M. Rubino, PhD(1,2) , L. Marcone, MSc(1,2) , C. Vavassori, PhD(3) , L. F. Pisani, PhD(3) , S. Serio, MSc(1,4) , A. Tramontano, PhD(1,5) , L. Lambroia, PhD(1,2) , C. Catalano, MSc(2,6) , G. Basso, PhD(7) , M. Climent, PhD(1,2) , M. Kallikourdis, PhD(2,6) , L. Elia, PhD(1,5) , A. Baggiano, MD, PhD(8) , R. Maragna, MD(8) , S. Mushtaq, MD(8) , G. Pontone, MD, PhD(8,9) , G. I. Colombo, MD, PhD(3†) , G. Condorelli, MD, PhD(1.2†)

1. Department of Cardiovascular Medicine, IRCCS Humanitas Research Hospital, 20089 Rozzano, Milan, Italy; 2. Department of Biomedical Sciences, Humanitas University, 20090 Pieve Emanuele, Milan, Italy;  3. Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy;  4. Institute of Genetics and Biomedical Research, UoS Milan, National Research Council, 20089 Rozzano, Milan, Italy; 5. Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy; 6. Adaptive Immunity Lab, IRCCS Humanitas Research Hospital, 20089 Rozzano, Milan, Italy; 7. Humanitas Genomics Facility, IRCCS Humanitas Research Hospital, 20089 Rozzano, Milan; 8. Department of Perioperative Cardiology and Cardiovascular Imaging, Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; 9. Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy.

Background: Atherosclerosis is driven primarily by high cholesterol and inflammation, which act synergistically to form plaques within arteries. Single-cell RNA sequencing (scRNA-seq) approach has improved knowledge on the role of specific cell subtypes in disease pathogenesis, including atherosclerosis. We sought to determine whether specific circulating immune cells and pathway activation levels could play a role in plaque formation and could be exploitable for therapeutics.

Methods: A cohort of 275 individuals undergoing coronary computed tomography angiography (CCCA) for suspected coronary artery disease (CAD) was divided in three categories: without CAD (NO-CAD); with nonobstructive CAD (NON-OB-CAD); and with obstructive CAD (OB-CAD); their peripheral blood mononuclear cells (PBMCs) were collected and subjected to scRNA-seq and flow cytometry. The functional role of specific PBMCs was investigated using public scRNA-seq datasets from human coronary and aortic plaques and on spatial transcriptomics and proteomics carried out on human carotid plaques. Main findings were confirmed in vitro using isolated PBMCs from healthy and diseased individuals.

Results: A significant increase in CD14⁺ monocytes was detected in OB-CAD patients, with up-regulation of interferon signaling in monocytes of CAD patients. Also, a peripheral CD14⁺ interferon responsive (IFNIC) monocyte could be identified. IFNICs were also detected in the atherosclerotic plaque core region using public scRNA-seq and spatial transcriptomics datasets. Monocyte pseudo-time-space analysis revealed an IFNIC-to-foam cell transition from distal to atherosclerotic core regions. Finally, in vitro validations showed the ability of the plasma isolated from CAD patients to induce a time-dependent transformation of the monocyte-derived macrophages.