Dendritic cell subset distributions in the aorta in healthy and atherosclerotic mice

Abstract

Dendritic cells (DCs) can be sub-divided into various subsets that play specialized roles in priming of adaptive immune responses. Atherosclerosis is regarded as a chronic inflammatory disease of the vessel wall and DCs can be found in non-inflamed and diseased arteries. We here performed a systematic analyses of DCs subsets during atherogenesis. Our data indicate that distinct DC subsets can be localized in the vessel wall. In C57BL/6 and low density lipoprotein receptor-deficient (Ldlr (-/-)) mice, CD11c(+) MHCII(+) DCs could be discriminated into CD103(-) CD11b(+)F4/80(+), CD11b(+)F4/80(-) and CD11b(-)F4/80(-) DCs and CD103(+) CD11b(-)F4/80(-) DCs. Except for CD103(-) CD11b(-) F4/80(-) DCs, these subsets expanded in high fat diet-fed Ldlr (-/-) mice. Signal-regulatory protein (Sirp)-α was detected on aortic macrophages, CD11b(+) DCs, and partially on CD103(-) CD11b(-) F4/80(-) but not on CD103(+) DCs. Notably, in FMS-like tyrosine kinase 3-ligand-deficient (Flt3l (-/-)) mice, a specific loss of CD103(+) DCs but also CD103(-) CD11b(+) F4/80(-) DCs was evidenced. Aortic CD103(+) and CD11b(+) F4/80(-) CD103(-) DCs may thus belong to conventional rather than monocyte-derived DCs, given their dependence on Flt3L-signalling. CD64, postulated to distinguish macrophages from DCs, could not be detected on DC subsets under physiological conditions, but appeared in a fraction of CD103(-) CD11b(+) F4/80(-) and CD11b(+) F4/80(+) cells in atherosclerotic Ldlr (-/-) mice. The emergence of CD64 expression in atherosclerosis may indicate that CD11b(+) F4/80(-) DCs similar to CD11b(+) F4/80(+) DCs are at least in part derived from immigrated monocytes during atherosclerotic lesion formation. Our data advance our knowledge about the presence of distinct DC subsets and their accumulation characteristics in atherosclerosis, and may help to assist in future studies aiming at specific DC-based therapeutic strategies for the treatment of chronic vascular inflammation.

Figure 1. Localization of CD11c⁺ cells in the aortic root.

(A) Aortic root sections of healthy and atherosclerotic apoE ^−/−CD11c-YFP mice (arrow heads indicate CD11c⁺ cells, green). Nuclei are counterstained with DAPI (blue; scale bars, 50 µm).

Figure 2. Identification of DC subsets in the aorta.

Representative FACS plots for identification of DC subsets in healthy, chow-fed Ldlr ^−/− mice. After exclusion of TCRβ⁺/CD19⁺ T and B cells, CD11c⁺ MHCII⁺ DCs were further subdivided into CD103⁺ and CD103⁻ DCs. CD103⁺ DCs do not express CD11b or F4/80 while CD103⁻ DCs were further subdivided into CD11b⁺ F4/80⁻, CD11b⁺ F4/80⁺ and CD11b⁻F4/80⁻ DCs. Macrophages were defined as CD11c⁻ MHCII⁺ CD11b⁺ F4/80⁺. Representative contour plots from 6–9 mice per group are shown.

Figure 3. Quantification of aortic DC subsets.

Aortic DC and macrophage numbers were quantified in the aorta of C57BL/6J and Ldlr ^−/− mice fed a normal chow, or in Ldlr ^−/− mice after 6 or 12 weeks of high fat diet-feeding by flow cytometry (n = 6–9 mice per group; data were obtained by pooling 2–3 aortae per experiment and then calculating the average number of cells per mouse and aorta, and represent 6–9 independent experiments). Data represent mean±SEM. *P<0.05.

Figure 4. Characterization of aortic DC subsets.

Representative co-immunofluorescence staining of aortic root sections of Ldlr ^−/− mice fed a high fat diet for 12 weeks, revealing cells showing staining for only CD11c (red, filled arrow heads) or F4/80⁺ (green, narrow arrows) as well as both CD11c and F4/80 (yellow, bold arrows). Nuclei are counterstained with DAPI (blue). Oil-red-O staining (red) for lipids in adjacent sections. Scale bars, 50 µm.

Figure 5. SIRPα expression on aortic DC subsets and macrophages.

Representative histograms of the expression of SIRPα on DC subsets and macrophages in the aorta of Ldlr^−/− mice fed a normal chow or after 12 weeks of high fat diet-feeding. Representative histograms from 6–9 mice per group are shown.

Figure 6. CD64 expression on aortic DC subsets and macrophages.

Representative histograms of CD64 expression on aortic DC subsets and macrophages. Representative histograms from 6–9 mice per group are shown.