Severity of SARS-CoV-2 infection is associated with high numbers of alveolar mast cells and their degranulation.

Background: The systemic inflammatory response post-SARS-CoV-2 infection increases pro-inflammatory cytokine production, multi-organ damage, and mortality rates. Mast cells (MC) modulate thrombo-inflammatory disease progression (e.g., deep vein thrombosis) and the inflammatory response post-infection. Objective: To enhance our understanding of the contribution of MC and their proteases in SARS-CoV-2 infection and the pathogenesis of the disease, which might help to identify novel therapeutic targets. Methods: MC proteases chymase (CMA1), carboxypeptidase A3 (CPA3), and tryptase beta 2 (TPSB2), as well as cytokine levels, were measured in the serum of 60 patients with SARS-CoV-2 infection (30 moderate and 30 severe; severity of the disease assessed by chest CT) and 17 healthy controls by ELISA. MC number and degranulation were quantified by immunofluorescent staining for tryptase in lung autopsies of patients deceased from either SARS-CoV-2 infection or unrelated reasons (control). Immortalized human FcεR1+c-Kit+ LUVA MC were infected with SARS-CoV-2, or treated with its viral proteins, to assess direct MC activation by flow cytometry. Results: The levels of all three proteases were increased in the serum of patients with COVID-19, and strongly correlated with clinical severity. The density of degranulated MC in COVID-19 lung autopsies was increased compared to control lungs. The total number of released granules and the number of granules per each MC were elevated and positively correlated with von Willebrand factor levels in the lung. SARS-CoV-2 or its viral proteins spike and nucleocapsid did not induce activation or degranulation of LUVA MC in vitro. Conclusion: In this study, we demonstrate that SARS-CoV-2 is strongly associated with activation of MC, which likely occurs indirectly, driven by the inflammatory response. The results suggest that plasma MC protease levels could predict the disease course, and that severe COVID-19 patients might benefit from including MC-stabilizing drugs in the treatment scheme.

Severity of SARS-CoV-2 infection is associated with high numbers of alveolar mast cells and their degranulation

Background The systemic inflammatory response post-SARS-CoV-2 infection increases pro-inflammatory cytokine production, multi-organ damage, and mortality rates. Mast cells (MC) modulate thrombo-inflammatory disease progression (e.g., deep vein thrombosis) and the inflammatory response post-infection. Objective To enhance our understanding of the contribution of MC and their proteases in SARS-CoV-2 infection and the pathogenesis of the disease, which might help to identify novel therapeutic targets. Methods MC proteases chymase (CMA1), carboxypeptidase A3 (CPA3), and tryptase beta 2 (TPSB2), as well as cytokine levels, were measured in the serum of 60 patients with SARS-CoV-2 infection (30 moderate and 30 severe;severity of the disease assessed by chest CT) and 17 healthy controls by ELISA. MC number and degranulation were quantified by immunofluorescent staining for tryptase in lung autopsies of patients deceased from either SARS-CoV-2 infection or unrelated reasons (control). Immortalized human FcεR1+c-Kit+ LUVA MC were infected with SARS-CoV-2, or treated with its viral proteins, to assess direct MC activation by flow cytometry. Results The levels of all three proteases were increased in the serum of patients with COVID-19, and strongly correlated with clinical severity. The density of degranulated MC in COVID-19 lung autopsies was increased compared to control lungs. The total number of released granules and the number of granules per each MC were elevated and positively correlated with von Willebrand factor levels in the lung. SARS-CoV-2 or its viral proteins spike and nucleocapsid did not induce activation or degranulation of LUVA MC in vitro. Conclusion In this study, we demonstrate that SARS-CoV-2 is strongly associated with activation of MC, which likely occurs indirectly, driven by the inflammatory response. The results suggest that plasma MC protease levels could predict the disease course, and that severe COVID-19 patients might benefit from including MC-stabilizing drugs in the treatment scheme.

Preferential uptake of SARS-CoV-2 by pericytes potentiates vascular damage and permeability in an organoid model of the microvasculature.

AIMS: Thrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection, however the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity. METHODS AND RESULTS: Immunofluorescence of post-mortem patient sections were used to assess pathophysiological aspects of COVID19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor ACE2 on pericytes when compared to vascular endothelium, and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability including NOTCH3, angiopoietin-2 and TEK. Activation of vascular organoids with IL-1ß did not have an additive effect on vascular permeability. Spike antigen was detected in some patients' lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and VWF deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin. CONCLUSIONS: Together our data indicates that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment result in pericyte infection, detachment, damage and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation. TRANSLATIONAL PERSPECTIVE: Endotheliitis is a serious complication of severe COVID-19 patients which remains poorly understood. We identify a pericyte mediated mechanism by which the vasculature becomes compromised, contributing to thrombotic complications, highlighting important avenues for the development of therapies.