Background: SARS-CoV-2 infection causes severe pneumonia (COVID-19), but the mechanisms of subsequent respiratory failure and complicating renal and myocardial involvement are poorly understood. In addition, a systemic prothrombotic phenotype has been reported in COVID-19 patients. Methods: A total of 62 subjects were included in our study (n=38 patients with RT-PCR confirmed COVID-19 and n=24 non-COVID-19 controls). We performed histopathological assessment of autopsy cases, surface-marker based phenotyping of neutrophils and platelets, and functional assays for platelet, neutrophil functions as well as coagulation tests. Results: We provide evidence that organ involvement and prothrombotic features in COVID-19 are linked by immunothrombosis. We show that in COVID-19 inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin. COVID-19 patients also present with neutrophil-platelet aggregates and a distinct neutrophil and platelet activation pattern in blood, which changes with disease severity. Whereas cases of intermediate severity show an exhausted platelet and hyporeactive neutrophil phenotype, severely affected COVID-19 patients are characterized by excessive platelet and neutrophil activation compared to healthy controls and non-COVID-19 pneumonia. Dysregulated immunothrombosis in SARS-CoV-2 pneumonia is linked to both ARDS and systemic hypercoagulability. Conclusions: Taken together, our data point to immunothrombotic dysregulation as a key marker of disease severity in COVID-19. Further work is necessary to determine the role of immunothrombosis in COVID-19.

This study uses genetic and pharmacologic approaches to demonstrate novel roles of eosinophils in the progression of atherosclerosis and arterial thrombosis.

Objective Infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) can lead to severe pneumonia, but also thrombotic complications and non‐pulmonary organ failure. Recent studies suggest intravascular neutrophil activation and subsequent immune cell–triggered immunothrombosis as a central pathomechanism linking the heterogenous clinical picture of coronavirus disease 2019 (COVID‐19). We sought to study whether immunothrombosis is a pathognomonic factor in COVID‐19 or a general feature of (viral) pneumonia, as well as to better understand its upstream regulation. Approach and results By comparing histopathological specimens of SARS‐CoV‐2 with influenza‐affected lungs, we show that vascular neutrophil recruitment, NETosis, and subsequent immunothrombosis are typical features of severe COVID‐19, but less prominent in influenza pneumonia. Activated neutrophils were typically found in physical association with monocytes. To explore this further, we combined clinical data of COVID‐19 cases with comprehensive immune cell phenotyping and bronchoalveolar lavage fluid scRNA‐seq data. We show that a HLADR low CD9 low monocyte population expands in severe COVID‐19, which releases neutrophil chemokines in the lungs, and might in turn explain neutrophil expansion and pulmonary recruitment in the late stages of severe COVID‐19. Conclusions Our data underline an innate immune cell axis causing vascular inflammation and immunothrombosis in severe SARS‐CoV‐2 infection.

Neutrophils provide a critical line of defense in immune responses to various pathogens, but also inflict self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients suffering from COVID-19. Here, we utilize state-of-the art mass spectrometry-based proteomics, transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic interleukin-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophildriven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces SARS-CoV-2 spike protein-induced, hACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8-axis as promising therapeutic target in severe SARS-CoV-2 infection.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Vaccines against SARS-CoV-2 are based on a range of novel platforms, with adenovirus-based approaches (like ChAdOx1 nCov-19) being one of them. Recently a novel complication of SARS-CoV-2 targeted adenovirus vaccines has emerged: immune thrombocytopenia (ITP), either isolated, or accompanied by thrombosis (then termed VITT). This complication is characterized by low platelet counts, and in the case of VITT also by platelet-activating platelet factor 4 (PF4) antibodies reminiscent of heparin-induced thrombocytopenia leading to a prothrombotic state with clot formation at unusual anatomic sites. Here, we detected anti-platelet antibodies targeting platelet glycoprotein receptors in 30% of patients with proven VITT (n=27), as well as 42% of patients with isolated thrombocytopenia after ChAdOx1 nCov-19 vaccination (n=26), indicating broad antiplatelet autoimmunity in these clinical entities. We employ in vitro and in vivo models to characterize possible mechanisms of these platelet-targeted autoimmune responses leading to thrombocytopenia. We show that intravenous but not intramuscular injection of ChAdOx1 nCov-19 triggers platelet-adenovirus aggregate formation and platelet activation. After intravenous injection, these aggregates are phagocytosed by macrophages in the spleen and platelet remnants are found in the marginal zone and follicles. This is followed by a pronounced B-cell response with the emergence of circulating antibodies binding to platelets. Our work contributes to the understanding of platelet associated complications after ChAdOx1 nCov-19 administration and highlights accidental intravenous injection as a potential mechanism of platelet targeted autoimmunity. Hence, preventing intravenous injection when administering adenovirus-based vaccines could be a potential measure against platelet associated pathologies following the vaccination.

Pericytes build together with endothelial cells the microvascular vessel wall. They have been mainly implicated in angiogenesis and maintenance of the blood brain barrier, but there is accumulating evidence for an immunological function of pericytes. Occupying a strategic position between the blood stream and the interstitial space, pericytes are able to sense environmental cues from both sides in an organ-specific manner and serve as gatekeepers for innate immune cells. In addition, pericytes are able to interact with leukocytes mediated by adhesion molecules as well as chemokines and are involved from the development of leukocytes in the bone marrow to their migration to injured or infected areas in target tissues. During disease processes pericytes regulate blood flow, vascular permeability, and leukocyte recruitment, but also contribute to maladaptive tissue responses, such as fibrosis making pericytes an attractive therapeutic target.

Vaccines against SARS-CoV-2 are based on a range of novel vaccine platforms, with adenovirus-based approaches (like ChAdOx1 nCov-19) being one of them. Recently a rare and novel complication of SARS-CoV-2 targeted adenovirus vaccines has emerged: thrombosis with thrombocytopenia syndrome (TTS). TTS is characterized by low platelet counts, clot formation at unusual anatomic sites and platelet-activating PF4-polyanion antibodies reminiscent of heparin-induced thrombocytopenia. Here, we employ in vitro and in vivo models to characterize the possible mechanisms of this platelet-targeted autoimmunity. We show that intravenous but not intramuscular injection of ChAdOx1 nCov-19 triggers platelet-adenovirus aggregate formation and platelet activation. After intravenous injection, these aggregates are phagocytosed by macrophages in the spleen and platelet remnants are found in the marginal zone and follicles. This is followed by a pronounced B-cell response with the emergence of circulating antibodies binding to platelets. Our work contributes to the understanding of TTS and highlights accidental intravenous injection as potential mechanism for post-vaccination TTS. Hence, safe intramuscular injection, with aspiration prior to injection, could be a potential preventive measure when administering adenovirus-based vaccines.