Macrophage Foam Cell-Targeting Immunization Attenuates Atherosclerosis - PubMed (original) (raw)
Macrophage Foam Cell-Targeting Immunization Attenuates Atherosclerosis
Fazhan Wang et al. Front Immunol. 2019.
Abstract
Background: Macrophage foam cells (FCs) play a crucial role in the initiation and progression of atherosclerosis. Reducing the formation or inducing the removal of FCs could ameliorate atherosclerosis. The present study examined whether the whole-cell vaccination using FCs could be used as novel prevention and treatment strategies to battle atherosclerosis. Methods: ApoE-/- mice with initial or established atherosclerosis were subcutaneously immunized three times with FCs in Freund's adjuvant. Results: Immunization with FCs resulted in an overt reduction of atherosclerotic lesion in the whole aorta and the aortic root with enhanced lesion stability. Subsequent study in mechanism showed that FCs vaccination dramatically increased CD4+ T cell and CD8+ T cell populations. Immunization with FCs significantly raised the plasma FCs-specific IgG antibodies. Of note, the FCs immune plasma could selectively recognize and bind to FC. FCs immune plasma significantly blocked the process of FCs formation, finally reduced the accumulation of FCs in plaque. Additionally, it was observed that FCs immunization down-regulated the expression level of atherosclerosis related pro-inflammatory cytokines, including IFN-γ, MCP-1, and IL-6 and enhanced the lesion stability with a significant increase in TGF-β1 level and collagen content. Conclusions: These findings demonstrate that the whole-cell vaccination using FCs significantly decreased lesion development and positively modulated lesion progression and stability by targeting FCs. The whole-cell FCs vaccine might represent a potential novel strategy for development of new antibodies and vaccines to the prevention or treatment of atherosclerosis.
Keywords: atherosclerosis; humoral immune responses; immunization; macrophage foam cells; whole-cell vaccine.
Figures
Figure 1
The oxLDL uptake by peritoneal macrophages treated with or without oxLDL (20, 50 or 100 μg/mL) for 24 h. Internalized oxLDL were stained with Oil Red O. Six random fields per condition were observed with OLYMPUS BX53 microscope. (A) A representative image is shown for each condition. Scale bars, 20 μm. (B) Quantification of intracellular oxLDL. The mean ± SEM of Oil Red O staining per condition is expressed in arbitrary units (au). Peritoneal macrophages or FCs were stained with APC anti-mouse F4/80. Flow cytometry histograms for macrophages (D), and FCs (E), respectively. Unlabeled macrophages were used as negative controls (C). The experiment was performed in triplicate. **p < 0.01; ***p < 0.001.
Figure 2
FCs immunization attenuates initial atherosclerotic lesion. (A) Schematic illustration of the prevention experimental setup. (B) Evaluation of en face lesion size of the whole aorta was carried out by Oil Red O staining. (C) Plaque size in the three-valve area of the aortic root was assessed by hematoxylin and eosin staining. (D) Collagen content in the aortic root was determined by Masson's Trichrome staining as the percentage of the total lesion area. Scale bars, 200 μm. All data are presented as mean ± SEM and are representative of all mice. n = 5–6 in each group; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 3
FCs vaccination attenuates advanced lesion. (A) Experimental design of the therapeutic study. (B) En face lesion size of the whole aorta. (C) Plaque size in the three-valve area of the aortic root. (D) Collagen content in the aortic root. Scale bars, 200 μm. All data are presented as mean ± SEM and are representative of all mice. n = 5–6 in each group; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
FCs vaccine decreases accumulation of FCs in plaque. (A) Serial cryosections of the aortic root were stained with hematoxylin and eosin, Mac-2, and Oil Red O, and hematoxylin, respectively. Representative photographs of corresponding staining were shown with co-location of Mac-2 and Oil Red O positive area outlined and FCs depicted by arrows. Scale bars, 100 μm. The FCs content in the aortic root of initial (B) and advanced (C) plaque were evaluated as the Mac-2 positive area per plaque area. Scale bars, 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
FCs immunization results in a specific antibody response in initial atherosclerosis. (A) ELISA was performed to measure the specific antibodies to FCs and macrophages as well as immunoglobulin isotypes including IgG1 and IgG2a titers. Data are shown at plasma dilutions of 1: 800 for each group (n = 8). OD, optical density. (B) Peritoneal macrophages and FCs were fixed and stained with PBS-immune, Macrophages-immune or FCs-immune plasma (dilution 1:50). Scale bars, 50 μm.
Figure 6
FCs immunization induces cellar response. (A) Macrophages and FCs vaccines significantly increased CD4+ T cell and CD8+ T cell populations in spleen compared with those PBS immunized mice. (B) Total splenocytes were cultured in vitro in the presence of FCs or Macrophages. Proliferation of CD3 positive T cell was measured by CFSE with peaks reflecting cell divisions.
Figure 7
FCs immune plasma blocks the process of FCs formation. (A) For the FCs formation inhibition study, the macrophages were incubated in duplicate with or without plasma (50 μL/mL) from FC-immune ApoE−/− mice for 3 h in the presence of DiI-oxLDL. (B) Further investigation of the effect of immune plasma on FCs formation. Macrophages were treated with or without plasma (50 μL/mL) from PBS-immune, macrophages-immune, or FC-immune ApoE−/− mice for 1 h. After washed, cells were cultured with 30 μg/mL DiI-oxLDL for 3 h (B). Internalized oxLDL was revealed by DiI fluorescence, and six random fields per condition were captured by High Content Screening. A representative image is shown for each condition. Scale bars, 50 μm. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 8
FCs vaccination modulates cytokines in initial atherosclerosis. The levels of IFN-γ (A), IL-6 (B), MCP-1 (C), and TGF-β1 (D) in immune ApoE−/− mouse plasma were determined at 1:2 dilution by ELISA according to the manufacturer's protocol. n = 8 in each group. Aortic roots were sectioned and stained with MCP-1. Representative photographs of MCP-1 immunostaining of PBS (E), macrophages (F), and FCs (G) immunized mice, respectively. Scale bars, 100 μm. (H) Quantification of MCP-1 expression in atherosclerotic lesion area of aortic root. n = 6 in each group; *p < 0.05; **p < 0.01; ***p < 0.001.
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