Ablation of CD8α(+) dendritic cell mediated cross-presentation does not impact atherosclerosis in hyperlipidemic mice

Abstract

Clinical complications of atherosclerosis are almost exclusively linked to destabilization of the atherosclerotic plaque. Batf3-dependent dendritic cells specialize in cross-presentation of necrotic tissue-derived epitopes to directly activate cytolytic CD8 Tcells. The mature plaque (necrotic, containing dendritic cells and CD8 Tcells) could offer the ideal environment for cross-presentation, resulting in cytotoxic immunity and plaque destabilization. Ldlr(-/-) mice were transplanted with batf3(-/-) or wt bone marrow and put on a western type diet. Hematopoietic batf3 deficiency sharply decreased CD8α(+) DC numbers in spleen and lymph nodes (>80%; P < 0,001). Concordantly, batf3(-/-) chimeras had a 75% reduction in OT-I cross-priming capacity in vivo. Batf3(-/-) chimeric mice did not show lower Tcell or other leukocyte subset numbers. Despite dampened cross-presentation capacity, batf3(-/-) chimeras had equal atherosclerosis burden in aortic arch and root. Likewise, batf3(-/-) chimeras and wt mice revealed no differences in parameters of plaque stability: plaque Tcell infiltration, cell death, collagen composition, and macrophage and vascular smooth muscle cell content were unchanged. These results show that CD8α(+) DC loss in hyperlipidemic mice profoundly reduces cross-priming ability, nevertheless it does not influence lesion development. Taken together, we clearly demonstrate that CD8α(+) DC-mediated cross-presentation does not significantly contribute to atherosclerotic plaque formation and stability.

Figure 1. Expression of cross presentation markers in human and mouse atherosclerosis.

(a) Total RNA was isolated from fresh-frozen human atherosclerotic plaques. Real-time PCR results of expression levels of BDCA3, IRF8, ADFP, Batf3, TAP1 and Necl2 are shown as mean ± SEM. All expression levels were first normalized for levels of β-actin expression, and are depicted as fold induction when compared to expression levels in early plaques. Samples were grouped based on histological qualification of plaque stage according to Virmani et al.. Early: Intimal Thickening/ Pathological Intimal Thickening (n = 5), Advanced: Thick/Thin Fibrous Cap Atheroma (n = 6), Unstable: Intra Plaque Hemorrhage (n = 5). *p < 0.05, ***p < 0.001. (b) Representative images of frozen human carotid plaque sections (n = 8–10) doublestained with antibodies against XCR1 (green) and CD11c (red) to identify cross-presenting DCs. Colocalization was determined using a Nuance Spectral Imaging System and is indicated in yellow. (c) Total RNA was isolated from fresh-frozen mouse aorta’s. Real-time PCR results of expression levels of Rab11b, TAP1 and XCR1 are shown as mean ± SEM. All expression levels were first normalized for levels of GAPDH expression, and are depicted as fold induction when compared to expression levels in early plaques. Early: 8 wk old C57Bl6 mice (n = 6), Advanced: >35 wk old C57Bl6 ApoE^−/− mice (n = 5) (d) Representative images of frozen mouse aortic root sections doublestained with antibodies against CD8α (red) and CD11c (blue) to identify cross-presenting DCs. Nuclei were lightly counterstained with MethylGreen. Arrow: doublestained cell.

Figure 2. Cross-presentation occurs under hyperlipidemic conditions.

Ldlr^−/− mice (n = 3) on a normal chow diet or fed a Western type diet (WTD) for three weeks were iv injected with irradiated OVA-expressing splenocytes and CFSE-labeled OT-I Tcells. After 72 hrs, spleens were harvested and cross-presentation was assessed by flow cytometry, quantifying the proportion of proliferating OT-I Tcells (cells with a diluted CFSE signal) within the total OT-I Tcell population, normalized for amount of injected cells. (a) Bar graph of proliferated OT-I Tcells (% of total OT-I Tcells) in spleen of chow or WTD-fed ldlr^−/− mice. (b) Representive CFSE dilution peaks of the OT-I Tcell population. Data are presented as mean ± SEM.

Figure 3. Batf3 deficiency results in severe CD8α⁺ DC depletion in the atherosclerosis model.

(a) Lethally irradiated ldlr^−/− mice were reconstituted with wt (n = 15) or batf3^−/− (n = 12) bone marrow, and after 6 weeks recovery, put on a WTD containing 0,25% cholesterol for 10 weeks. (b) Representative flow cytometry gating of CD8α⁺ DC population (Lin⁻, CD11c^high, MHCII^high, CD8α⁺). (c) Bar graph of CD8α⁺ DCs as percentage of cDCs. (d) Bar graph of CD103⁺ DCs as percentage of cDCs. (e) Body weight at sacrifice. (f) Total cholesterol content in serum at sacrifice. Data are presented as mean ± SEM, **p < 0,01, ***p < 0,001.

Figure 4. Cross-presentation is affected in batf3^−/− chimeric mice.

Batf3^−/− chimeric or wt ldlr^−/− mice (n = 7) were iv injected with necrotic OVA-expressing splenocytes and CFSE-labeled OT-I T cells. After 72 hrs, spleens were harvested and cross-presentation was assessed by flow cytometry, quantifying the proportion of proliferating OT-I Tcells (cells with a diluted CFSE signal) within the total OT-I Tcell population, normalized for amount of injected cells. (a) Bar graph of proliferated OT-I Tcells (% of total OT-I Tcells) in spleen. (b) Representive CFSE dilution peaks of the OT-I Tcell population. (c) Correlation analysis between amount of residual CD8α⁺ DCs and the remaining cross-presentation capacity in batf3^−/− chimeras. Data are presented as mean ± SEM, ***p < 0,001.

Figure 5. Batf3 deficiency does not influence atherosclerotic plaque size.

Aortic arch and root were dissected from wt (n = 15) or batf3^−/− (n = 12) ldlr^−/− mice and analyzed by histology. (a) Aortic arch and root were H&E stained for plaque size analysis. (b, c) Plaque area, necrotic core area and percentage necrotic core relative to plaque area are did not differ in the brachiocephalic artey (b) and aortic root (c). Data are presented as mean ± SEM.

Figure 6. Batf3 deficiency does not influence atherosclerotic plaque composition.

Aortic arch and root were dissected from wt (n = 15) or batf3^−/− (n = 12) ldlr^−/− mice and analyzed by immunohistochemistry. (a) Representative images of Macrophages (Mac3 staining), vascular smooth muscle cells (αSMA staining), T cells (CD3 staining), collagen (Sirius Red staining) and apoptosis (cleaved caspase 3 staining) in the aortic roots of wt and batf3^−/− chimeric mice. (b) Quantification of immunohistochemical stainings shown in (a). Data are presented as mean ± SEM.

Figure 7. Tcell numbers are unchanged in batf3^−/− chimeras.

Tcell subset numbers were analyzed in the aorta-draining lymph node by flow cytometry. (a) CD25⁺, FoxP3⁺ regulatory Tcells cell are presented relative to the CD4⁺ Tcell population. (b) Naïve (CD62L^hi, CD44^lo), central memory (CD62L^hi, CD44^hi) and effector memory (CD62L^lo, CD44^hi) populations are presented as percentages of CD8⁺ Tcells. (c) Naïve (CD62L^hi, CD44^lo), and effector memory (CD62L^lo, CD44^hi) populations are presented as percentages of CD4⁺ Tcells. Data are presented as mean ± SEM.