Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. METHODS: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. RESULTS: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. CONCLUSIONS: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

Cell specific pro- and antiinflammatory lipidmediator shift in critically ill Covid19 patients

Introduction The COVID-19 pandemic had a significant impact on morbidity and mortality in Germany challenging intensive care unit (ICU) capacities across the country. To delineate the high variability in disease severity, clinical presentation and outcome, we focused on cellular regulators of inflammation and resolution on a single cell level to gain a deeper understanding of the patient's individual inflammatory response and their impact on survival. Methods Written informed consent was obtained from all patients and healthy controls. The study was approved by the local ethical review board (Az249/20 S-EB). To characterize the peripheral immune landscape, we performed a 14 parameter flow cytometric analysis of PBMCs of 32 critically ill CoV2 patients and a targeted HPLC-MS/MS of previously sorted PBMCs. All data was analyzed and correlated to clinical parameters and patients' outcomes (Fig. 4). Results As known [1], computational analysis of flow cytometry revealed a strong decrease of B Cell and CD8+ T Cell ratios and an increase of monocytes in critically ill CoV19 patients compared to control (Fig. 1A). Interestingly, non-survivors displayed an increased ratio of CD16+ monocytes and proinflammatory IL- 1beta in monocytes, B and T cells, while HLADR receptors were downregulated correlating with clinical outcome (Fig. 1B). Not unexpectedly, we saw a major increase in proinflammatory lipidmediators, such as PGJ2, PGF2, TxB2 (Fig. 1C). Additionally, our analysis revealed that not only the amount, but also the source of those mediators was shifted from CD16 to classical CD14 monocytes, even more pronounced in non-survivors. CD16 monocytes of CoV2 patients, however, lost the ability to generate proresolving lipidmediators depending on cytochrome p450 (Cyp450) or soluble epoxide hydrolase (sEH) TxB2 (Fig. 1D). Conclusions Even though a lot of insight into CoV2 has been gained over the last 2 years, relatively little is known about the impact of immune changes in critically ill patients. With this study, we are the first to attribute lipid mediators to specific cell types. Our findings show that TxB2 in critically ill CoV2 patients, which correlates with mortality in CoV2 [2], is produced mainly in CD14 monocytes. We further report that specifically non-survivors display increased ratios of non-classical CD16 monocytes, which are impaired to generate a major class of lipidmediators depending on Cyp450. In conclusion, these data provide evidence that not only the absolute amount of pro- and anti-inflammatory mediators, but also the cellular source of these mediators remains key to fully understand their role in critically ill CoV2 patients. (Figure Presented).

First Experience Addressing the Prognostic Utility of Novel Urinary Biomarkers in Patients With COVID-19.

Urine 11-dehydro-thromboxane B2 (u11-dh-TxB2), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and liver-type fatty acid binding protein levels (L-FABP) at the time of hospitalization were higher in coronavirus disease 2019 (COVID-19) patients with adverse events vs without events. Higher u11-dh-TxB2 and L-FABP levels were associated with longer hospitalization, more thrombotic events, and greater mortality, providing evidence for potential utility as early prognostic biomarkers for COVID-19.