SARS-CoV-2 derived peptides potentiate proinflammatory and pro-coagulant responses to the TLR3 ligand poly(I:C) inhuman endothelial cells and mice

Background : COVID-19 disease, caused by SARS-CoV-2 infection, is associated with cytokine storm and thrombosis. The doublestranded RNA (dsRNA) mimetic poly(I:C) induces cytokines and Tissue Factor (TF) expression in endothelial cells. We demonstrated that host-encoded antimicrobial peptides (AMP) can organize dsRNA into pro-inflammatory nanocrystals with optimal spacing to stimulate toll-like receptor-mediated immune responses. Aims : We determined if pro-inflammatory sequences in the SARSCoV-2 proteome enhanced host inflammatory and pro-coagulant responses to poly(I:C) in-vitro and in-vivo . Methods : We identified pro-inflammatory sequences from the SARS-CoV-2 proteome using a machine learning-based classifier trained on innate immune peptide sequences. We identified multiple peptide sequences encoded by ORF1ab, ORF3, S, and N genes. We tested these peptides for their ability to enhance poly(I:C) induction of TF activity in Human umbilical vein endothelial cells (HUVECs) by stimulating with poly(I:C) (5 μg/ml) and/or the peptides. TF activity in cell lysates was determined using two-stage FXa generation assay 6 h after stimulation. Peptide 6 (2.2 μM), derived from ORF1ab showed the highest potency. Mice were administered poly(I:C) (100 μg) or poly(I:C) and peptide 6 (344 μg) intravenously. Cytokine levels in HUVEC supernatants (IL-8) or mouse plasma (IL-6 and CXCL1) and plasma Thrombin-antithrombin complex (TAT) levels were analyzed by ELISA. Results : Co-stimulation of HUVECs with poly(I:C) and peptide 6 significantly increased IL-8 release ( P < 0.0001) and TF activity ( P < 0.0001) compared to poly(I:C) alone. Mice co-treated with poly(I:C)/peptide 6 had significantly increased plasma levels of CXCL1 (mouse homolog of IL-8) ( P = 0.005) and IL-6 ( P = 0.015) at 2 h compared to poly(I:C)-treated control mice. We also observed an increase in TAT complexes, a marker of activation of coagulation in mice receiving poly(I:C)/peptide 6 compared to poly(I:C) alone, although this increase did not reach statistical significance. Conclusions : Our data demonstrate that SARS-CoV-2 derived AMPlike peptides, such as peptide 6 (encoded in ORF1ab), enhance proinflammatory and pro-coagulant responses to dsRNA in human endothelial cells and inflammation in mice.