We believe that the link between the spleen and cardiovascular disease will be important for future research in connection to the activation of T cells known to be important for atherosclerosis development. Materials and Methods Experimental Animals. marginal zone macrophages (MZM) that express specific receptors, including the oxLDL-binding class A scavenger receptor macrophage receptor with collagenous structure (MARCO) (32). In atherosclerosis, splenocyte transfer experiments from aged Apoe?/? to young splenectomized Apoe?/? mice show that this spleen confers an atheroprotective effect and that this is usually mediated by B cells (10). Experiments in which B-cellCdeficient LDLr?/? mice develop a more severe disease than B-cellCsufficient mice have further exhibited 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 Sunitinib Malate effects of immunization with apoptotic cells on atherosclerosis development, focusing on subpopulations of splenic B cells. Our results show that hyperlipidemia associated with atherosclerosis by itself Sunitinib Malate activates B cells in the spleen to produce large numbers of Sunitinib Malate 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 KMT3B antibody 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 accompanied by increased activation and proliferation in the peripheral B-cell compartment, in part compensating for the decreased output (37). Next we determined whether there was any evidence for B-cell activation other than the changes in naive populations. After activation, B cells can become IL-10Cproducing cells Sunitinib Malate (B10) before differentiating further to AFC (38). As these cells have been shown to suppress inflammation 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 produce IL-10 in young as well as old Apoe?/? mice compared with WT mice. The frequency of B10 cells significantly decreased with age, suggesting this population was not responsible for B-cellCdependent disease protection in previously published B-cell transfers (Fig. 1and and and Fig. S2). However, in the Vh5 (7183) and Vh7 (S107) families, specific clones were expanded in Apoe?/? compared with WT mice (Fig. 2= 7C10) are plotted. Anti-PC (= 7C10) are plotted (and and 0.05, ** .