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. 2012 Oct;4(10):1072-86.
doi: 10.1002/emmm.201201374.

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

Larissa Cardilo-Reis  1 , Sabrina Gruber, Sabine M Schreier, Maik Drechsler, Nikolina Papac-Milicevic, Christian Weber, Oswald Wagner, Herbert Stangl, Oliver Soehnlein, Christoph J Binder
Affiliations

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

Larissa Cardilo-Reis et al. EMBO Mol Med. 2012 Oct.

Abstract

Atherosclerotic lesions are characterized by the accumulation of oxidized LDL (OxLDL) and the infiltration of macrophages and T cells. Cytokine expression in 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-γ, is well established, the role of Th2 cytokines is less clear. Therefore, we characterized the role of the Th2 cytokine interleukin (IL)-13 in murine atherosclerosis. Here, we report that IL-13 administration favourably modulated the morphology of already established atherosclerotic lesions by increasing lesional 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-γ-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.

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Figures

Figure 1
Figure 1. IL-13 administration alters plaque morphology of established atherosclerotic lesions

  1. LDLR−/− mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n = 11) or IL-13 (n = 13) during the last 5 weeks. Equal extent of atherosclerotic lesion size in cross-sections of the aortic origin in injected LDLR−/− mice. Values represent µm2/section throughout the entire aortic origin (300 µm). Original magnification 50×.

  2. 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. *p = 0.035. Original magnification 100×.

  3. Decreased macrophage content in lesions of LDLR−/− mice injected with IL-13. Sections were stained with the macrophage-specific anti-mac-3 antibody and values represent the numbers of mac-3+ cells/mm2 of total lesion area. ***p = 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.

Figure 2
Figure 2. IL-13 administration has no effect on macrophage egress from established atherosclerotic lesions
ApoE−/− mice were fed an 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-liposome depletion 24 h before. One week later, one group of mice was sacrificed for baseline (n = 7) measurements of bead and macrophage content and the remaining mice received biweekly intraperitoneal injections with either PBS (n = 7) or IL-13 (n = 8) until sacrifice.

  1. Diagram of experimental design.

  2. Similar content of fluorescent beads per plaque area in the aortic root of PBS and IL-13 administration, respectively, compared to baseline.

  3. Decreased macrophage content in lesions of ApoE−/− mice injected with IL-13. Sections were stained with the macrophage-specific anti-mac-2 antibody and values represent the percentages of mac-2+ cells/total DAPI+ cells. ***p = 0.0001. All data are shown as mean ± SEM values of all mice of each group.

Figure 3
Figure 3. IL-13 administration reduces VCAM-1-dependent monocyte recruitment in established atherosclerotic lesions

  1. Cx3cr1gfp/wt ApoE−/− mice were fed an atherogenic diet for 6 weeks and received biweekly intraperitoneal injections with PBS (n = 8) or IL-13 (n = 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. *p = 0.0185, Mann–Whitney test.

  2. ApoE−/− mice were fed an atherogenic diet for 6 weeks and received biweekly intraperitoneal injections with PBS (n = 8) or IL-13 (n = 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. ***p = 0.0001. Representative microscopy images are shown. Bar: 100 µm. All data are shown as mean ± SEM values of all mice of each group.

Figure 4
Figure 4. IL-13 administration skews macrophage phenotype towards alternatively activated (M2) macrophages in vivo

  1. LDLR−/− mice were fed an atherogenic diet for 16 weeks and received biweekly intraperitoneal injections with PBS (n = 11) or IL-13 (n = 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. *p = 0.04.

  2. Decreased numbers of M1 macrophages in lesions of LDLR−/− mice injected with IL-13. Sections were stained with an antibody against iNOS, which is specifically expressed by M1 macrophages and values represent the numbers of iNOS+ cells/mm2 of total lesion area. *p = 0.012.

  3. 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 specifically expressed by M2 macrophages. Values represent the numbers of Ym-1+ cells/mm2 of total lesion area. *p = 0.024.

  4. Increased ratio of M2:M1 macrophages in lesions of LDLR−/− mice injected with IL-13. ***p = 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×.

Figure 5
Figure 5. Alternatively activated macrophages (M2) exhibit increased clearance of OxLDL in vitro

  1. A. Thioglycollate-elicited macrophages were stimulated with IFN-γ 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 µg/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. *p = 0.04, ***p = 0.0001.

  2. B. Increased HDL-dependent cholesterol efflux by M2-derived foam cells. M1 and M2 macrophages were incubated with CuOx-LDL plus 1 µM of [3H]-cholesterol and HDL-dependent efflux was assayed as described in Materials and Methods Section. Data represent percentages of HDL-dependent efflux/total efflux. *p = 0.010, t-test.

  3. C. Increased ABCA1 and ABCG1 expression in M2-derived foam cells. Shown is a representative Western blot for the presence of ABCA1, ABCG1 and β-actin in lysates of cells that were treated as indicated.

  4. D,E. The quantification of the band intensity of ABCA1 (D) and ABCG1 (E) related to β-actin. *p = 0.04, **p = 0.0075, ***p = 0.0001. All data in (B–E) are shown as mean ± SEM values of three independent experiments performed in triplicates.

  5. 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 (n = 11) or IL-13 (n = 13) during the last 5 weeks. Sections were stained with an antibody against ABCA1 and values represent the numbers of ABCA1+ cells/mm2 of total lesion area, *p = 0.031. Data are shown as mean ± SEM values of all mice of each group. Images show representative ABCA1 staining. Original magnification 400×.

Figure 6
Figure 6. Increased atherosclerosis in IL-13-deficient LDLR−/− mice

  1. LDLR−/− mice were reconstituted with bone marrow from either IL-13+/+ mice (n = 12) or IL-13−/− mice (n = 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 µm2/section throughout the entire aortic origin, (400 µm). **p = 0.0023. Images show representative H&E stains. Original magnification 50×.

  2. Increased necrotic core area in lesions of recipients of IL-13−/− bone marrow. Values represent percentages of necrotic core area/total lesion area. *p = 0.016.

  3. 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.

  4. 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 specifically expressed by M2 macrophages and values represent number of Ym-1+ cells/mm2 of cellular lesion area. **p = 0.0043, Mann–Whitney test.

  5. 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.

Figure 7
Figure 7. Effect of IL-13 deficiency on splenic cytokine production and antibody isotype levels

  1. LDLR−/− mice were reconstituted with bone marrow from either IL-13+/+ mice (n = 12) or IL-13−/− mice (n = 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-γ by splenocytes stimulated with anti-CD3/CD28 in vitro. Data are presented as ng/ml cytokine of splenocyte cultures ***p = 0.0001.

  2. 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. *p = 0.029.

  3. Decreased ratio of IgG1:IgG2c antibodies in IL-13-deficient LDLR−/− mice. *p = 0.04. All data are shown as mean ± SEM values of all mice of each group.

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