Abstract 4370467: Downregulation of CCR6 in severe coronary artery disease impairs marginal zone B cell mediated atheroprotective IgM production

Abstract

Introduction: Oxidized low density lipoproteins (OxLDL) present in atherosclerotic plaques. Epidemiological studies demonstrate that IgM antibody against malondialdehyde LDL (MDA-LDL), the major subtype of OxLDL, is inversely associated with severity of coronary artery disease (CAD), heart attack and death. In human, circulating marginal zone B cells (MZB) were recently identified as producers of these atheroprotective IgM to MDA-LDL. The frequency of circulating MZB cells correlates with CAD severity. We further discover that MZB cells from subjects with high CAD burden produce less atheroprotective IgM to MDA-LDL. This study explores phenotypic changes in MZB cells in relation to CAD severity and investigates underlying mechanisms Hypothesis: The chemokine receptor CCR6 regulates the production of atheroprotective IgM against MDA-LDL by MZB cells and is downregulated in subjects with severe CAD. Methods: Humanized mice were adoptively transferred with sort-purified human MZB cells from low and high CAD severity subjects. Single cell multi-omics (CITE-Seq) was performed on B cells from both low and high CAD groups. In-vitro migration study, imaging-flow cytometry, as well as CRISPR-mediated gene knock down were utilized to further investigate mechanisms. Results: Humanized mice receiving MZB cells from low-CAD subjects (n = 7) produced higher levels of atheroprotective IgM than those receiving cells from high-CAD subjects (n = 6). Single-cell analysis of B cells from low (n = 30) and high (n = 30) CAD subjects revealed higher CCR6 expression in MZB cells from the low-CAD group. In vitro activation of MZB cells with MDA antigen led to downregulation of both CD24 and CCR6. Imaging flow cytometry and proximity ligation assays suggested co-localization of CD24 and CCR6. Blocking CD24 with a monoclonal antibody reduced surface CCR6 expression and impaired CCL20-induced migration. Finally, CCR6-knockdown MZB cells (n = 4) exhibited reduced splenic migration and IgM production in humanized mice compared to wild-type controls (n = 6). Conclusion: MZB cells from individuals with severe CAD exhibit reduced CCR6 expression and impaired atheroprotective IgM production. CCR6 is critical for splenic migration of human MZB cells and subsequent IgM production.