1Universitätsklinikum Frankfurt Med. Klinik III - Kardiologie, Angiologie Frankfurt am Main, Deutschland; 2Goethe University Institute for Medical Virology Frankfurt, Deutschland; 3Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration and Department of Cardiology Frankfurt am Main, Deutschland; 4Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; 5Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration Frankfurt am Main, Deutschland; 6Universitätskliniku Frankfurt Infectious Diseases Frankfurt, Deutschland; 7Max-Planck-Institut für Herz- und Lungenforschung Bad Nauheim, Deutschland
Background: COVID-19 is characterized by emergency hematopoiesis with a dysregulated myeloid compartment, comprising proinflammatory and immunosuppressive immune cells. Preexisting cardiovascular disease (CVD) is a major risk factor for severe and fatal COVID-19 outcomes. Individuals with atherosclerosis are known to have a pro-inflammatory immune cell phenotype. However, the mechanisms of how CVD causes worse outcomes during SARS-Cov-2 infection remain unknown.
Methods: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) derived from hospitalized SARS-Cov-2 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. Cytoscape was used to predict transcription factors (TF) that may contribute to the observed transcriptomic changes. For in-vitro experiments, monocytes from patients with and without CVD were stimulated with SARS-CoV-2 and Assay for Transposase-Accessible Chromatin (ATAC) sequencing was performed.
Results: Baseline characteristics between the two groups were similar (mean age, oxygen requirement and serum levels of CRP and interleukin-6 (IL-6)). Following previous COVID-19 scRNA-seq studies, we found low-density neutrophils (LDNs), neutrophil-like plasmablasts and mostly classical monocytes in the myeloid compartment. Upregulated genes in myeloid cells were associated with inflammasome signaling (IL18R, IL18RAP), alarmins (S100A9, S100A12, HMGB2), toll-like receptor signaling (TLR2, TLR4, IRAK3) and the complement cascade (C3AR1). We also found an upregulation of genes associated with interferon (IFN) signaling (STAT 1, JAK2, TRIM 22, IFI44L, IFI44, IRF1). We also unexpectedly found an upregulation of immunosuppressive genes (IL1R2, ARG1, and ANNEX). This phenotype of concomitant hyperinflammation and immunosuppressive immune cell dysregulation has been described for severe COVID-19 in previous studies.
We performed an in-silico TF activity analysis of our scRNA dataset to explore potential regulatory mechanisms underlying the dysfunctional myeloid phenotype. TF activity analysis implicated STAT1, STAT2, STAT3, and IRF1 as key upstream regulators of the observed transcriptional phenotype in myeloid cells, suggesting that they are activated by type I IFNs known to be responsible for hyperinflammation in severe COVID-19.
To gain further mechanistic insight, monocytes from patients with and without CVD were isolated and stimulated with SARS-CoV-2 ex-vivo. Unbiased ATAC-sequencing revealed increased chromatin accessibility of Typ I IFN-related genes (IFI44, IFNA1) in monocytes from CVD patients after infection with SARS-CoV-2, confirming our in-silico analysis.
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 in this study have previously been associated with severe COVID-19 and inferior outcomes. In myeloid cells from CVD patients infected with SARS-CoV-2 ex-vivo, we observed an increased gene accessibility of Typ I IFN-related genes, pointing to an increased Type I IFN response as an underlying mechanism.
The enhanced inflammatory response may contribute to the worse outcome of patients with CVD and might be addressed by tailored anti-inflammatory drugs.