Xcr1+ type 1 conventional dendritic cells are essential mediators for atherosclerosis progression
- School of Basic Medicine, Xinxiang Medical University, China
- School of Medical Technology, Xinxiang Medical University, China
- Centre d'Immunologie de Marseille-Luminy, INSERM, CNRS, Aix-Marseille Université, France
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, China
- Hunan Key Laboratory of Disease Models for Integrative Medicine,Center of Disease Model and Immunology, Hunan Academy of Chinese Medicine, China
Figures
Figure 1
Progressive accumulation of Xcr1+ cDC1 cells in human and mouse advanced atherosclerotic lesions.
(A) Representative images of immunohistochemical staining demonstrate Xcr1 expression in normal areas and plaque areas of the femoral artery from three clinical samples. (B) Quantitative analysis of the Xcr1-positive area in normal area and plaque regions. (C) Representative immunofluorescence images depict DAPI (blue), Xcr1 (green), and CD11c (red) within the lesions of aortic root of Apoe–/– mice fed with 16 week normal diet (ND) or high-fat diet (HFD). (D) Quantitative analysis of Xcr1 and CD11c double positive area in lesion area (n=6). Scale bars, 100 μm. (E) Representative images depicting immunohistochemical staining of Xcr1 in lesions of the aortic root of Apoe–/– mice fed a HFD for varying durations. (F) Quantitative analysis of the Xcr1-positive area in the lesion regions. (8 week HFD, n=11; 12 week HFD, n=15; 16 week HFD, n=29). Data represent as mean ± SEM. *p<0.05. ***p<0.001.****p<0.0001.
Figure 2 with 2 supplements
Selective expression of Xcr1Cre-Gfp in cDC1 cells.
(A) Diagram illustrating the bone marrow transfer process. (B) and (C) Representative flow cytometric analysis and corresponding quantification of GFP+RFP+ in cDC1 and non-cDC1 populations within the cDC1 and non-cDC1 populations of the spleen. These data were obtained from Apoe–/– mice that had received bone marrow transplants from either wild-type (WT) or Xcr1Cre-Gfp Rosa26LSL-RFP donors and were maintained on a HFD for 16 weeks (WT, n=4; Xcr1Cre-Gfp Rosa26LSL-RFP, n=3). (D) and (E) Representative flow cytometric analysis and quantification of the percentage of GFP+ cells among pDC, cDC1, and cDC2 cells populations in the spleen. (F) Quantification of the percentage of pDC, cDC1, and cDC2 cells in spleen. (D-F, Apoe–/–, Xcr1Cre-Gfp Apoe–/–, and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed with a high-fat diet (HFD) for 16 weeks, n=3). Data represent as mean ± SEM. **** p<0.0001; NS, non-significant.
Figure 2—figure supplement 1
Schematic diagram illustrating the knock-in of the 5’HA-iCre-P2A-EGFP-P2A-3’HA vector into the mouse Xcr1 locus.
Figure 2—figure supplement 2
Flow cytometric analysis of cDC1 cells in the lymph nodes and spleens of Apoe–/– mice and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed a 7 weeks normal diet (ND).
(A) Representative flow cytometric analysis of cDC1 cells in lymph nodes. (B) Quantification of the frequencies and number of cDC1 cells in lymph nodes. (C) Representative flow cytometric analysis of cDC1 cells in the spleen. (D) Quantification of the frequencies and number of cDC1 cell in the spleens. Apoe–/– mice and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed with 7 week ND, n=7. Data represent the mean ± SEM. ***p<0.001.
Figure 3 with 1 supplement
Specific depletion of Xcr1+ cDC1 cells in Apoe–/– mice reduces atherosclerosis progression with no effect on macrophages.
(A) Oil Red O (ORO) staining of the descending aortas. Scale bar, 5 mm. (B) Quantification of the lesion area in the descending aorta (Apoe–/– mice, n=9; Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, n=6). (C) ORO and H&E staining of the aortic roots of representative mice from each group. (D), Quantification of lesion area in the aortic root. (Apoe–/– mice, n=7; Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, n=8). Scale bar, 200 μm. (E) and (F) H&E staining and quantification of necrotic core area in the lesions of aortic root. (Apoe–/– mice, n=7; Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, n=8). Scale bar, 200 μm. (G and H) Representative flow cytometric analysis and quantification of macrophages in the aorta. (Apoe–/– mice, n=8; Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, n=6).(I and J), Representative immunohistochemical staining images of CD68 and quantification of CD68-positive area in the lesions of the aortic root. (Apoe–/– mice, n=6; Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, n=8). Scale bar, 100 μm. Apoe–/– and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice were fed on a 16 week high-fat diet (HFD) to develop atherosclerosis. Data represent as mean ± SEM. NS, non-significant. **p<0.01. ***p<0.001.
Figure 3—figure supplement 1
Lipid profile, body weight and Oil Red O (ORO) staining of the liver in Apoe–/– mice and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed a 16 week high-fat diet (HFD).
(A) Concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in the serum. (B) Body weight measurements of both groups of mice. (C) ORO staining of liver from each group. Scale bar, 100 μm. (D) Quantification of ORO-positive area in liver. Apoe–/– mice (n=7) and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice (n=8) were fed on a 16 week HFD to develop atherosclerosis. Data represent the mean ± SEM. NS, non-significant.
Figure 4 with 3 supplements
Specific depletion of Xcr1+ cDC1 cells in Apoe–/– mice decreases T cell activation within aorta.
(A) Representative flow cytometric analysis of cDC1 cells in aortas. (B and C) Quantification of the frequencies and absolute counts of cDC1 cells in aorta (n=4). (D) Representative flow cytometric analysis T cells in aortas. (E and F) Quantification of the frequencies and absolute counts of total T, CD4+, and CD8+ T cells in aortas. (G) Quantification of the frequencies of CD4+CD69+ and CD8+ CD69+ in aortas. (H) Quantification of the MFI of CD44 in CD4+ and CD8+ T cells in aortas. (D-G, Apoe–/– mice, n=8, Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, n=7, fed on a high-fat diet HFD for 16 weeks). Data represent as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, NS, non-significant.
Figure 4—figure supplement 1
Flow cytometric analysis of cDC1 cells in the lymph nodes and spleens of Apoe–/– and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed a 16 week high-fat diet (HFD).
(A) Representative flow cytometric analysis of cDC1 cells in lymph nodes. (B) Quantification of the frequencies and numbers of cDC1 cells in lymph nodes. (C) Representative flow cytometric analysis of cDC1 cells in the spleens. (D) Quantification of the frequencies and numbers of cDC1 cells in the spleens. Apoe–/– mice (n=7) and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice (n=8) were fed on a 16 week HFD. Data represent the mean ± SEM. ***p<0.001.
Figure 4—figure supplement 2
Flow cytometric analysis of T cells in the lymph nodes and spleens of Apoe–/– mice and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed a 16 week high-fat diet (HFD).
(A) Representative flow cytometric analysis of CD4+, CD8+, CD4+CD69+, and CD8+CD69+ T cells in lymph nodes. (B and C) Quantification of the frequencies and numbers of CD4+, CD8+, CD4+ CD69+, and CD8+ CD69+ T cells in lymph nodes. (D) Representative flow cytometric analysis and quantification of CD4+, CD8+, CD4+ CD69+, and CD8+ CD69+ T cells in the spleens. (E and F) Quantification of the frequencies and numbers of CD4+, CD8+, CD4+ CD69+, and CD8+ CD69+ T cells in the spleens. Apoe–/– mice (n=7) and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice (n=8) were fed on a 16 week HFD. Data represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, NS, non-significant.
Figure 4—figure supplement 3
T cell analysis in the lymph nodes and spleens of Apoe–/– mice and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed a 7 week normal diet (ND).
(A) Representative flow cytometric analysis and quantification of CD4+, CD4+ memory, CD8+, and CD8+ memory T cells in lymph nodes. (B) Quantification of the frequencies of CD4+, CD8+, CD4+CD69+, and CD8+CD69+ T cells in the lymph nodes. (C) Representative flow cytometric analysis and quantification of CD4+, CD4+ memory, CD8+, and CD8+ memory T cells in the spleens. (D) Quantification of the frequencies of CD4+, CD8+, CD4+ CD69+, and CD8+ CD69+ T cells in the spleens. (E and F) Quantification of the numbers of CD4+ and CD8+ T cells in the lymph nodes and spleens. Apoe–/– mice and Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice fed with a 7 week ND (n=6). Data represent the mean ± SEM. *p<0.05, **p<0.01, NS, non-significant.
Figure 5 with 4 supplements
Sc-RNA sequencing analysis of the aorta, lymph nodes and spleen in Apoe–/– mice fed with a 20 week high-fat diet (HFD).
(A) UMAP plot delineates 10 annotated cell types of Xcr1+ cDC1 cells from aorta, lymph node and spleen in Apoe–/– mice maintained on a HFD or normal diet (ND) for 20 weeks. (B) Heatmap statistic map displays the top ten up-regulated genes across various clusters. (C) The proportion of 10 clusters among groups. (D and E) UMAP plot representing Xcr1+ cDC1 cells from aorta, lymph nodes, spleen, and a merged plot in Apoe–/– mice maintained on a HFD for 20 weeks.
Figure 5—figure supplement 1
Depletion of Xcr1+ cDC1 cells of bone marrow reduces cDC1 cells without affecting macrophages in atherosclerotic lesion of Apoe–/– mice.
(A) Diagram of the bone marrow transfer process. Apoe–/– mice transplanted with bone marrow from wild-type (WT) or Xcr1Cre-Gfp Rosa26LSL-DTA donors (n = 7). (B) Body weight of each group. (C) Serum concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). (D) Representative Oil Red O (ORO) staining images of liver sections and quantification of the ORO positive area percentage in the liver. Scale bar, 50 μm. (E) Representative flow cytometric analysis of pDC, cDC1 and cDC2 cells in the aortas, Lin- means CD3- CD19- Ly6G- NK1.1-. (F) Flow cytometric analysis of macrophages in the aortas. Data represent as mean ± SEM. ***p<0.001, NS, non-significant.
Figure 5—figure supplement 2
The single-cell RNA sequencing process.
Figure 5—figure supplement 3
Sc-RNA sequencing analysis of the aorta, lymph nodes and spleen in Apoe–/– mice.
(A) UMAP plot delineates 10 annotated cell types of Xcr1+ cDC1 cells from aorta, lymph node and spleen in Apoe–/– mice maintained on a high-fat diet (HFD) or normal diet (ND) for 20 weeks. (B-G), Heatmap statistic map showing marker genes in different clusters. Ccr7 is the mature gene marker (B). Sell+Cd8a- cDC1s are defined as Pre-cDC1s (C). Stmn1 and Top2a are the proliferating markers (D). Itgae and Nr4a2 genes marked the transition from early to late immature cDC1s (E). Cxcl9 and Cxcl10 genes marked the transition from late immature to early mature cDC1s (F). Cd63 and Fscn1 are the late mature markers (G).
Figure 5—figure supplement 4
Single-cell RNA sequencing of splenic cDC1s from both the high-fat diet (HFD) and normal diet (ND) groups.
(A) UMAP plot of splenic cDC1s showing annotated clusters. (B) The proportions of cDC1 maturation states across diet groups. (C-H) Heatmap statistic map depicting representative markers defining cDC1 maturation clusters. Ccr7 is the mature gene marker (C). Sell+Cd8a- cDC1s are defined as Pre-cDC1s (D). Top2a, Stmn1, and Mki67 are the proliferating markers (E). Itgae, Nr4a2, Nr4a3, and Egr3 genes marked the transition from early to late immature cDC1s (F). Cxcl9 and Cxcl10 genes marked the transition from late immature to early mature cDC1s (G). Cd63 and Fscn1 are the late mature markers (H). (I-J) Heatmap statistic map of upregulated genes (Tnfaip3 and Serinc3), and downregulated genes (Apol7c and Tifab) in the HFD spleen compared to the ND spleen.
Figure 6
Xcl1 deficiency reduces atherosclerotic lesion formation in Apoe–/– mice.
(A) Oil Red O (ORO) staining of the descending aortas. Scale bar, 5 mm. (B) Quantification of the lesion area in aortas. (C), ORO and H&E staining of aortic roots. Scale bar, 200 μm. (D) Quantification of the lesion area in aortic roots. (E and F) Representative liver images and quantification of ORO-positive area in livers (n=24 regions from 8 mice, 3 regions per mice). Scale bar, 100 μm. (G) The concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in the serum. (H), Body weight of mice before and after feeding with 19 week high-fat diet (HFD). (I) Representative immunohistochemical staining of CD68 in aortic roots. (J) Quantification of CD68 positive area in the lesions of aortic roots (n=24 lesions from 8 mice, 3 lesions per mice). Scale bar, 100 μm. Apoe–/– mice and Xcl1–/– Apoe–/– mice fed a HFD for 19 weeks (n=8 per group). Data represent as mean ± SEM. **p<0.01, ***p<0.001, NS, non-significant.
Figure 7 with 2 supplements
Xcl1 deficiency reduces CD8+ T cells in the aortas from Apoe–/– mice.
(A) Representative flow cytometric analysis of cDC, cDC1, and cDC2 cells in aortas. (B and C) Quantification of the frequencies and absolute counts of cDC, cDC1, and cDC2 cells in aortas (n=7). (D) Representative flow cytometric analysis of T cell subsets in aortas, including CD3e+ CD5+, CD4+, CD4+ CD69+, CD8+, and CD8+ CD69+ T cells. (E) Quantification of the frequencies of CD4+CD69+ and CD8+ CD69+ in aortas. (F), Quantification of the Mean Fluorescence Intensity (MFI) of CD44 in CD4+ and CD8+ T cells in aortas. (G and H) Quantification of the frequencies and absolute counts of CD3e+ CD5+, CD4+, and CD8+ cells in aortas. (D-H, Apoe–/– mice (n=5) and Xcl1–/–Apoe–/– mice (n=6) fed with a 16 week high-fat diet HFD). Data represent as mean ± SEM. * p<0.05, NS, non-significant.
Figure 7—figure supplement 1
Dendritic cell (DC) analysis in lymph nodes and spleens of Apoe–/– and Xcl1–/– Apoe–/– mice fed with 19 week high-fat diet (HFD).
(A) Representative flow cytometric analysis and quantification of pDC, cDCs, cDC1 and cDC2 cells in lymph nodes. (B) Quantification of absolute counts of pDC, cDCs, cDC1 and cDC2 cells in lymph nodes. (C) Representative flow cytometric analysis of pDC, cDC1 and cDC2 cells in spleens. (D) Quantification of absolute counts of pDC, cDCs, cDC1 and cDC2 cells in spleens. Apoe–/– (n=9) and Xcl1–/– Apoe–/– mice (n=7 or 8) fed a HFD for 19 weeks. Data represent the mean ± SEM. NS, non-significant.
Figure 7—figure supplement 2
T cell analysis in lymph node and spleen of Apoe–/– and Xcl1–/– Apoe–/– mice fed with 16 week high-fat diet (HFD).
(A) and (B) Representative flow cytometric analysis and quantification of absolute counts of CD4+, CD8+, CD4+CD69+, and CD8+CD69+ T cells in lymph nodes (Apoe–/–, n=9; Xcl1–/– Apoe–/–, n=7). (C) and (D) Representative flow cytometric analysis and quantification of absolute counts of CD4+, CD8+, CD4+CD69+, and CD8+CD69+ T cells in spleens (Apoe–/–, n=10; Xcl1–/– Apoe–/–, n=7). Data represent the mean ± SEM. NS, non-significant.
Additional files
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Supplementary file 1
The top 10 highly expressed marker genes in ten clusters.
- https://cdn.elifesciences.org/articles/107742/elife-107742-supp1-v1.xls
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Supplementary file 2
Upregulated genes in Xcr1+ cDC1 cells from aorta vs spleen, and aorta vs lymph node in Apoe–/– mice fed a high-fat diet (HFD) for 20 weeks.
- https://cdn.elifesciences.org/articles/107742/elife-107742-supp2-v1.xls
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Supplementary file 3
The sequences of the sgRNA used for generation of Xcr1Cre-Gfp knock-in mice and Xcl1 knockout mice.
- https://cdn.elifesciences.org/articles/107742/elife-107742-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107742/elife-107742-mdarchecklist1-v1.docx
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Xcr1+ type 1 conventional dendritic cells are essential mediators for atherosclerosis progression
eLife 14:RP107742.
https://doi.org/10.7554/eLife.107742.3
