biostudies-arrayexpressDavid JohnHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16472Single-cell RNA sequencing revealed a dysregulated myeloid immune response with hyperinflammatory and immunosuppressive features in patients with ASCVD and moderate COVID-19. Assay for Transposase-Accessible Chromatin sequencing and in-vitro experiments with isolated monocytes infected with SARS-CoV-2 showed epigenetic priming of monocytes from patients with ASCVD towards increased expression of inflammatory mediators and type I interferon signalling. In a German nationwide cohort (NAPKON), using multiplex cytokine assays, enzyme-linked immunosorbent assays, and bulk-RNA-sequencing, we confirmed that patients with ASCVD display an exaggerated inflammatory response during moderate COVID-19.biostudies-arrayexpressSequencing - Indexed libraries were equimolarly pooled and sequenced on 2 Illumina NovaSeq 6000 using paired-end 26×98 bp as sequencing mode by GenomeScan (Leiden, the Netherlands).Library Construction - Cellular suspensions of thawed, MetOH-fixed PBMCs were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer’s protocol based on the 10X Genomics proprietary technology. All scRNA-seq libraries were prepared using Chromium Single- Cell 3′ v3 Reagent Kit (10X Genomics) according to manufacturer’s protocol. Briefly, the initial step consisted of performing an emulsion where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10X cell barcodes, unique molecular identifiers, and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Bulk cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well Sample Block (98 °C for 3 minutes; cycled 14×: 98°C for 15 s, 67°C for 20 s, and 72°C for 1 minute; 72°C for 1 minute; held at 4°C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single-Cell 3′ v3 Reagent Kit as follows: fragmentation, end repair, and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation clean up with SPRIselect; sample index polymerase chain reaction; and clean up with SPRI select beads. Library quantification and quality assessment were performed using Bioanalyzer Agilent 2100 using a High-Sensitivity DNA chip (Agilent Genomics)Sample Collection - Blood was obtained from hospitalized SARS-CoV-2-infected patients with moderate COVID-19 according to the WHO Clinical Progression Scale (WHO-CPS)5 , not requiring intensive-care treatment, with (n=5) and without (n=6) preexisting atherosclerotic cardiovascular disease. ASCVD was defined as a history of myocardial infarction, severe carotid artery stenosis (> 70% narrowing) or atherosclerotic stroke, according to the European Society of Cardiology. As a comparision group patients with comparable age and disease severity without a prior history of cardio- or cerebrovascular illness were included. Patients with end-stage renal failure, malignancies, rheumatological conditions and diseases requiring immunosuppression were not included. All patients were hospitalized for moderate COVID-19 pneumonia requiring oxygen supplementation but no invasive ventilation or intensive care unit treatment. Patients were included between July 03, 2020, and November 11, 2020.Nucleic Acid Extraction - Blood was sampled in EDTA tubes on 3 after hospitalization before vaccines against SARS-CoV-2 were available. Peripheral blood mononuclear cells (PBMCs) were isolated via density centrifugation (800g for 20 min) of PBS-diluted whole blood over Ficoll-Paque. The PBMC layer was carefully collected, washed with PBS, resuspended in 80% MetOH and frozen at -80°C. Written informed consent was obtained from all patients. The samples were acquired as part of the CAPNETZ Study, which was approved by the local ethics review board (protocol number 11/17) and complies with the Declaration of Helsinki.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - scRNA-seq expression data were processed using STARsolo (version 2.7.3a) to perform quality control, barcode processing and single-cell 3′ gene counting. Sequencing reads were aligned to the human reference genome (GRCh38). STARsolo was run with default parameters, and the quality and content of the sequenced libraries were assessed. Further analysis was performed using R and the R package Seurat (version 4.1).Data Transformation - Data were filtered based on the number of genes detected per cell (cells with fewer than 200 genes per cell were filtered). Data was scaled and regressed against the number of UMIs. Data was subjected to principal component analysis and unsupervised clustering by the Louvain clustering method. Cell clusters were visualised using Uniform Manifold Approximation and Projection (UMAP).UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000<h4>Aims</h4>Patients with cardiovascular disease (CVD) have an increased risk of developing severe respiratory infections, including COVID-19. However, the underlying molecular mechanisms are not completely understood. It has been previously shown that CVD predisposes to an altered responsiveness to subsequent inflammatory triggers by an imprinted epigenetic memory in innate immune cells. Therefore, we hypothesized that patients with pre-existing atherosclerotic cardiovascular disease (ASCVD) and COVID-19 display a dysregulated inflammatory response compared to patients without ASCVD due to epigenetically altered immune cells leading to increased disease severity.<h4>Methods and results</h4>Single-cell RNA sequencing revealed a dysregulated myeloid immune response with hyperinflammatory and immunosuppressive features in patients with ASCVD and moderate COVID-19. Assay for Transposase-Accessible Chromatin sequencing and in vitro experiments with isolated monocytes infected with SARS-CoV-2 showed epigenetic priming of monocytes from patients with ASCVD towards increased expression of inflammatory mediators and type I interferon signalling. In a German nationwide cohort (NAPKON), using multiplex cytokine assays, enzyme-linked immunosorbent assays, and bulk-RNA sequencing, we confirmed that patients with ASCVD display an exaggerated inflammatory response during moderate COVID-19.<h4>Conclusion</h4>This study demonstrates that patients with ASCVD show a dysregulated myeloid immune response in moderate COVID-19 disease. Mechanistically, epigenetic imprinting sensitizes myeloid cells of patients with ASCVD to an exaggerated type I interferon-associated immune response.RNA-seq of coding RNA from single cellsHomo sapiensAtherosclerosis licenses for an exceeding immune response in COVID-19 disease by interferon priming in circulating myeloid cellsDavid Johneberzammer J, Abplanalp WT, Grikscheit K, Solomonidis EG, Glaser SF, Schuhmacher B, Merten M, Jeremijev G, Wilken-Schmitz A, Korth L, John D, Günther S, Kuenne C, Looso M, Valasarajan C, Benes V, Jung F, Niehaus V, Anton G, Stellbrink C, Römmele C, Göpel S, Pullamsetti SS, Vehreschild J, Vehreschild M, Ciesek S, Leistner DM, Zeiher AM, Dimmeler S, Cremer S.falseAtherosclerosis licenses for an exceeding immune response in COVID-19 disease by interferon priming in circulating myeloid cellsSingle-cell RNA sequencing revealed a dysregulated myeloid immune response with hyperinflammatory and immunosuppressive features in patients with ASCVD and moderate COVID-19. Assay for Transposase-Accessible Chromatin sequencing and in-vitro experiments with isolated monocytes infected with SARS-CoV-2 showed epigenetic priming of monocytes from patients with ASCVD towards increased expression of inflammatory mediators and type I interferon signalling. In a German nationwide cohort (NAPKON), using multiplex cytokine assays, enzyme-linked immunosorbent assays, and bulk-RNA-sequencing, we confirmed that patients with ASCVD display an exaggerated inflammatory response during moderate COVID-19.2025-01-15T00:00:00Z2026-05-27T12:37:17.881Z2026-01-02T14:54:23.497ZE-MTAB-1647241389000ERP187207EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_000418410.1093/cvr/cvaf268