Blunted sFasL signalling exacerbates TNF‐driven neutrophil necroptosis in critically ill COVID‐19 patients

Abstract

Objectives. Critically ill coronavirus disease 2019 patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. Methods. Regulated cell death phenotype of neutrophils isolated from critically ill COVID-19 patients or healthy donors and stimulated with COVID-19 or healthy plasma ex vivo was assessed by flow cytometry, time-lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID-19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID-19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID-19 patients. Results. COVID-19 plasma induced a necroptosis-sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damageassociated molecular patterns (DAMPs), increased receptorinteracting serine/threonine-protein kinase (RIPK) 1 levels and mixed lineage kinase domain-like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID-19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF-a axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID-19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF-driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. Conclusion. Our results suggest a novel role for sFasL signalling in the TNF-a-induced RCD programme in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

Hyperinflammatory environment drives dysfunctional myeloid cell effector response to bacterial challenge in COVID-19

“…A recent single-cell transcriptomic study proposed that premature neutrophils in severe COVID-19 might be programmed towards an anti-inflammatory state or even exert suppressive functions, similar to myeloid-derived suppressor cells (MDSCs) [20]. Furthermore, we recently demonstrated that a subpopulation of neutrophils in critically ill COVID-19 patients undergoes TNF-α-driven necroptosis [54], with concomitant release of various damage associated molecular patterns and alarmins, such as S100A8/A9, which are known to contribute to pathological damage [22,55,56].…”

Intervertebral disc cell chondroptosis elicits neutrophil response inStaphylococcus aureusspondylodiscitis

“…Next, isolated cells were filtered through strainer cap tubes (Falcon) and remaining red blood cells were subsequently lysed with water. The cells were stained with Annexin V-FITC/7AAD as described previously (57). Cells were acquired on an Attune NxT (Thermofisher).…”

“…Cell culture supernatants were filtered through a 0.22 μm filter and cytokine levels were analyzed on a Luminex™ MAGPIX™ (Thermofisher) as previously described (57,59). Analysis was performed using the xPONENT® software.…”

“…Neutrophils from healthy donors were isolated with the EasySep™ Direct Human Neutrophil Isolation Kit (StemCell Technologies) as previously described (57,59). Neutrophils were resuspended in DMEM/F12 and counted on an Attune NxT.…”

“…The Caspase-Glo® 3/7, Caspase-Glo® 8 and Caspase-Glo® 9 (all Promega) kits were used as previously described (57). The Caspase-1 activity assay kit (Novus Biologicals) and the mitochondrial ToxGlo TM (Promega) assay were used accordingly to the manufacturer's instruction.…”