Conflict of interest: JDL is the founder of Amyndas Pharmaceuticals, which develops complement inhibitors for therapeutic purposes; he has a broad portfolio of patents describing the use of complement inhibitors for therapeutic purposes (www.lambris.com/ patents), some of which are developed by Amyndas (US patents 8946145/9371365, 9630992) and Apellis (US patents 6319897, 7989589, 7888323). JDL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (i.e., 4(1MeW)7W/ POT-4/APL-1 and PEGylated derivatives such as pegcetacoplan and APL-9).

Emerging data indicate that complement and neutrophils are involved in the maladaptive host immune response that fuels hyper-inflammation and thrombotic microangiopathy increasing the mortality rate in coronavirus disease 2019 (COVID-19). Here, we investigated the interaction between complement and the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 clinical samples, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, TF activity and sC5b-9 were detected in patients. Neutrophils yielded high tissue factor (TF) expression and released NETs carrying functionally active TF. Confirming our ex vivo findings, treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. Serum isolated from COVID-19 patients induces complement activation in vitro, which is consistent with high complement activity in clinical samples. Complement inhibition at the level of C3 with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis that reveals the pivotal role of complement and NETs in COVID-19 immmunothrombosis. This study supports emerging strategies against SARS-CoV-2 infection that exploit complement therapeutics or NETosis inhibition.

Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis, leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies, and surgical specimens of abdominal aortic aneurysms (AAAs) from patients with EH were used. Cell-based assays, NETs/human aortic endothelial cell cocultures, and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly diagnosed patients with EH. Stimulation of control neutrophils with plasma from patients with untreated EH generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via an ROS/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced substantially in patients with EH starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAAs. These data reveal the important pathogenic role of an Ang II/ROS/NET/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.

Ankylosing spondylitis (AS) is an inflammatory disease characterized by excessive bone formation. We investigated the presence of neutrophil extracellular traps (NETs) in AS and how they are involved in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin‐17A (IL‐17A). Peripheral neutrophils and sera were obtained from patients with active AS and healthy controls. NET formation and neutrophil/NET‐associated proteins were studied using immunofluorescence, immunoblotting, qPCR, and ELISA. In vitro co‐culture systems of AS NET structures and MSCs isolated from controls were deployed to examine the role of NETs in the differentiation of MSCs toward osteogenic cells. Analysis was performed using specific staining and qPCR. Neutrophils from patients with AS were characterized by enhanced formation of NETs carrying bioactive IL‐17A and IL‐1β. IL‐17A‐enriched AS NETs mediated the differentiation of MSCs toward bone‐forming cells. The neutrophil expression of IL‐17A was positively regulated by IL‐1β. Blocking IL‐1β signaling on neutrophils with anakinra or dismantling NETs using DNase‐I disrupted osteogenesis driven by IL‐17A‐bearing NETs. These findings propose a novel role of neutrophils in AS‐related inflammation, linking IL‐17A‐decorated NETs with the differentiation of MSCs toward bone‐forming cells. Moreover, IL‐1β triggers the expression of IL‐17A on NETs offering an additional therapeutic target in AS.