Methods: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) derived from hospitalized SARS-Cov2 infected patients in an uncomplicated phase of the disease not requiring intensive-care treatment with (n=5) and without (n=6) preexisting atherosclerosis was performed. Single-cell transcriptomes were analyzed by R using the Seurat package. For in-vitro experiments monocytes from patients with and without CVD were stimulated with SARS-CoV-2 and analyzed by qPCR.
Results: Baseline characteristics between the two groups were similar (atherosclerosis vs. no atherosclerosis: mean age 75 vs. 70 years, oxygen requirement 2,2 vs. 3,2 l/min, CRP 10,7 vs. 6,6 mg/dl, IL-6 61,6 vs. 60,6 pg/ml, all p>0.05). In accordance with previous COVID-19 scRNA-seq studies, we found low-density neutrophils, immature neutrophils, neutrophil like plasmablasts and mostly classical monocytes in the myeloid compartment. Low-density neutrophils from patients with atherosclerosis demonstrated an increased expression of proinflammatory (IL18R1: 3,3, S100A12: 1,7, TLR2: 1,5, S100A9: 1,4, TLR4:1,4, all fold change, p<1,3x10-98) and immunosuppressive genes (IL1R2: 2,6, ARG1: 1,7, ANXA1: 1,6, all fold change, p <4,1x10-67). Interestingly, we found an enrichment of proinflammatory COVID-19 specific neutrophil like plasmablasts in patients with atherosclerosis (p=0.049). This subpopulation of cells also showed an increased expression of inflammatory genes (S100A12: 2,5, S100A9: 2,5, S100A8: 1,8, IL18R1: 3,9, all fold change, p<1,1x10-54). In accordance, monocytes from patients with atherosclerotic disease showed an enrichment of inflammatory (S100A9:1,6, TLR2: 1,5, IL13RA1: 1,3, CCR2: 1,2 , all fold change, p < 1,3x10-60) and immunomodulatory genes (IL1R2: 3,5, CD163: 2,2, all fold change, p<2,7x10-87). To gain insight on the mechanisms how cardiovascular disease promotes an overshooting inflammatory response, monocytes from patients with and without CVD were isolated and stimulated with SARS-CoV-2 in vitro. We analyzed proinflammatory markers of trained immunity and found a significant upregulation of Il1b (p<0.05) and an increased IL-6 expression in monocytes derived from CVD patients after infection with SARS-Cov-2. Validating the scRNA-seq data, we also replicated an increased expression of CD163 in monocytes derived from patients with CVD (p<0.05).
Conclusions: Our data show for the first time that patients with atherosclerosis have a dysregulated myeloid immune response already in the uncomplicated phase of SARS-CoV-2 infection. Upregulated genes and cell populations found in this study have previously been associated with severe COVID-19 and inferior outcomes. SARS-CoV-2 induces mediators of trained immunity in monocytes from patients with CVD. This might be due to epigenetic memory in myeloid cells.
The enhanced inflammatory response may contribute to the worse outcome of patients with CVD and might be addressed by tailored anti-inflammatory drugs
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