Journal: EBioMedicine

Article Title: Batf3-dependent CD8α + Dendritic Cells Aggravates Atherosclerosis via Th1 Cell Induction and Enhanced CCL5 Expression in Plaque Macrophages

doi: 10.1016/j.ebiom.2017.04.008

Figure Lengend Snippet: Lack of CD8α + DCs led to decreased IFN-γ and CCL5 expression in the aorta. The Apoe −/− mice (n = 8) and the Batf3 −/− Apoe −/− mice (n = 8) were fed either a chow diet or Western diet for 12 weeks. (a) Analysis of aortic Ifng , Foxp3 , and Tbx21 mRNA expression by qPCR. (b) An aortic tissue ELISA analysis of IFN-γ. Data represent pg/mg aortic protein. (c) qPCR analysis of Il12a , Tnf , Il6 , Tgfb1 , and Il10 mRNA expression. (d) qPCR analysis of Ccl5 expression. (e) ELISA analysis of the CCL5 concentration in the aortic tissue. Data represent pg per mg aortic protein. Data are presented as mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Data are representative of three independent experiments. See also Fig. S6.

Article Snippet: For the immunohistofluorescence analysis, the cryosections were stained with an antibody against CD45 (104; eBioscience Cat# 47-0451-82, RRID: AB_1548781 ), Mac3 (M3/84; BD Biosciences Cat# 550292, RRID: AB_393587 ), CCL5 (Bioss Inc. Cat# bs-1324R-Biotin, RRID: AB_11099534 ).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: EBioMedicine

Article Title: Batf3-dependent CD8α + Dendritic Cells Aggravates Atherosclerosis via Th1 Cell Induction and Enhanced CCL5 Expression in Plaque Macrophages

doi: 10.1016/j.ebiom.2017.04.008

Figure Lengend Snippet: IFN-γ promoted CCL5 expression on macrophages, promoting leukocytes infiltration into the plaque area. (a) Female Apoe −/− mice (n = 15) were put on a Western diet for eight weeks, then the aortic cells were pooled to prepared as described in the methods and stained with an antibody against CD45. Then, CD45 + leukocytes and CD45 − non-leukocytes were sorted by FACS, and qPCR was used to analyze Ccl5 mRNA expression. (b and c) Immunofluorescence of the aortic root. The Apoe −/− mice (n = 8) and the Batf3 −/− Apoe −/− mice (n = 8) were fed a Western diet for 12 weeks. The cryosections of the aortic root were stained with an antibody against CD45 (red), MAC3 (red), and CCL5 (green). The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images were viewed and captured with a Laser Scanning Confocal Microscope. Scale bars: 100 μm, dashed lines indicate the internal elastic lamina, arrows pointing to representative colocalized cells. (d) Primary splenic macrophages were sorted as described in supplementary data, and then treated with100 ng/mL IFN-γ for 6 h. Ccl5 mRNA expression was analyzed by qPCR. The Apoe −/− mice (n = 8) and the Batf3 −/− Apoe −/− mice (n = 8) were fed a Western diet for 6 weeks, and cryosections of the aortic root were performed. (e) H&E staining. Scale bars: 100 μm. (f) Immunohistochemistry. Representative images of leukocytes (CD45), T cells (CD3), DCs (CD11c), and macrophages (Mac3) in the aortic are shown. Scale bars: 200 μm. Data are presented as mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. ** P < 0.01; *** P < 0.001; ns, not significant. Data are representative of three independent experiments.

Article Snippet: For the immunohistofluorescence analysis, the cryosections were stained with an antibody against CD45 (104; eBioscience Cat# 47-0451-82, RRID: AB_1548781 ), Mac3 (M3/84; BD Biosciences Cat# 550292, RRID: AB_393587 ), CCL5 (Bioss Inc. Cat# bs-1324R-Biotin, RRID: AB_11099534 ).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Microscopy, Immunohistochemistry