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

Atherosclerosis is characterized by lipid accumulation within plaques, leading to foam cell formation and inflammatory response within the aortic lesions. Lipid disorders have been extensively investigated, however the cellular and molecular mechanisms that trigger the inflammatory response in atherosclerotic plaques remain far from being fully understood. Xcr1+ cDC1 cells are newly identified antigen-presenting cells in activating immune cells. However, the role of cDC1 cells in atherosclerosis development remains highly controversial. We first confirmed the presence of cDC1 within human atherosclerotic plaques and discovered a significant association between the increasing cDC1 numbers and atherosclerosis progression in mice. Subsequently, we established Xcr1Cre-Gfp Rosa26LSL-DTA Apoe–/– mice, a novel and complex genetic model, in which cDC1 was constitutively depleted in vivo during atherosclerosis development. Intriguingly, we observed a notable reduction in atherosclerotic lesions in hyperlipidemic mice, alongside suppressed T cell activation of both CD4+ and CD8+ subsets in the aortic plaques. Notably, aortic macrophages and serum lipid levels were not significantly changed in the cDC1-depleted mice. Single-cell RNA sequencing revealed heterogeneity of Xcr1+ cDC1 cells across the aorta and lymphoid organs under hyperlipidemic conditions. As Xcr1 is the sole receptor for Xcl1, we next explored to target Xcr1+ cDC1 cells via Xcl1 by establishing Xcl1–/–Apoe–/– mice. Xcl1–/–Apoe–/– mice exhibited decreased atherosclerotic plaque formation and reduced aortic cDC1 accumulation, indicating that Xcl1 contributes to cDC1-mediated atherosclerotic lesion development. Our results reveal crucial roles of cDC1 in atherosclerosis progression and provide insights into the development of immunotherapies by targeting cDC1 through Xcl1. Overall design: Eight-week-old male Apoe–/– mice were fed with 20-week ND or HFD. The single cell suspension of spleen, lymph node and aorta were obtained according to the flow cytometry analysis section. Single cell suspension from spleens and lymph nodes were firstly removed the CD5+, CD19+ and CD11b+ cells via SAV conjugated beads. Then two million live cells from spleen and lymph node were stained with mix1 (CD45.2-PE-Cy7, CD11c-PE-Cy5.5, CD172a-FITC, MHCII- APC-eFluor780 and Xcr1-APC), and all cells from aorta were stained with mix2 (CD45.2-PE-Cy7, CD11b-super bright 600, CD11c-PE-Cy5.5, MHCII- FITC, CD3- Alexa Fluor 700, CD19- Alexa Fluor 700, Ly6G- Alexa Fluor 700 and Xcr1-APC) for 30 minutes at 4 °C. After washing with 1 mL FACS buffer two times, cells from different organs were stained with different anti-CD45 antibodies conjugated with barcode oligos from BD™ Ms Single Cell Sample Multiplexing Kit (BD Bioscience, Cat: 633793). Finally, cells were resuspended with Sytox blue and CD11c+ MHCII+ Xcr1+ cells were sorted and subjected to the BD Rhapsody Express system. Then cDNA and sample tag libraries were built with BD Rhapsody whole transcriptome amplification (WTA) reagent kit (BD Bioscience, Cat: 633733, 633773, 633801, 664887) following manufacturer's instructions, and sequenced on an illumine Novaseq 600 sequencer. Pair-end Fastq files of sample tag and WTA data were processed via BD RhapsodyTM analysis pipeline v2.0, and the resultant dataset was mainly analysis using SeqGeqTM software, which contain Lex-BDSMK and Seurat v4.04 plugin components. This experiment was performed once, and both raw data and processed data were uploaded into GEO (Accession number: GSE279370).