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.

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.