Platelet-monocyte interaction amplifies thromboinflammation through tissue factor signaling in COVID-19.

Accumulating evidence into the pathogenesis of COVID-19 highlights a hypercoagulability state with high risk of life-threatening thromboembolic complications. However, the mechanisms of hypercoagulability and their link to hyperinflammation remain poorly understood. Here, we investigate functions and mechanisms of platelet activation and platelet-monocyte interactions in inflammatory amplification during SARS-CoV-2 infection. We used a combination of immunophenotyping, single-cell analysis, functional assays, and pharmacological approaches to gain insights on mechanisms. Critically ill patients with COVID-19 exhibited increased platelet-monocyte aggregates formation. We identified a subset of inflammatory monocytes presenting high CD16 and low HLA-DR expression as the subset mainly interacting with platelets during severe COVID-19. Single-cell RNA-sequencing analysis indicated enhanced fibrinogen receptor Mac-1 in monocytes from patients with severe COVID-19. Monocytes from patients with severe COVID-19 displayed increased platelet binding and hyperresponsiveness to P-selectin and fibrinogen with respect to tumor necrosis factor-α and interleukin-1ß secretion. Platelets were able to orchestrate monocyte responses driving tissue factor (TF) expression, inflammatory activation, and inflammatory cytokines secretion in SARS-CoV-2 infection. Platelet-monocyte interactions ex vivo and in SARS-CoV-2 infection model in vitro reciprocally activated monocytes and platelets, inducing the heightened secretion of a wide panel of inflammatory mediators. We identified platelet adhesion as a primary signaling mechanism inducing mediator secretion and TF expression, whereas TF signaling played major roles in amplifying inflammation by inducing proinflammatory cytokines, especially tumor necrosis factor-α and interleukin-1ß. Our data identify platelet-induced TF expression and activity at the crossroad of coagulation and inflammation in severe COVID-19.

VIP plasma levels associate with survival in severe COVID-19 patients, correlating with protective effects in SARS-CoV-2-infected cells.

Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.

Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes.

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/ß3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.