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Background: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. Methods: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry–based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. Results: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3 + T cells in the atherosclerotic lesions suggested a T-cell–related effect of homoarginine supplementation, which was mainly attributed to CD4 + T cells. Macrophages, dendritic cells, and B cells were not affected. CD4 + T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. Conclusions: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

RATIONALE: B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes (OSE) on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a, and the factors maintaining the BM B-1a population remain unexplored. The chemokine receptor CXCR4 has been implicated in human CVD and B cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human CVD is unknown. OBJECTIVE: To characterize the BM B-1a IgM repertoire and to determine whether CXCR4 regulates B-1 production of atheroprotective IgM in mice and humans. METHODS AND RESULTS: Single-cell sequencing demonstrated that BM B-1a cells from aged ApoE(−/−) mice with established atherosclerosis express a unique repertoire of IgM antibodies containing increased N-additions and a greater frequency of unique CDR-H3 sequences compared to peritoneal (PerC) B-1a cells. Some CDR-H3 sequences were common to both compartments suggesting B-1a migration between compartments. Indeed, mature PerC B-1a cells migrated to BM in a CXCR4-dependent manner. Furthermore, BM production of anti-OSE IgM and plasma IgM levels were reduced in ApoE(−/−) mice with B cell-specific knockout of CXCR4, and overexpression of CXCR4 on B-1a cells increased bone marrow localization and plasma anti-OSE IgM, including IgM against malondialdehyde(MDA)-modified LDL. Finally, in a 50-subject human cohort, we find that CXCR4 expression on circulating human B-1 cells positively associates with plasma levels of anti-MDA-LDL IgM antibodies and inversely associates with human coronary artery plaque burden and necrosis. CONCLUSIONS: These data provide the first report of a unique BM B-1a cell IgM repertoire and identifies CXCR4 expression as a critical factor selectively governing BM B-1a localization and anti-OSE IgM production. That CXCR4 expression on human B-1 cells was greater in humans with low coronary artery plaque burden suggests a potential targeted approach for immune modulation to limit atherosclerosis.