Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19

Abstract

Background: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown.

Methods: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19.

Results: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression.

Conclusions: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

Keywords: immunity; inflammation; paralysis; systems biology; thrombosis.

Figure 1.. Circulating mediators at the time of first contact demonstrates an exaggerated immune and vascular responses in patients hospitalized with COVID-19.

Serum proteomic signatures were determined using Olink PEA platform (184 plasma markers) on COVID-19 subjects at admission. A) Heatmap of 44 samples (rows) and 184 protein markers (columns) visually demonstrating clustering of COVID-19 subjects based on COVID-19 severity. Proteomic data were scaled within each mediator. Rows were split by K-means clustering. B) Violin plots display medians (solid lines) and interquartile ranges (boxplots). IL-6 and TF demonstrate an increase with increasing COVID-19 severity. Data were analyzed for statistical significance using Kruskal-Wallis tests with Dunn’s tests for multiple comparisons between all groups. adjusted p-values: *p <0.001; #p=0.002; **p=0.05.

Figure 2.. Principal component analysis of mediators from hospitalized COVID-19 subjects.

A) An unsupervised principal component analysis was performed on mediator levels measured in serum from COVID-19 subjects (n=35) collected at the time of admission. The PCA map is annotated with the peak disease severity of each patient (blue = mild, grey = moderate, red = severe). B) Individual loading values of each mediator for PC1 (Dim1) and PC2 (Dim2).

Figure 3.. Correlation matrices demonstrating significant associations between Inflammatory and vascular markers.

Heatmaps of Pearson correlation matrix coefficients (r) computed between blood proteomic markers (184) determined via Olink proteomic platform from serum samples at the time of admission. A) The correlation matrix from all COVID-19 subjects (n=35) and all proteomic markers and correlation matrices of proteomic markers from Mild and Severe COVID-19 subjects were analyzed independently. B) Because of the heterogeneity in COVID-19 subjects, the subsetting cohort reveals novel, significant interactions between TF and other circulating markers that were masked when all proteomic data is aggregated (significance set at 0.1 for what is displayed on the correlation matrix; marker 1 [not displayed in panel B table] is TF).

Figure 4.. CD14+ monocytes from Severe COVID-19 subjects have increased expression of TF.

PBMC from COVID-19 subjects were isolated and samples were barcoded and stained (24 surface markers), and mass cytometry was performed. A) Representative PBMC antibody surface-staining results and gating strategy for mass cytometry analysis. B) Expression matrix derived from %positive activation markers based on immune cell identities. C) Representative viSNE plot of PBMC from Severe COVID-19 and age-matched control subjects demonstrating increased TF expression on monocyte subset in severe COVID-19 subjects. Circles represent CD14+ monocytes. D) Biaxial plot of CD14 vs CD142 expression and quantification of CD142 percent positive monocytes out of total CD14+ monocytes demonstrating an increase in TF Severe COVID-19 subjects. * p-value = 0.02.

Figure 5.. Immune “stress” tests reveal immune paralysis in Severe COVID-19 patients.

Bulk transcriptomics (using RNA-seq) and bioinformatic analysis of circulating PBMCs from Severe COVID-19 (n=3) and age-matched control subjects (n=3). A) Biological processes analysis of differentially regulated biological pathways between severe COVID-19 and age-matched control subjects demonstrating differential expression of apoptosis and immune response processes. B) Venn diagram of differentially expressed genes between stimulated PBMC from Severe COVID-19 and control subjects; all contrasts are in relation to non-stimulated PBMC from control subjects demonstrating minimal TLR transcriptional induction in Severe COVID-19 subjects. C) Transcriptomic analysis of key immune response genes demonstrates evidence of immune paralysis. D). ELISA of inflammatory cytokines in supernatants of stimulated PBMC also suggests severe blunting of PBMC inflammatory cytokine release in response to TLR activation. Adjusted p-values: *p=0.01; **p=0.002; #p=0.03; ##p=0.002.

Figure 6.. Sera from Severe COVID-19 patients induce CD142/TF expression in human monocytes.

THP1 – Dual Monocytes were treated with sera from Mild and Severe COVID-19 patients at a 1:5 concentration for 4 hours. Cells were then stained with anti-human CD142-PE antibody and analyzed via flow cytometry for CD142/TF expression. (A) CD142/TF mean fluorescence intensity for control, Mild, and Severe sera-treated groups. (B) Quantification of CD142 percent positive monocytes demonstrating an increase in TF in Severe COVID-19 patients. No treatment TF control set to 5% positive. *P < 0.001 (ANOVA). **P = 0.006 (ANOVA).