Figure 2

Hif1α deletion in cDCs promotes atherosclerotic plaque formation. Hif1α^−/− → Ldlr^−/− and WT → Ldlr^−/− mice were fed a WD for 12 weeks. (a) Flow cytometry analysis of aortic-infiltrated cDCs for Hypoxiprobe staining. (b) Aorta was enzymatically digested and the presence of CD45⁺ hematopoietic cells, cDCs, CD4⁺ T cells, CD8⁺ T cells and FoxP3⁺ Tregs were quantified by flow cytometry. Representative dot plot from aorta infiltrated CD45⁺ cells. Bars represent mean ± SEM (n = 5). (c) IFNγ and IL17 production was analysed by flow cytometry in spleen’s and dLN’s CD4⁺ T cell. Dot plot is representative of 3 independent experiments. Bars represent mean ± SEM (n = 6). (d) Representative Sirius red staining of aortic root. (e) Representative images of en face oil red O staining of atherosclerotic lesions in aorta and (f) quantification of atherosclerotic lesion size. Bars indicate mean ± SEM (n = 10). (g) Total cholesterol in obese mice (n = 10). (h) Quantitative RT-PCR analysis for gene expression in perivascular fat from obese Hif1α^−/−. Expression levels of all genes were normalized against GAPDH RNA and compared to WT mice, which was set as 1. Error bars indicate the geometric mean of 7 biological replicates. (i) For in vivo antigen presentation, CFSE-labelled OTII cells were transferred i.v. one day before immunization with 200 μg OVA i.p. Three days later, frequency and division of proliferating OT-II T cells were analysed in aorta, LN, VAT and Spleen from Hif1α^−/− and WT mice by flow cytometry. Dot plots show gating strategy for CFSE⁺ OTII cells. Histograms show dilution of CFSE⁺ OTII gated cells and are representative of 3 independent experiments. Statistical analysis was performed with Student’s test, *p < 0.05, **p < 0.01 and ***p < 0.0005.