Background: Atherosclerosis is a chronic inflammatory disease driven by the accumulation of LDL-cholesterol and inflammatory leukocytes in the subendothelial space of medium to large-sized arteries. While the aortic leukocyte composition in murine atherosclerotic plaques has been extensively studied, little is known about the diversity and spatial distribution of the different leukocyte subsets in the healthy aorta. This study aims to provide a detailed characterization of the leukocyte composition across the different layers of the vascular wall in healthy mice.
Methods: Aortas were excised from healthy male and female 8-week-old C57BL/6J wild type and Tomato-reporter (CD4-tdT) mice. The adventitia (ADV) was carefully separated from the intima and media (IM), and the leukocyte composition within the different aortic layers was studied by nuclear and single-cell RNA sequencing, flow cytometry, and confocal microscopy.
Results: The ADV harboured a higher total leukocyte count compared to the IM layers, with F4/80+ macrophages being particularly enriched in the ADV. Myeloid cells, B cells, and several T cell subsets were identified within the ADV as well as in the IM layers of the aorta by flow cytometry and scRNA-seq. Presence of CD4+ T cells was confirmed in the IM layers across the aortic arch, thoracic, and abdominal regions of CD4-tdT reporter mice by confocal microscopy. To exclude contamination by circulating leukocytes, an anti-CD45 APC antibody was injected in the tail vein of C57BL/6 mice 5 minutes prior to aortic dissection. Most of the T cells found in the IM layers of the aorta remained unstained, indicating that detected T cells were non-circulating. Moreover, aortic T cells exhibited phenotypic markers indicative of tissue residency (CD69, CD103, and CD49a), alongside reduced CD25 expression compared to circulating T cells.
Conclusion: Our detailed characterization of the cellular composition of the healthy mouse aorta reveals a strikingly diverse leukocyte repertoire in both the ADV and IM layers. We have also identified T cells in the ADV as well as in the IM of healthy mice, providing a basis for further research into their potential contribution to vascular homeostasis and their role in the pathogenesis of cardiovascular diseases.
