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

Abstract

Dendritic cells (DCs) play an important role in controlling T cell-mediated adaptive immunity in atherogenesis. However, the role of the basic leucine zipper transcription factor, ATF-like 3 (Batf3)-dependent CD8α⁺ DC subset in atherogenesis remains unclear. Here we show that Batf3^-/-Apoe^-/- mice, lacking CD8α⁺ DCs, exhibited a significant reduction in atherogenesis and T help 1 (Th1) cells compared with Apoe^-/- controls. Then, we found that CD8α⁺ DCs preferentially induce Th1 cells via secreting interleukin-12 (IL-12), and that the expression of interferon-gamma (IFN-γ)or chemokine (C-C motif) ligand 5 (CCL5) in aorta were significantly decreased in Batf3^-/-Apoe^-/- mice. We further demonstrated that macrophages were the major CCL5-expressing cells in the plaque, which was significantly reduced in Batf3^-/-Apoe^-/- mice. Furthermore, we found CCL5 expression in macrophages was promoted by IFN-γ. Finally, we showed that Batf3^-/-Apoe^-/- mice displayed decreased infiltration of leukocytes in the plaque. Thus, CD8α⁺ DCs aggravated atherosclerosis, likely by inducing Th1 cell response, which promoted CCL5 expression in macrophages and increased infiltration of leukocytes and lesion inflammation.

Keywords: Atherosclerosis; Batf3; CCL5; CD8α(+) DCs; Th1 cells.

Fig. 1

Batf3 deficiency effectively depleted CD8α⁺ DCs in the spleen and CD103⁺ DCs in the aorta, and alleviated atherosclerosis in Apoe^−/− mice. The Apoe^−/− mice (n = 8) and the Batf3^−/− Apoe^−/− mice(n = 8)were fed a WD for 12 weeks. (a) The single cell suspensions of the spleen and aorta were prepared, the proportion and the cells number of CD8α⁺ DCs in the spleen or CD103⁺ DCs in the aorta was detected by flow cytometry. (b) Cryosections of the aortic root were performed Oil Red O staining to quantify the lesion area. Scale bars: 200 μm. (c) Aorta en face staining of Oil Red O. Representative images of the aortic root. Data are presented as mean ± SD. Differences of P < 0.05 were considered to be statistically significant. ***P < 0.001; ns, not significant. Data are representative of three independent experiments. See also Fig. S1 and S2.

Fig. 2

Ablation of CD8α⁺ DCs induces decreased Th1 cells but not Th17 cells and Treg cells responses in the spleen. (a) Flow cytometry analysis of the spleen CD4⁺ IFN-γ⁺ (Th1) cells and CD4⁺ IL-17⁺ (Th17) cells. The Apoe^−/− mice (n = 8) and the Batf3^−/− Apoe^−/− mice (n = 8) were fed either a chow diet or Western diet for 12 weeks. The splenocytes were stimulated with PMA (20 ng/mL), ionomycin (1 μg/mL), and Brefeldin A (5 μg/mL), at 37 °C, 5% CO₂ for 4 h, and then assayed for assessment of Th1 cell and Th17 cell responses by flow cytometry. (b) Flow cytometry analysis of the spleen CD4⁺ Foxp3⁺ Tregs. Intracellular staining was performed on splenocytes for foxp3 detection using the Transcription factor staining buffer set according to the manufacturers' instructions. Data are presented as mean ± SD. Differences of 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. See also Fig. S3.

Fig. 3

CD8α⁺ DCs from Apoe^−/− mice fed a western diet preferentially induced Th1 cell differentiation in vitro. (a) Flow cytometry analysis of a coculture of DC subsets as indicated, and eFluor 450 labeled naïve CD4⁺ T cell in the presence of OVA_323–339 (500 ng/mL), four days after coculture. e450 dilution peaks of the CD4⁺ T cells were analyzed. (b) Coculture of DC subsets as denoted, and naïve CD4⁺ T cells in the presence of OVA_323–339 peptide (500 ng/mL). Four days after coculture, Th1 cell polarization was analyzed by intracellular cytokine (ICC) staining and then by Flow cytometry analysis. (c) ELISA analysis of the IFN-γ content from the cocultured supernatants. A volume of 100 μL from supernatants was used to detect the IFN-γ content using an IFN-γ ELISA kit (eBioscience) according to the manufacturers' instructions. Data are presented as the mean ± SD. Differences of P < 0.05 are considered to be statistically significant. *P < 0.05; **P < 0.01; ns, not significant. Data are representative of three independent experiments. See also Fig. S4.

Fig. 4

CD8α⁺ DCs expressed higher levels of CD86 and IL-12p35 with the WD. The Apoe^−/− mice were fed on a chow diet (Apoe^−/− CD, n = 8) or a western diet (Apoe^−/− WD, n = 8) for 12 weeks, and the splenocytes were used for analysis. (a) CD8α⁺ DCs from the chow diet(CD8 +/CD) and western diet(CD8 +/WD)were sorted from the spleen as described previously by FACS, the RNA was extracted, and Cd86, Il12a, Cd36 and Cd70 mRNA expression was analyzed by qPCR (data are represented normalized to Gapdh and relative to the CD8 +/CD control). (b) Flow cytometry analysis of mean fluorescent intensity (MFI) of CD8α⁺ DCs surface markers CD86, CD40, CD80, B7-DC, and B7-H2. Gray solid area, fluorescent minus one (FMO) staining control. (c) IL-12p70 in the cocultured supernatants of CD8α⁺ DCs and naïve CD4⁺ CD62L⁺ T by ELISA. Sorted splenic CD8α⁺ DCs from the CD8 +/CD or CD8 + WD group was cocultured with naïve CD4⁺ CD62L⁺ T cells as described above, and 100 μL supernatants were used to detect the IL-12p70 content using an IL-12p70 ELISA kit (eBioscience). Data are presented as the mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. *P < 0.05; **P < 0.01; ns, not significant. Data are representative of three independent experiments. See also Fig. S5.

Fig. 5

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 Il10mRNA 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.

Fig. 6

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.