Immunology and Inflammation

Xcr1⁺ type 1 conventional dendritic cells are essential mediators for atherosclerosis progression

Tianhan Li
Liaoxun Lu
Juanjuan Qiu
Xin Dong
Le Yang
Kexin He
Yanrong Gu
Binhui Zhou
Tingting Jia
Toby Lawrence
Marie Malissen
Guixue Wang
Rong Huang
Hui Wang
Bernard Malissen author has email address
Yinming Liang author has email address
Lichen Zhang author has email address

School of Basic Medicine, Xinxiang Medical University, Xinxiang, China
School of Medical Technology, Xinxiang Medical University, Xinxiang, China
Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix-Marseille Université, Marseille, 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, Chongqing, China
Center of Disease Model and Immunology, Hunan Academy of Chinese Medicine, Changsha, China

https://doi.org/10.7554/eLife.107742.1

Open access
Copyright information

Figures and data

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 cardiovascular disease patients. 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 chow diet or 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. (HFD-8W mice, n = 11; HFD-12W mice, n = 15; HFD-16W mice, n = 29). Data represent as mean ± SEM. *** P<0.001.

Selective expression of Xcr1^Cre-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 WT or Xcr1^Cre-Gfp Rosa26^LSL-RFP donors and were maintained on a HFD for 16 weeks (WT, n = 4; Xcr1^Cre-Gfp Rosa26^LSL-RFP, n = 3). D through F, Eight-week-old ApoE^−/−, Xcr1^Cre-Gfp ApoE^−/− and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed with a HFD for 16 weeks (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. Data represent as mean ± SEM. **** P<0.0001; NS, non-significant.

Specific depletion of Xcr1⁺ cDC1 cells in ApoE^−/− mice reduces atherosclerosis progression with no effect on macrophages.
A through I, Eight-week-old ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice were fed on a 16-week HFD to develop atherosclerosis. A, ORO staining of the descending aortas. Scale bar, 5 mm. B, Quantification of the lesion area in the descending aorta (ApoE^−/− mice, n = 9; Xcr1^Cre-Gfp Rosa26^LSL-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; Xcr1^Cre-Gfp Rosa26^LSL-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; Xcr1^Cre-Gfp Rosa26^LSL-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; Xcr1^Cre-Gfp Rosa26^LSL-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; Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice, n = 8). Scale bar, 100 μm. Data represent as mean ± SEM. NS, non-significant.

Specific depletion of Xcr1⁺ cDC1 cells in ApoE^−/− mice decreases T cell activation within aorta.
A through D, Eight-week-old ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed a HFD for 16 weeks. A, Representative flow cytometric analysis cDC1 cells in aortas. B, Quantification of cDC1 cells in aorta (n = 4). C, Representative flow cytometric analysis T cells in aortas D, Quantification of T cell populations, including CD4⁺, CD4⁺CD69L⁺, CD8b⁺ and CD8b⁺CD69L⁺ T cells in aortas (n = 7). Data represent as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001, ns, non-significant.

Sc-RNA sequencing analysis of the aorta, lymph nodes and spleen in ApoE^−/− mice fed with a 20-week HFD.
A, UMAP plot delineates 10 annotated cell types of CD11c⁺ MHCII⁺ Xcr1⁺ cDC1 cells from aorta, lymph node and spleen in ApoE^−/− mice maintained on a HFD for 20 weeks. B, Heatmap statistic map displays the top ten up-regulated genes across various clusters. C, Histogram plot illustrates the proportion of different cell types among groups. D and E, UMAP plot representing CD11c⁺ MHCII⁺ Xcr1⁺ cDC1 cells from aorta, lymph nodes, spleen, and a merged plot in ApoE^−/− mice maintained on a HFD for 20 weeks.

Xcl1 deficiency reduces atherosclerotic lesion formation in ApoE^−/− mice.
A through I, Eight-week-old ApoE^−/− mice and Xcl1^−/− ApoE^−/− mice fed a HFD for 19 weeks (n = 8 per group). A, 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 TC, TG, LDL and HDL in the serum. H, Body weight of mice before and after feeding with 19-week 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. Data represent as mean ± SEM. ** P<0.01, *** P<0.001, NS, non-significant.

Xcl1 deficiency reduces CD8⁺ T cells frequency in the aorta from.
A through D, Eight-week-old ApoE^−/− mice and Xcl1^−/−ApoE^−/− mice fed with a 16-week HFD. (n = 7). A and B, Representative flow cytometric analysis and quantification of pDC, cDC1 and cDC2 cells in aortas. C and D, Representative flow cytometric analysis and quantification of T cell subsets, including CD4⁺, CD4⁺ CD69L⁺, CD8b⁺ and CD8b⁺ CD69L⁺ T cells in aortas. Data represent as mean ± SEM. * P<0.05, NS, non-significant.

Schematic diagram illustrating the knock-in of the 5’HA-iCre-P2A-EGFP-P2A-3’HA vector into the mouse Xcr1 locus.

Flow cytometric analysis of cDC1 cells in the lymph nodes and spleens of ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed a 7-weeks chow diet.
A through D, ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed with a 7-week chow diet. (n = 7). A, Representative flow cytometric analysis of cDC1 cells in lymph nodes. B, Quantification of the frequencies and number of cDC1 cell in lymph nodes. C, Representative flow cytometric analysis cDC1 cells in the spleens. D, Quantification of the frequencies and number of cDC1 cell in the spleens. Data represent the mean ± SEM. *** P<0.001.

Lipid profile, body weight and ORO staining of the liver in ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed a 16-week HFD.
A through D, Eight-week-old ApoE^−/− mice (n =7) and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice (n =8) were fed on a 16-week HFD to develop atherosclerosis. A, Concentrations of TC, TG, LDL and 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. Data represent the mean ± SEM. ** P<0.01, *** P<0.001, NS, non-significant.

Flow cytometric analysis of cDC1 cells in the lymph nodes and spleens of ApoE^−/− and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed a 16-week HFD.
A through D, Eight-week-old ApoE^−/− mice (n =7) and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice (n =8) were fed on a 16-week HFD to develop atherosclerosis. 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. Data represent the mean ± SEM. ** P<0.01, *** P<0.001, NS, non-significant.

Flow cytometric analysis of T cells in the lymph nodes and spleens of ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed a 16-week HFD.
A through F, Eight-week-old ApoE^−/− mice (n =7) and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice (n =8) were fed on a 16-week HFD to develop atherosclerosis. 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. Data represent the mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001, NS, non-significant.

T cells analysis in the lymph nodes and spleens of ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed a 7-week chow diet.
A through F, ApoE^−/− mice and Xcr1^Cre-Gfp Rosa26^LSL-DTA ApoE^−/− mice fed with a 7-week chow diet. (n = 7). A, Representative flow cytometric analysis and quantification of CD4⁺, CD4⁺ CD44⁺ CD62L⁻, CD8⁺ and CD8⁺ CD44⁺ CD62L⁻ 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⁺ CD44⁺ CD62L⁻, CD8⁺ and CD8⁺ CD44⁺ CD62L⁻ 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. Data represent the mean ± SEM. * P<0.05, ** P<0.01, NS, non-significant.

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 WT or Xcr1^Cre-Gfp Rosa26^LSL-DTA donors before and after 16 weeks on a HFD (n = 7). B, Body weight of each group. C, Serum concentrations of TC, TG, LDL and HDL. D, Representative 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, Representative flow cytometric analysis of macrophages in the aortas of each group. Data represent as mean ± SEM. *** P<0.001, NS, non-significant.

The single-cell RNA sequencing process.

DC cells analysis in lymph nodes and spleens of ApoE^−/− and Xcl1^−/− ApoE^−/− mice fed with 19-week HFD.
A through D, Eight-week-old ApoE^−/− (n = 9) and Xcl1^−/− ApoE^−/− mice (n = 7) fed a HFD for 19 weeks. 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. Data represent the mean ± SEM. NS, non-significant.

T cells analysis in lymph node and spleen of ApoE^−/− and Xcl1^−/− ApoE^−/− mice fed with 16-week HFD.
A through D, Eight-week-old ApoE^−/− (n = 9) and Xcl1^−/− ApoE^−/− mice (n = 7) fed a HFD for 19 weeks. 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. C and D, Representative flow cytometric analysis and quantification of absolute counts of CD4⁺, CD8⁺, CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells in spleens. Data represent the mean ± SEM. NS, non-significant.

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