efﬂux in macrophage foam cells, macro- increased necrotic core formation, consistent with advanced phages were ﬁrst loaded with CuOx-LDL plus [ H]-cholesterol plaque progression (Fig 6B). Nevertheless, the relative macro- and then stimulated with IFN-g and IL-13, respectively. phage content was equivalent between the two groups (Fig 6C) Importantly, HDL-dependent efﬂux was signiﬁcantly increased with signiﬁcantly less M2 macrophages in lesions of IL-13 in IL-13-stimulated foam cells using this experimental setup as bone marrow chimeras (Fig 6D). The predominant M1 well (Fig S7 of Supporting Information). We therefore macrophage areas were similar between the two groups investigated the expression levels of the two most important (Fig 6E). transporters responsible for cholesterol efﬂux in macrophages, To investigate potential immunological differences parallel- ATP-binding cassette A1 (ABCA1) and G1 (ABCG1), by ing this increased lesion formation, total splenocytes of mice immunoblotting and found that upon stimulation with CuOx- from both groups were stimulated with anti-CD3 and anti-CD28 LDL the expression of both ABC transporters was signiﬁcantly for 72 h to induce maximal T-cell activation in vitro.As / / up-regulated in foam cells derived from IL-13-activated macro- expected, splenocytes from IL-13 LDLR bone marrow phages compared to IFN-g-activated foam cells (Fig 5C–E) and chimeras produced only minimal amounts of IL-13. Moreover, non-activated foam cells (unpublished observation). This was the production of IL-4 and IL-10, but not IL-5 and IFN-g were also conﬁrmed by quantitative PCR on the messenger RNA signiﬁcantly diminished in these mice (Fig 7A and Table SI of (mRNA) level (Fig S8A and S8B of Supporting Information). In Supporting Information). This selective decrease in Th2 contrast, IFN-g-activated macrophages exhibited only a sig- cytokine production was also reﬂected by a signiﬁcant increase niﬁcant up-regulation of ABCG1 protein following CuOx-LDL in Th1-dependent IgG2c antibodies in serum of the IL-13 stimulation, albeit to a lesser degree than IL-13-activated LDLR bone marrow chimeric mice, while total IgG1 antibody macrophages (Fig 5E). Consistent with that, IL-13 stimulation levels were not different (Fig 7B and Table SII of Supporting also resulted in the increased expression of the nuclear receptor Information). IgM levels were similar between both groups LXRa, which is the main transcription factor controlling ABCA1 (Table SI of Supporting Information). These changes in T-cell- and G1 expression (Fig S8C of Supporting Information). dependent IgG levels resulted in a signiﬁcantly decreased To correlate these in vitro ﬁndings with effects of IL-13 on IgG1:IgG2c ratio indicating an overall Th1-biased response macrophage function in vivo, lesions of cholesterol-fed LDLR (Fig 7C). Similar results were obtained for MDA-LDL-speciﬁc mice that received either IL-13 or PBS (see Fig 1) were analyzed IgG1 and IgG2c titers, respectively (Fig S10A of Supporting for ABCA1 expression by immunohistochemistry. Remarkably, Information). Importantly, the numbers of splenic T cells and atherosclerotic lesions of IL-13-treated mice showed a sig- B cells (including B1 and B2 cells) were not different between niﬁcantly higher number of ABCA1 expressing cells compared the two groups (Table SI of Supporting Information). Consistent to lesions from control mice (Fig 5F). with an inherent Th1 bias, non-atherosclerotic IL-13-deﬁcient Taken together, our data demonstrate that macrophages mice that were used as bone marrow donors were also found to alternatively activated by IL-13 have an overall increased display decreased IgG1 and increased IgG2c levels compared capacity of OxLDL clearance, as they display increased uptake as to wild type controls (Fig S10B of Supporting Information). well as increased cholesterol efﬂux capacities without enhan- These data indicate that IL-13 deﬁciency results in an overall cing foam-cell formation. This should result in a more efﬁcient pro-inﬂammatory environment that inhibits alternative activa- removal of pro-inﬂammatory OxLDL and consequently a less tion of lesional macrophages and promotes atherosclerotic inﬂammatory environment in atherosclerotic lesions. lesion development. IL-13 deficiency accelerates atherosclerosis Finally, to demonstrate the role of IL-13 in the development of DISCUSSION atherosclerotic lesions, we transplanted lethally irradiated / þ/þ LDLR mice with bone marrow from either IL-13 or In the current study, we demonstrate a previously unrecognized IL-13 mice. Four weeks after bone marrow transplantation atheroprotective role of IL-13 in murine models of atherogen- (BMT) and successful replenishment, mice were switched to an esis. It was assumed that IL-13 would have a similar pro- atherogenic diet for the subsequent 16 weeks to induce atherogenic role in atherosclerosis as IL-4, because both atherosclerosis (Fig S9 of Supporting Information). At time of cytokines share similar functions by engaging the same receptor sacriﬁce, the two groups of mice were not different regarding complexes. Our data now point to a differential role in body weight, TC and TG levels (Table SI of Supporting atherosclerosis, which can be explained by unique functions EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1079 Research Article www.embomolmed.org IL-13 protects from atherosclerosis A B -/- IL-13→LDLR ** 35 ** -/- 45 ** -/- ** +/+ ** ** ** 15 +/+ ** 0 50 100 150 200 250 300 350 400 -/- IL-13→LDLR µµµµm +/+ -/- C D E 200 ** 0 0 -/- -/- -/- IL-13→LDLR +/+ -/- IL-13→LDLR +/+ -/- IL-13→LDLR +/+ -/- Figure 6. Increased atherosclerosis in IL-13-deficient LDLR mice. / þ/þ / A. LDLR mice were reconstituted with bone marrow from either IL-13 mice (n¼ 12) or IL-13 mice (n¼ 14) and fed an atherogenic diet for 16 weeks. Increased extent of atherosclerotic lesion size in cross-sections of the aortic origin in mice reconstituted with IL-13 bone marrow. Values represent mm /section throughout the entire aortic origin, (400 mm). p¼ 0.0023. Images show representative H&E stains. Original magnification 50. B. Increased necrotic core area in lesions of recipients of IL-13 bone marrow. Values represent percentages of necrotic core area/total lesion area. p¼ 0.016. / þ/þ C. Lesional macrophage content between recipients of IL-13 or IL-13 bone marrow. Sections were stained with the macrophage-specific anti-mac-3 antibody and values represent the percentages of mac-3 area/cellular lesion area. D. Decreased lesional M2 macrophage content in lesions of recipients of IL-13 bone marrow. Sections were stained with an antibody against Ym-1, which is þ 2 specifically expressed by M2 macrophages and values represent number of Ym-1 cells/mm of cellular lesion area. p¼ 0.0043, Mann–Whitney test. / þ/þ E. Relative lesional M1 macrophage content between recipients of IL-13 or IL-13 bone marrow. Sections were stained with an antibody against iNOS, which is specifically expressed by M1 macrophages and values represent the percentages of iNOS area/cellular lesion area. All data are shown as mean SEM values of all mice of each group. of IL-4 and IL-13 as a consequence of the exclusive engagement and Th1/Th2-dependent IgG isotype levels in sera of these mice. of the alternative receptors IL-4Ra/gc and IL-13Ra2, respec- Our data are consistent with the known impairment in Th2-cell tively, or by differences in ligand afﬁnity for the same IL-4Ra/ development in IL-13-deﬁcient mice (McKenzie et al, 1998a). IL-13Ra1 receptor complex (Kelly-Welch et al, 2003; LaPorte Likely this overall shift in the immune response contributed in et al, 2008). This is exempliﬁed by the ability of only IL-4 to part to the pro-atherogenic effect observed. The profound effects differentiate naı¨ve CD4 T cells into Th2 cells (Seder & Paul, of IL-13 deﬁciency on atherosclerotic lesion formation discour- 1994) or the non-redundant role of IL-13 in parasite expulsion, aged detailed analyses of plaque morphology, as a meaningful allergic inﬂammation and asthma (Bancroft et al, 1998; Grunig interpretation of potential differences in content of lesions of et al, 1998; Liang et al, 2011). different size and stage is virtually impossible. Speciﬁcally, we found that cholesterol-fed chimeric IL-13 To study the atheroprotective effect of IL-13 directly, we LDLR mice develop nearly twofold larger lesions in the aortic examined the impact of exogenous IL-13 administration on / / origin than LDLR mice that were reconstituted with wild type established atherosclerotic lesions in cholesterol-fed LDLR bone marrow. This pro-atherogenic effect of IL-13 deﬁciency mice. To avoid hepatic ﬁbrosis (Wynn, 2008), we chose a low was accompanied by an overall Th1-biased immune phenotype, dose of IL-13 administration to achieve only three times higher as judged by cytokine release of stimulated splenocyte cultures serum levels than found in atherosclerotic mice. Importantly, 1080 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 4 2 Lesion area (x10 µµµµm ) Macrophage content (%) M2 macrophages + 2 (Ym1 cells/mm ) M1 macrophages Necrotic core (%) (% iNOS area/cellular area) www.embomolmed.org Research Article Larissa Cardilo-Reis et al. A B 45 200 2.5 *** 175 n.s 2.0 1.5 1.0 0.5 0 0 -/- -/- -/- IL-13→LDLR IL-13→LDLR +/+ -/- +/+ -/- +/+ -/- +/+ -/- IL-13→LDLR +/+ -/- +/+ -/- αCD3/CD28 IgG1 IgG2c - αCD3/CD28 - 20 3.5 17.5 3.0 *** *** 2.5 12.5 2.0 1.5 7.5 1.0 0.5 2.5 0 0 -/- -/- -/- IL-13→LDLR IL-13→LDLR IL-13→LDLR +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- αCD3/CD28 αCD3/CD28 - - Figure 7. Effect of IL-13 deficiency on splenic cytokine production and antibody isotype levels. / þ/þ / A. LDLR mice were reconstituted with bone marrow from either IL-13 mice (n¼ 12) or IL-13 mice (n¼ 14) and fed an atherogenic diet for 16 weeks. At time of sacrifice, spleens and blood were collected from all mice. Recipients of IL-13-deficient bone marrow show a decreased production of Th2 cytokines (IL-13, IL-4 and IL-10) but not IFN-g by splenocytes stimulated with anti-CD3/CD28 in vitro. Data are presented as ng/ml cytokine of splenocyte cultures p¼ 0.0001. B. Increased levels of total IgG2c antibodies in sera of IL-13-deficient LDLR mice. Data are presented as mg/ml of indicated serum IgG isotypes. p¼ 0.029. C. Decreased ratio of IgG1:IgG2c antibodies in IL-13-deficient LDLR mice. p¼ 0.04. All data are shown as mean SEM values of all mice of each group. this interventional strategy did not result in an alteration of the In this regard it is important to point out that Fisher and Th1/Th2 phenotype of the immune response. Nevertheless, we colleagues recently reported that regression of atherosclerotic discovered that IL-13 administration resulted in signiﬁcantly lesions is associated with the up-regulation of markers of increased lesional collagen content, which is consistent with the alternative macrophage activation (M2) (Feig et al, 2011a,b; known strong pro-ﬁbrotic role of IL-13 (Wynn, 2008), as well as Rayner et al, 2011). M2 macrophages have been documented in signiﬁcantly decreased lesional macrophage content. These murine and human lesions (Bouhlel et al, 2007; Chinetti- alterations in plaque morphology are strongly reminiscent of Gbaguidi et al, 2011; El Hadri et al, 2012; Feig et al, 2012; changes that were reported to occur as a result of lesion Khallou-Laschet et al, 2010). We now show that IL-13 regression induced by lowering serum cholesterol or increasing administration also induces relative and absolute increases in serum HDL in mice (Reis et al, 2001; Rong et al, 2001; Williams M2 macrophages in cholesterol-fed LDLR mice and that the et al, 2008). Of interest, the changes in our model of IL-13 same lesions that show increased collagen and a decreased administration occurred without changes in serum cholesterol macrophage content have signiﬁcantly increased numbers of levels. It remains to be shown whether IL-13 is also alternatively activated macrophages and furthermore, conco- mechanistically involved in atherosclerotic lesion stabilization mitantly decreased numbers of classically activated macro- during lipid lowering or active lesion regression. phages (M1). Because our quantitative assessment of lesional EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1081 IL-4 (ng/mL) IL-13 (ng/mL) IFN-γγγγ (ng/mL) IL-10 (ng/mL) Ratio IgG1/IgG2c Total IgG (mg/mL) Research Article www.embomolmed.org IL-13 protects from atherosclerosis macrophages suggests the presence of still ‘uncommitted’ ment seems to be largely a consequence of decreased macrophages (i.e. iNOS and Ym-1 ), we believe that IL-13 endothelial VCAM-1 expression, which we observed in athero- primarily acts on this particular population of existing sclerotic mice treated with IL-13. A mechanistic role for VCAM-1 macrophages. In contrast, lesions of cholesterol-fed chimeric is further supported by the fact that only recruitment of / / IL-13 LDLR mice displayed decreased numbers of M2 monocytes, but not neutrophils, was affected by the IL-13 macrophages. Although this dual classiﬁcation pattern of intervention. Furthermore, no difference between the recruit- lo hi macrophages has been considered overly simplistic, it helped ment of nonclassical Ly6C or classical Ly6C monocytes was characterizing macrophage heterogeneity and plasticity within observed, which is typically dependent on the expression of atherosclerotic plaques (Feig et al, 2011a,b; Khallou-Laschet speciﬁc chemokines (Combadiere et al, 2008; Ingersoll et al, et al, 2010; Stoger et al, 2010). Our data show that the 2011; Tacke et al, 2007). In agreement with that, we did not macrophage phenotype in atherosclerotic lesions can be observe an alteration of CCL2 presentation by endothelial cells modulated by IL-13 independent of cholesterol-lowering. in the carotid arteries of atherosclerotic ApoE mice injected We also addressed the functional consequences of increased with IL-13. Finally, in analogy to the parallels with lesion numbers of M2 macrophages with respect to uptake of OxLDL regression discussed above, Potteaux et al recently reported that and cholesterol efﬂux, rate limiting steps in foam-cell formation decreased monocyte recruitment during lesion regression was during atherogenesis (Steinberg & Witztum, 2010). Using also associated with decreased endothelial VCAM-1 expression murine primary macrophages differentially activated with (Potteaux et al, 2011). Endothelial VCAM-1 expression is a key either IL-13 or IFN-g, respectively, to model potential extremes event during atherosclerotic lesion formation (Cybulsky et al, of cytokine exposure inside the plaques, we observed an 2001; Dansky et al, 2001). However, it is unlikely that IL-13 increased capacity of IL-13-stimulated macrophages to take up administration had a direct effect on VCAM-1 expression, OxLDL. This is consistent with previous studies showing that as a previous study demonstrated that IL-13 in fact promoted M2 macrophages exhibit increased expression of scavenger the up-regulation of VCAM-1 on activated-endothelial cells receptor CD36 and possess higher phagocytic activity (Berry in vitro (Bochner et al, 1995; Woltmann et al, 2000). It is et al, 2007; Gordon & Martinez, 2010). Previously, IL-4/IL-13 known that VCAM-1 is strongly induced by OxLDL in stimulation has been shown to activate PPARg, leading to endothelial cells in vitro and at lesion prone-sites even before upregulation of CD36 through the generation of endogenous the appearance of visible lesions (Cybulsky & Gimbrone, 1991; ligands in murine and human macrophages (Huang et al, 1999; Khan et al, 1995). Therefore, considering our data, we would Rey et al, 1998), which might be predicted to lead to enhanced speculate that a more likely explanation for the decreased foam-cell formation. Indeed, we could also demonstrate VCAM-1 expression is a reduced intimal content of OxLDL as a increased expression of CD36, but not SRA-1 or LOX-1, in consequence of enhanced IL-13-induced M2 macrophage- IL-13-activated macrophage foam cells. We also found that mediated clearance. OxLDL-loaded IL-13-activated macrophages exhibited a higher In conclusion, our data indicate a key role for IL-13 in halting cholesterol-efﬂux capacity and had increased expression of the progression of atherogenesis and promoting plaque ABCA1 and ABCG1 compared to IFN-g-activated macrophages, stabilization. We provide evidence that IL-13 leads to decreased resulting in no net increase in cholesterol accumulation. Our VCAM-1 mediated monocyte recruitment to atherosclerotic data is supported by a previous report demonstrating decreased lesions, enhanced phenotypic modulation towards the repara- ABCA1 expression and cholesterol-efﬂux of IFN-g-treated tive and atheroprotective M2 phenotype, and enhanced collagen murine foam cells compared to unstimulated foam cells deposition. Even in the absence of decreased plasma cholesterol (Panousis & Zuckerman, 2000). If translatable to the in vivo levels, the changes induced by IL-13 are strongly reminiscent situation, our data suggest an enhanced ability of IL-13- of effects seen during plaque regression in response to stimulated M2 type macrophages to clear OxLDL from the cholesterol lowering. Thus, our ﬁndings identify a potential extracellular environment and efﬁciently promote efﬂux of free new target for the prevention and treatment of atherosclerosis. cholesterol via ABCA1/G1 pathways without enhancing foam- cell formation—a protective response that would be desirable in a lesional macrophage. In fact, we did ﬁnd increased expression MATERIALS AND METHODS of ABCA1 in lesions of IL-13-treated mice. It is noteworthy that Chinetti-Gbaguidi et al recently reported that IL-4-activated An expanded Materials and Methods Section is available in the human monocyte-derived macrophages are less prone to foam- Supporting Information. cell formation, although they found lower efﬂux capacity and increased cholesterol esteriﬁcation in these cells compared to Animal and intervention studies untreated cells. This comparison to ‘neutral’ monocytes and LDL receptor-deficient mice (LDLR ) and C57BL/6J were from The differences between IL-4 and IL-13 may explain the discrepan- Jackson Laboratories (Bar Harbor, Maine, USA); IL-13 mice were a cies (Chinetti-Gbaguidi et al, 2011). kind gift of Dr. Thomas Wynn (NIAID/NIH, Bethesda, USA). All mice A prominent consequence of IL-13 administration was were on a C57BL/6J background (tenth generation) and were bred decreased recruitment of monocytes to carotid arteries of in-house. All experimental protocols were approved by the institu- atherosclerotic ApoE mice, whereas no effect on lesional tional animal experimentation committee and the Austrian Ministry of macrophage egression was observed. The diminished recruit- Science. 1082 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. The paper explained PROBLEM: Atherosclerosis is a chronic inflammatory disease of the vessel activation states of plaque macrophages, and we could show that wall and the underlying cause of heart attacks and a majority of IL-13-activated macrophages (M2) possess more efficient, strokes. Atherosclerotic lesions are characterized by infiltrating beneficial lipid handling capacities compared to IFN-g-activated macrophages that take up accumulating lipids resulting in the macrophages (M1) in vitro. Importantly, atherosclerosis-prone formation of pro-inflammatory foam cells. Innate and adaptive mice that were incapable of secreting IL-13 by hematopoietic immunity modulate the development and progression of cells developed significantly bigger and more advanced ather- atherosclerotic lesions, which provides potential therapeutic osclerotic plaques. targets in addition to existing cholesterol lowering strategies. The functions of a number of cytokines, including the pro- IMPACT: inflammatory effects of IFN-g, are well established. However, the Our findings indicate a protective role for IL-13 in athero- role of interleukin-13 (IL-13) is still unknown. sclerosis. Remarkably, IL-13 has the capacity to alter plaque morphology in the presence of high serum cholesterol levels RESULTS: towards more stable, less vulnerable plaques. Because IL-13 In our study, we show that IL-13 administration limits the leads to alternative macrophage activation in atherosclerotic recruitment of macrophages to and promotes collagen produc- lesions, and these macrophages have anti-atherogenic proper- tion in established lesions of atherosclerosis-prone mice. These ties, our data identify macrophage polarization by IL-13 as novel effects are paralleled by IL-13-dependent changes in the point for therapeutic intervention. Bone marrow transplantation studies were performed as previously California, USA), smooth-muscle cell actin (Sigma-Aldrich), CD3 (DAKO, described (Binder et al, 2004). Thirty 8-week-old male LDLR mice Glostrup, Denmark), iNOS (ABCAM, Cambridge, UK), CD206 (BioLegend, were given a single dose of 9-Gy lethal irradiation and irradiated mice San Diego, California, USA), Ym1/2 (a kind gift of Dr. Shioko Kimura, were injected intravenously with 2 10 bone marrow cells harvested NIH/NCI, Bethesda, USA) and ABCA1 (Novus Biological, Littleton, / þ/þ from either IL-13 (n¼ 15) or IL-13 (n¼ 15) mice. Mice were fed Colorado, USA). The photographed images were analyzed using ImageJ regular chow diet for 4 weeks after BMT to allow for bone marrow 1.41 software. reconstitution and then switched to an atherogenic diet containing 21% fat and 0.2% cholesterol (TD88137, Ssniff Spezialdia ¨ten GmbH, Macrophage foam-cell assays Soest, Germany) for an additional 16 weeks to induce lesion For all in vitro experiments, C57BL/6J mice, 12–16 weeks of age, were formation. injected intraperitoneally with 2 ml of 3% thioglycollate (Difco, For the atherosclerosis intervention study, twenty-four 12-week-old Thermo Fischer Scientific). After 3 days, thioglycollate-elicited macro- female LDLR mice were fed an atherogenic diet (Ssniff) for a total of phages were harvested and differentiated into classically activated 16 weeks to induce lesion formation. At week 11, mice were divided macrophages (M1) with 100 ng/ml IFN-g (R&D systems) or alterna- randomly into two groups and injected intraperitoneally with PBS tively activated macrophages (M2) with 5 ng/ml IL-13 (R&D systems) (n¼ 11) or IL-13 (50 ng/mouse R&D systems, Minneapolis, Minnesota, for 16 h. Following differentiation, cells were stimulated with USA; n¼ 13) twice per week for the following remaining 5 weeks. 50 mg/ml CuOx-LDL in the presence or absence of 10 mg/ml of HDL in full culture medium containing 1% mouse serum for 24 h to induce Evaluation and phenotypic analysis of atherosclerotic lesions foam-cell formation. Subsequently, cholesterol efflux, RNA/protein The extent of atherosclerosis was determined in a blinded fashion isolation and cellular cholesterol quantification experiments were in en face preparations of the entire aorta, as well as in cross performed as described in the Supporting Information. For primer sections through the aortic origin, by computer-assisted image sequences see Table SII of Supporting Information. analysis as previously described (Binder et al, 2004). Lesion phenotype was determined by the content of collagen, size of necrotic core area, Monocyte labeling and macrophage egression assessment and the presence of macrophages, smooth-muscle cells, T cells, Twenty-two male, 8-week-old, ApoE mice (C57BL/6J background) classically activated (M1) macrophages, alternatively activated (M2) were fed a standard western diet containing 21% fat and 0.2% macrophages, and ABCA1 expressing cells in lesions of equal size. For cholesterol (Altromin Spezialfutter GmbH & Co.KG, Lage, Germany) for the collagen content, sections were stained with Sirius Red, and for the 6 weeks to induce lesion formation. At week 3, circulating classical hi assessment of necrotic cores, sections were stained with a modified Ly6C monocytes were labelled by intravenous (i.v) injection of 1 mm elastic-trichrome stain. For the presence of macrophages, smooth- Fluoresbrite green fluorescent (YG) plain microspheres (Polysciences muscle cells, T cells, M1 macrophages, M2 macrophages and ABCA1 Inc., Warrington, Pennsylvania, USA), 24 h after clodronate-liposome expressing cells immunohistochemistry was performed using anti- depletion of monocytes. Latex-beads monocytes were allowed to accumulate within atherosclerotic plaques for 1 week. At week 4, mice bodies against mouse Mac-3 (BD-Biosciences Pharmingen, San Diego, EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1083 Research Article www.embomolmed.org IL-13 protects from atherosclerosis were divided into three groups. One group was sacrificed for the Academy of Sciences (CJB), the Fondation Leducq (CJB), the quantification of bead and macrophage (mac-2 cells) content Austrian Science Fund (FWF) Project P22838 (HS) and the (baseline, n¼ 7); the remaining two groups received biweekly Deutsche Forschungsgemeinschaft (FOR809 and SFB914 TP intraperitoneal injections of either PBS (n¼ 7) or IL-13 (50 ng/mouse, B08 to CW and OS). R&D systems n¼ 8) for the remaining 2 weeks. All animal experiments were approved by the local ethical committee (Regierung von Supporting Information is available at EMBO Molecular Oberbayern). Macrophage emigration from atherosclerotic lesions Medicine Online. was analyzed as described previously (Potteaux et al, 2011). The authors declare that they have no conflict of interest. Intravital microscopy gfp/wt / Sixteen male, 8-week-old, Cx3cr1 ApoE mice (C57BL/6J background, for leukocyte endothelial interactions) and 16 male, References 8-week-old, ApoE mice (C57BL/6J background, for endothelial Ait-Oufella H, Taleb S, Mallat Z, Tedgui A (2011) Recent advances on the adhesion molecules expression) were fed an atherogenic diet contain- role of cytokines in atherosclerosis. Arterioscler Thromb Vasc Biol 31: 969- ing 21% fat and 0.2% cholesterol (Altromin) for 6 weeks to induce lesion formation. At week 4, they were randomly divided into two Bancroft AJ, McKenzie AN, Grencis RK (1998) A critical role for IL-13 in groups and injected intraperitoneally with PBS (n¼ 8) or IL-13 resistance to intestinal nematode infection. J Immunol 160: 3453-3461 Barlow JL, McKenzie AN (2011) Nuocytes: expanding the innate cell repertoire (50 ng/mouse, R&D systems, n¼ 8) twice per week for the remaining in type-2 immunity. J Leukoc Biol 90: 867-874 2 weeks. At the end of this period, mice were anaesthetized with Berry A, Balard P, Coste A, Olagnier D, Lagane C, Authier H, Benoit-Vical F, ketamine/xylazine and leukocyte endothelial interactions (Drechsler ´ ´ Lepert JC, Seguela JP, Magnaval JF et al (2007) IL-13 induces expression of et al, 2010) and expression of endothelial adhesion molecules (Engel CD36 in human monocytes through PPArg activation. Eur J Immunol 37: et al, 2011) were analyzed by intravital microscopy of the left carotid 1642-1652 artery as described previously. To permit discrimination of phagocyte Binder CJ, Chang MK, Shaw PX, Miller YI, Hartvigsen K, Dewan A, Witztum JL (2002) Innate and acquired immunity in atherogenesis. Nat Med 8: 1218- subsets a PE-conjugated antibody to Gr1 (eBioscience) was introduced via an intravenous catheder 5 min prior to recording. All animal Binder CJ, Hartvigsen K, Chang MK, Miller M, Broide D, Palinski W, Curtiss LK, experiments were approved by the local ethical committee (Regierung Corr M, Witztum JL (2004) IL-5 links adaptive and natural immunity speciﬁc von Oberbayern). for epitopes of oxidized LDL and protects from atherosclerosis. J Clin Invest 114: 427-437 Statistical analysis Bochner BS, Klunk DA, Sterbinsky SA, Coffman RL, Schleimer RP (1995) IL-13 Statistical analyses were performed by unpaired Student t-test for all selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells. J Immunol 154: 799-803 results of in vivo studies and by one-way analysis of variance (with Bouhlel MA, Derudas B, Rigamonti E, Dievart R, Brozek J, Haulon S, Zawadzki C, Bonferoni post-test analysis) for all in vitro data (unless indicated Jude B, Torpier G, Marx N et al (2007) PPARgamma activation primes human differentially) to determine statistical significance between the groups. monocytes into alternative M2 macrophages with anti-inﬂammatory Data are presented as mean SEM and p< 0.05 was considered properties. Cell Metab 6: 137-143 significant. Buono C, Come CE, Stavrakis G, Maguire GF, Connelly PW, Lichtman AH (2003) Inﬂuence of interferon-gamma on the extent and phenotype of diet- induced atherosclerosis in the LDLR-deﬁcient mouse. Arterioscler Thromb Vasc Biol 23: 454-460 Author contributions Buono C, Binder CJ, Stavrakis G, Witztum JL, Glimcher LH, Lichtman AH (2005) The study was conceived by CJB. LCR and CJB designed the T-bet deﬁciency reduces atherosclerosis and alters plaque antigen-speciﬁc study and analyzed the data with contributions of OW. LCR immune responses. Proc Natl Acad Sci USA 102: 1596-1601 and SG performed most of the experiments with contributions Chiaramonte MG, Schopf LR, Neben TY, Cheever AW, Donaldson DD, Wynn TA of NPM. SMS and HS contributed to the cholesterol efﬂux (1999) IL-13 is a key regulatory cytokine for Th2 cell-mediated pulmonary experiments. MD and OS performed and analyzed carotid granuloma formation and IgE responses induced by Schistosoma mansoni eggs. J Immunol 162: 920-930 intravital microscopy and macrophage egression experiments Chinetti-Gbaguidi G, Baron M, Bouhlel MA, Vanhoutte J, Copin C, Sebti Y, with contributions of CW. LCR and CJB wrote the manuscript Derudas B, Mayi T, Bories G, Tailleux A et al (2011) Human atherosclerotic with contributions of HS and OS. All authors read and approved plaque alternative macrophages display low cholesterol handling but high the manuscript. phagocytosis because of distinct activities of the PPARgamma and LXRalpha pathways. Circ Res 108: 985-995 Chomarat P, Banchereau J (1998) Interleukin-4 and interleukin-13: their Acknowledgements similarities and discrepancies. Int Rev Immunol 17: 1-52 Combadiere C, Potteaux S, Rodero M, Simon T, Pezard A, Esposito B, Merval R, We are grateful to Maria Ozsvar-Kozma and Laura Go¨derle for Proudfoot A, Tedgui A, Mallat Z (2008) Combined inhibition of CCL2, precise and immeasurable technical support, Gernot Schab- CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost bauer and Pavel Uhrin for assistance with animal experiments, abolishes atherosclerosis in hypercholesterolemic mice. Circulation 117: and the staff of the Department of Laboratory Medicine. We 1649-1657 thank Joseph L. Witztum and Esther Lutgens for critical Cybulsky MI, Gimbrone MA Jr., (1991) Endothelial expression of a reading of the manuscript. This work was supported by grants mononuclear leukocyte adhesion molecule during atherogenesis. Science from the GenAU program (DRAGON) (CJB), the Austrian 251: 788-791 1084 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086 www.embomolmed.org Research Article Larissa Cardilo-Reis et al. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos vascular cell adhesion molecule-1 gene expression in human vascular JC, Connelly PW, Milstone DS (2001) A major role for VCAM-1, but not ICAM- endothelial cells. J Clin Invest 95: 1262-1270 1, in early atherosclerosis. J Clin Invest 107: 1255-1262 King VL, Szilvassy SJ, Daugherty A (2002) Interleukin-4 deﬁciency decreases Dansky HM, Barlow CB, Lominska C, Sikes JL, Kao C, Weinsaft J, Cybulsky MI, atherosclerotic lesion formation in a site-speciﬁc manner in female LDL Smith JD (2001) Adhesion of monocytes to arterial endothelium and initiation receptor/ mice. Arterioscler Thromb Vasc Biol 22: 456-461 of atherosclerosis are critically dependent on vascular cell adhesion King VL, Cassis LA, Daugherty A (2007) Interleukin-4 does not inﬂuence molecule-1 gene dosage. Arterioscler Thromb Vasc Biol 21: 1662-1667 development of hypercholesterolemia or angiotensin II-induced Davenport P, Tipping PG (2003) The role of interleukin-4 and interleukin-12 in atherosclerotic lesions in mice. Am J Pathol 171: 2040-2047 the progression of atherosclerosis in apolipoprotein E-deﬁcient mice. Am J Kumar RK, Herbert C, Yang M, Koskinen AM, McKenzie AN, Foster PS (2002) Pathol 163: 1117-1125 Role of interleukin-13 in eosinophil accumulation and airway remodelling Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O (2010) in a mouse model of chronic asthma. Clin Exp Allergy 32: 1104-1111 Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Kuperman DA, Schleimer RP (2008) Interleukin-4, interleukin-13, signal Circulation 122: 1837-1845 transducer and activator of transcription factor 6, and allergic asthma. Curr El Hadri K, Mahmood DF, Couchie D, Jguirim-Souissi I, Genze F, Diderot V, Mol Med 8: 384-392 Syrovets T, Lunov O, Simmet T, Rouis M (2012) Thioredoxin-1 promotes LaPorte SL, Juo ZS, Vaclavikova J, Colf LA, Qi X, Heller NM, Keegan AD, Garcia KC anti-inﬂammatory macrophages of the M2 phenotype and antagonizes (2008) Molecular and structural basis of cytokine receptor pleiotropy in the atherosclerosis. Arterioscler Thromb Vasc Biol 32: 1445-1452 interleukin-4/13 system. Cell 132: 259-272 Engel D, Beckers L, Wijnands E, Seijkens T, Lievens D, Drechsler M, Gerdes N, Liang HE, Reinhardt RL, Bando JK, Sullivan BM, Ho IC, Locksley RM (2011) Soehnlein O, Daemen MJ, Stan RV et al (2011) Caveolin-1 deﬁciency Divergent expression patterns of IL-4 and IL-13 deﬁne unique functions in decreases atherosclerosis by hampering leukocyte inﬂux into the arterial allergic immunity. Nat Immunol 13: 58-66 wall and generating a regulatory T-cell response. FASEB J 25: 3838-3848 Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF, Soubrier F, Feig JE, Parathath S, Rong JX, Mick SL, Vengrenyuk Y, Grauer L, Young SG, Fisher Esposito B, Duez H, Fievet C et al (1999) Protective role of interleukin-10 in EA (2011a) Reversal of hyperlipidemia with a genetic switch favorably atherosclerosis. Circ Res 85: e17-24 affects the content and inﬂammatory state of macrophages in McKenzie G, Emson C, Bell S, Anderson S, Fallon P, Zurawski G, Murray R, atherosclerotic plaques. Circulation 123: 989-998 Grencis R, McKenzie AN (1998a) Impaired development of Th2 cells in IL- Feig JE, Rong JX, Shamir R, Sanson M, Vengrenyuk Y, Liu J, Rayner K, Moore K, 13-deﬁcient mice. Immunity 9: 423-432 Garabedian M, Fisher EA (2011b) HDL promotes rapid atherosclerosis McKenzie GJ, Bancroft A, Grencis RK, McKenzie AN (1998b) A distinct role for regression in mice and alters inﬂammatory properties of plaque monocyte- interleukin-13 in Th2-cell-mediated immune responses. Curr Biol 8: 339- derived cells. Proc Natl Acad Sci USA 108: 7166-7171 342 Feig JE, Vengrenyuk Y, Reiser V, Wu C, Statnikov A, Aliferis CF, Garabedian MJ, Oliphant CJ, Barlow JL, McKenzie AN (2011) Insights into the initiation of type Fisher EA, Puig O (2012) Regression of atherosclerosis is characterized by 2 immune responses. Immunology 134: 378-385 broad changes in the plaque macrophage transcriptome. PLoS One 7: Oriente A, Fedarko NS, Pacocha SE, Huang SK, Lichtenstein LM, Essayan DM e39790 (2000) Interleukin-13 modulates collagen homeostasis in human skin and Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A (2006) IL-13 keloid ﬁbroblasts. J Pharmacol Exp Ther 292: 988-994 signaling through the IL-13alpha2 receptor is involved in induction of Panousis CG, Zuckerman SH (2000) Interferon-gamma induces TGF-beta1 production and ﬁbrosis. Nat Med 12: 99-106 downregulation of Tangier disease gene (ATP-binding-cassette transporter Gautier EL, Jakubzick C, Randolph GJ (2009) Regulation of the migration and 1) in macrophage-derived foam cells. Arterioscler Thromb Vasc Biol 20: survival of monocyte subsets by chemokine receptors and its relevance to 1565-1571 atherosclerosis. Arterioscler Thromb Vasc Biol 29: 1412-1418 Peters W, Charo IF (2001) Involvement of chemokine receptor 2 and its ligand, Gordon S, Martinez FO (2010) Alternative activation of macrophages: monocyte chemoattractant protein-1, in the development of mechanism and functions. Immunity 32: 593-604 atherosclerosis: lessons from knockout mice. Curr Opin Lipidol 12: 175-180 Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, Pinderski Oslund LJ, Hedrick CC, Olvera T, Hagenbaugh A, Territo M, Berliner JA, Sheppard D, Mohrs M, Donaldson DD, Locksley RM et al (1998) Requirement Fyfe AI (1999) Interleukin-10 blocks atherosclerotic events in vitro and for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261- in vivo. Arterioscler Thromb Vasc Biol 19: 2847-2853 2263 Potteaux S, Gautier EL, Hutchison SB, van Rooijen N, Rader DJ, Thomas MJ, Gupta S, Pablo AM, Jiang X, Wang N, Tall AR, Schindler C (1997) IFN-gamma Sorci-Thomas MG, Randolph GJ (2011) Suppressed monocyte recruitment potentiates atherosclerosis in ApoE knock-out mice. J Clin Invest 99: 2752- drives macrophage removal from atherosclerotic plaques of Apoe/ mice 2761 during disease regression. J Clin Invest 121: 2025-2036 Hansson GK (2005) Inﬂammation, atherosclerosis, and coronary artery Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van Gils JM, disease. N Engl J Med 352: 1685-1695 Rayner AJ, Chang AN, Suarez Y et al (2011) Antagonism of miR-33 in mice Hansson GK, Hermansson A (2011) The immune system in atherosclerosis. Nat promotes reverse cholesterol transport and regression of atherosclerosis. J Immunol 12: 204-212 Clin Invest 121: 2921-2931 Huang JT, Welch JS, Ricote M, Binder CJ, Willson TM, Kelly C, Witztum JL, Reis ED, Li J, Fayad ZA, Rong JX, Hansoty D, Aguinaldo JG, Fallon JT, Fisher EA Funk CD, Conrad D, Glass CK (1999) Interleukin-4-dependent production of (2001) Dramatic remodeling of advanced atherosclerotic plaques of the PPAR-gamma ligands in macrophages by 12/15-lipoxygenase. Nature 400: apolipoprotein E-deﬁcient mouse in a novel transplantation model. J Vasc 378-382 Surg 34: 541-547 Ingersoll MA, Platt AM, Potteaux S, Randolph GJ (2011) Monocyte trafﬁcking in Rey A, M’Rini C, Sozzani P, Lamboeuf Y, Beraud M, Caput D, Ferrara P, Pipy B acute and chronic inﬂammation. Trends Immunol 32: 470-477 (1998) IL-13 increases the cPLA2 gene and protein expression and the Kelly-Welch AE, Hanson EM, Boothby MR, Keegan AD (2003) Interleukin-4 and mobilization of arachidonic acid during inﬂammatory process in mouse interleukin-13 signaling connections maps. Science 300: 1527-1528 peritoneal macrophages. Biochem Biophys Acta 1393: 244-252 Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Clement M, Rong JX, Li J, Reis ED, Choudhury RP, Dansky HM, Elmalem VI, Fallon JT, Breslow Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A et al (2010) Macrophage JL, Fisher EA (2001) Elevating high-density lipoprotein cholesterol in plasticity in experimental atherosclerosis. PLoS One 5: e8852 apolipoprotein E-deﬁcient mice remodels advanced atherosclerotic lesions Khan BV, Parthasarathy SS, Alexander RW, Medford RM (1995) Modiﬁed low by decreasing macrophage and increasing smooth muscle cell content. density lipoprotein and its constituents augment cytokine-activated Circulation 104: 2447-2452 EMBO Mol Med (2012) 4, 1072–1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. 1085 Research Article www.embomolmed.org IL-13 protects from atherosclerosis Seder R, Paul W (1994) Acquisition of lymphokine-producing phenotype by Tedgui A, Mallat Z (2006) Cytokines in atherosclerosis: pathogenic and CD4þ T cells. Annu Rev Immunol 12: 635-673 regulatory pathways. Physiol Rev 86: 515-581 Steinberg D, Witztum JL (2010) Oxidized low-density lipoprotein and Tellides G, Tereb DA, Kirkiles-Smith NC, Kim RW, Wilson JH, Schechner JS, atherosclerosis. Arterioscler Thromb Vasc Biol 30: 2311-2316 Lorber MI, Pober JS (2000) Interferon-gamma elicits arteriosclerosis in the Stoger JL, Goossens P, de Winther MP (2010) Macrophage heterogeneity: absence of leukocytes. Nature 403: 207-211 relevance and functional implications in atherosclerosis. Curr Vasc Williams KJ, Feig JE, Fisher EA (2008) Rapid regression of atherosclerosis: Pharmacol 8: 233-248 insights from the clinical and experimental literature. Nat Clin Pract Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jaffer FA, Libby P, Weissleder R Cardiovasc Med 5: 91-102 (2006) Monocyte accumulation in mouse atherogenesis is progressive and Woltmann G, McNulty CA, Dewson G, Symon FA, Wardlaw AJ (2000) proportional to extent of disease. Proc Natl Acad Sci USA 103: 10340-10345 Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek R, Llodra J, Garin A, Liu J, eosinophils, but not neutrophils, to human umbilical vein endothelial cells Mack M, van Rooijen N et al (2007) Monocyte subsets differentially employ under ﬂow. Blood 95: 3146-3152 CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques. J Wynn TA (2008) Cellular and molecular mechanisms of ﬁbrosis. J Pathol 214: Clin Invest 117: 185-194 199-210 1086 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. EMBO Mol Med (2012) 4, 1072–1086

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Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

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Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

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