For lipid staining, sections were blocked with goat serum and stained with the lipid dye Nile red (Sigma-Aldrich), together with an anti-F4/80 antibody (BM8) (Biolegend). Apoe?/? mice show that the spleen confers an atheroprotective effect and that this is mediated by B Lycoctonine cells (10). Experiments in which B-cellCdeficient LDLr?/? mice develop a more severe disease than B-cellCsufficient mice have further demonstrated a protective role for B cells (12). Removal of the spleen has been shown to deplete B1a cells from the peritoneum, and it was recently shown that transfer of these cells has an atheroprotective effect in splenectomized mice (11, 33), Thus, B1a cells have the ability to play an atheroprotective role in the absence of a spleen and as producers of natural T15 antibodies (11, 16, 26). However, MZB are also missing after splenectomy, and so far, dissection of B cells in the spleen of atherosclerotic Apoe?/? mice has not been done. We therefore set out to characterize the protective splenic B-cell response. Because cells of the marginal zone express an array of specific receptors for modified self-antigens, we hypothesized that an immune activation in this region could be the origin of the protective B-cell response in atherosclerosis. This would be in line with our previous data showing that apoptotic cells, carrying oxidation-specific epitopes (16), are trapped in the marginal zone (34). Because oxidation-specific epitope-bearing antigens give rise to antibodies binding oxLDL (25, 35), we also investigated the effects of immunization with apoptotic cells on atherosclerosis development, focusing on Lycoctonine subpopulations of Rabbit Polyclonal to MMP-9 splenic B cells. Our results show that hyperlipidemia associated with atherosclerosis by itself activates B cells in the spleen to produce large numbers of antibody-forming cells (AFC) secreting antibodies against oxidation-specific epitopes. We also find lipid accumulation and inflammasome activation in phagocytes that could drive this B-cell activation. Finally, we show that we can accelerate the protective response by administration of apoptotic cells, which results in reduced lesion size and cholesterol drop in serum. Results B-Cell Activation and Population Dynamics During Atherogenesis. To explore the effect that hyperlipidemia has on B cells in the spleen, young (6C8 wk) and old (21C22 wk) Apoe?/? mice and age-matched wild-type C57BL/6 (WT) mice were investigated for B-cell precursors [transitional type 1 (T1) and type 2 (T2)] and naive B-cell populations (B1a, MZB, and FOB) (Fig. 1). The bone marrow-derived T1 precursors decreased with age in both strains, but to a significantly lesser extent in Apoe?/? mice compared with in WT mice (Fig. 1= 7C10) are plotted. In addition, germinal center and antibody-secreting foci formation was imaged by immunofluorescence as PNA+B220lo (and < 0.05, **< 0.01, and ***< 0.001 with a MannCWhitney test. Investigating B1a and B1b cells in the peritoneal cavity, we found an increase in WT but no significant change in Apoe?/? mice, showing that the reduction in B1 cells was not general (Fig. S1). The relative decrease seen in B-cell precursors is in line with previous data showing that decreased output from the bone marrow in older mice is definitely accompanied by improved activation and proliferation in the peripheral B-cell compartment, in part compensating for the decreased output (37). Next we identified whether there was any evidence for B-cell activation other than the changes in naive populations. After activation, B cells can become IL-10Cgenerating cells (B10) before differentiating further to AFC (38). As these cells have been shown to suppress Lycoctonine swelling in models of chronic inflammatory diseases, we investigated whether they accumulated in the spleen of Apoe?/? mice. To our surprise, B cells were less prone to create IL-10 in young as well as older Apoe?/? mice compared with WT mice. The rate of recurrence of B10 cells significantly decreased with age, suggesting this human population was not responsible for B-cellCdependent disease safety in previously published B-cell transfers (Fig. 1and and and Fig. S2). However, in the Vh5 (7183) and Vh7 (S107) family members, specific clones were expanded in Apoe?/? compared with WT mice (Fig. 2= 7C10) are plotted. Anti-PC (= 7C10) are plotted (and and <.