Deletion of Batf3-dependent antigen-presenting cells does not affect atherosclerotic lesion formation in mice

Abstract

Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs) bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3) is required for the development of classical CD8α+ and CD103+ DCs. By crossing mice deficient in Batf3 with atherosclerosis-prone low density lipoprotein receptor (Ldlr-/-)-deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α+ and CD103+ antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.

Fig 1. Deficiency in Batf3 does not alter atherosclerotic lesion development.

(a,b) Single cell suspensions from aortae and splenocytes were obtained from Ldlr^-/- and Ldlr^-/-Batf3^-/- mice fed a HFD for 8 weeks and analyzed by flow cytometry. CD11c⁺ MHCII⁺ DCs were gated and further discriminated by expression of CD11b and CD103 in the aorta (a) or CD8α in the spleen (b). (c-d) Quantification of plaque area in Oil-Red-O stained aortae and in Aldehyde Fuchsin-stained aortic roots in atherosclerotic Ldlr^-/- (n = 12) and Ldlr^-/-Batf3^-/- mice (n = 10) fed a HFD for 8 weeks; representative images of the aorta and aortic root sections are shown. Data ara presented as mean ± SEM; ns, non significant.

Fig 2. Plaque composition is not altered by Batf3 deficiency.

Quantification of the area positive for Mac-2 (a) representative images of immunofluorescence staining are shown; scale bars: 50μm; cell nuclei were counterstained with DAPI (blue), α-smooth muscle actin (b), Sirius-red (c), and of the necrotic core (d) in the aortic root from Ldlr^-/- (n = 12) and Ldlr^-/-Batf3^-/- mice (n = 10) fed with 8 weeks of HFD. Data ara presented as mean ± SEM; ns, non significant.

Fig 3. Immune responses are mildy alter in the spleen of Batf3-deficient mice after 8 weeks of HFD.

(a-d) Flow cytometric analyses of Ly6G⁺ neutrophils (a), Ly6C^high and Ly6C^low monocytes (b), and of CD3⁺ T cells (c) among CD45⁺ leukocytes, and of frequencies of CD4⁺ and CD8⁺ T cells among CD3⁺ T cells (d) in spleens from atherosclerotic Ldlr^-/- (n = 12) and Ldlr^-/-Batf3^-/- mice (n = 10) fed a HFD for 8 weeks. Data ara presented as mean ± SEM; **p<0.01; ***p<0.001; ns, non significant.

Fig 4. T cell activation is impaired in the spleen of Batf3^-/- mice after 8 weeks of HFD.

Flow cytometric analyses of spleen cells obtained from atherosclerotic Ldlr^-/- (n = 12) and Ldlr^-/-Batf3^-/- mice (n = 10) fed a HFD for 8 weeks. (a) Frequencies of activated CD44^highCD62L^low CD4⁺ and (b) CD8⁺ T cells (representative dot plots are shown; values indicate gated events among CD4⁺ T cells). (c) Frequencies of FoxP3⁺CD25⁺CD4⁺ Tregs, (d) IFNγ⁺CD4⁺ T cells (representative dot plots are shown, values indicate gated events among CD4⁺ T cells), (e) IL-17a⁺CD4⁺ T cells and (f) IFNγ⁺CD8⁺ T cells. Data ara presented as mean ± SEM; *p<0.5;,**p<0.01; ns, non significant.