B cell depletion reduces the development of atherosclerosis in mice

Abstract

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell-depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti-oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell-derived IFN-gamma secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody-mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.

Figure 1.

CD20 mAb (α-CD20) treatment depletes B cells and reduces the development of atherosclerosis. (a and b) Efficiency of B cell (B220⁺ or IgM⁺B220⁺) depletion in blood (gated on lymphocytes, low forward scatter/low side scatter; a) and spleens (gated on total viable splenocytes; b) of Apoe^−/− mice fed a Western diet for either 6 or 12 wk and treated with α-CD20 (blue) or a control antibody (magenta). Data are representative of four (spleen) or eight (blood) mice per group and per experiment, and two separate experiments. (c–f) Reduction of atherosclerosis development after α-CD20 therapy in four different experiments using Apoe^−/− or Ldlr^−/− mice fed either a chow diet (CD) or a Western diet (WD). Representative photomicrographs of oil red O–stained aortic sinuses are shown for each experimental setting along with quantification of intimal lesion size. Horizontal bars indicate median values. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 200 µm.

Figure 2.

CD20 mAb (α-CD20) reduces the production of anti-oxLDL antibodies. (a and b) Antibody (IgG and IgM) responses to copper-oxidized (CuOx) or malondialdehyde (MDA)-modified LDLs in the circulating blood of Apoe^−/− mice fed a Western diet for either 6 (a) or 12 wk (b) and treated with α-CD20 or a control antibody. MDA 1 indicates IgG1 type antibodies against MDA-LDL, and MDA 2c indicates IgG2c type antibodies. Data are representative of 6 (6 wk), 12 (Ctr; 12 wk), and 13 (α-CD20; 12 wk) mice per group and two different experiments. Mean values ± SEM are represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. R.L.U., relative light units.

Figure 3.

CD20 mAb (α-CD20) reduces systemic T cell activation and limits T cell accumulation within atherosclerotic lesions. (a) Representative photomicrographs and quantitative analysis of CD3 staining in atherosclerotic lesions of Apoe^−/− mice fed a Western diet for 12 wk and treated with CD20 mAb (α-CD20) or control antibody (Ctr). Horizontal bars indicate median values. Data are representative of one experiment with eight mice per group. Bars, 100 µm. (b and c) Representative histograms of CD69 and CD44 expression on gated CD4⁺ spleen cells of Apoe^−/− mice fed a Western diet for 12 wk and treated with CD20 mAb (α-CD20; blue) or control antibody (Ctr; magenta). CD44 expression was divided into low, intermediate, or high. Data are representative of four mice per group and two separate experiments. (d) Percentage of BrdU-positive cells determined using flow cytometry among spleen- or subcutaneous lymph node–derived CD4⁺ populations. BrdU was administered the week before animal sacrifice (after 6 wk of a Western diet), as described in Materials and methods. Data are representative of three mice per group and two separate experiments. (e) Representative histograms of CD40 expression on gated CD11c⁺ spleen cells of Apoe^−/− mice fed a Western diet for 12 wk and treated with CD20 mAb (α-CD20; blue) or control antibody (Ctr; magenta). Data are representative of four mice per group and two separate experiments. Mean values ± SEM are represented. *, P < 0.05; **, P < 0.01.

Figure 4.

Treatment with CD20 mAb (α-CD20) induces a switch toward lower IFN-γ but higher IL-17 production. (a) Representative examples and quantitative analysis of intracellular IFN-γ staining of isolated splenocytes from Apoe^−/− mice fed a Western diet for 6 wk and treated with CD20 mAb (α-CD20; blue) or control antibody (Ctr; magenta). Plots are gated on CD4⁺ cells. Numbers in each quadrant indicate percentages of cells. Histograms depict mean values ± SEM. Similar results were obtained after 12 wk of a high fat diet (not depicted). (b) Quantitative analysis of IL-17 and IFN-γ production (ratio of Il-17/IFN-γ is depicted) in supernatants of purified spleen-derived T cells (Apoe^−/− mice fed a Western diet for 12 wk) after stimulation with anti-CD3 antibody in the presence of purified CD11c⁺ cells. Horizontal bars indicate median values. Data are representative of four to five mice per group and two separate experiments. (c) Representative examples and quantitative analysis of intracellular IL-17 staining among spleen CD3⁺ cells, determined using flow cytometry on isolated splenocytes from Ldlr^−/− mice fed a Western diet for 6 wk and treated with CD20 mAb (α-CD20; blue) or control antibody (Ctr; magenta). Mean values ± SEM are represented. Data are representative of five mice per group and one experiment. **, P < 0.01; ***, P < 0.001.

Figure 5.

IL-17A neutralization abrogates CD20 antibody–mediated atheroprotection. (a) Representative photomicrographs of oil red O–stained aortic sinuses and quantitative analysis of intimal lesion size in Apoe^−/− mice fed a Western diet for 6 wk and treated with control antibodies for both CD20 and IL-17 (Ctr group), CD20 mAb and a control antibody for IL-17 (α-CD20 group), or neutralizing anti–L-17A mAb and a CD20 mAb (α-CD20 + α–IL-17A group). Horizontal bars indicate median values. *, P < 0.05. Bars, 200 µm. This experiment was repeated in c. (b) Quantitative analysis of IFN-γ, IL-10, and TGF-β mRNA expression (relative to GAPDH) in the thoracic aortas of Apoe^−/− mice fed a Western diet for 6 wk and treated with CD20 mAb or CD20 mAb and anti–L-17A mAb. Mean values ± SEM are representative of five to seven mice per group and one experiment. *, P < 0.05. (c) Quantitative analysis of intimal lesion size in an additional set of Apoe^−/− mice fed a Western diet for 6 wk and treated with control antibodies for both CD20 and IL-17 (Ctr group; n = 8), CD20 mAb and a control antibody for IL-17 (α-CD20 group; n = 7), or neutralizing anti–L-17A mAb and a CD20 mAb (α-CD20 + α–IL-17A group; n = 9). Data represent mean values ± SEM. *, P < 0.05; **, P < 0.01.