Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2–activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1β, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

The hypernociceptive effects of cytokines [TNF-␣, keratinocytederived chemokine (KC), and IL-1␤] and their participation in carrageenan (Cg)-induced inflammatory hypernociception in mice were investigated. Nociceptor sensitization (hypernociception) was quantified with an electronic version of the von Frey filament test in WT and TNF receptor type 1 knockout mice (TNF-R1 ؊/؊ ). TNF-␣-induced hypernociception was abolished in TNF-R1 ؊/؊ mice, partially inhibited by pretreatment with IL-1 receptor antagonist (IL-1ra) or indomethacin and unaffected by Ab against KC (AbKC) or guanethidine. IL-1ra and indomethacin pretreatment strongly inhibited the hypernociception induced by IL-1␤, which was not altered by AbKC or guanethidine or by knocking out TNF-R1. KC-induced hypernociception was abolished by AbKC, inhibited by pretreatment with indomethacin plus guanethidine, and partially inhibited by IL-1ra, indomethacin, or guanethidine. In contrast, KC-induced hypernociception was not altered by knocking out TNF-R1. Cg-induced hypernociception was abolished by administration of indomethacin plus guanethidine, diminished in TNF-R1 ؊/؊ mice, and partially inhibited in WT mice pretreated with AbKC, IL-1ra, indomethacin, or guanethidine. TNF-␣, KC, and IL-1␤ concentrations were elevated in the skin of Cg-injected paws. The TNF-␣ and KC concentrations rose concomitantly and peaked before that of IL-1␤. In mice, the cytokine cascade begins with the release of TNF-␣ (acting on TNF-R1 receptor) and KC, which stimulate the release of IL-1␤. As in rats, the final mediators of this cascade were prostaglandins released by IL-1␤ and sympathetic amines released by KC. These results extend to mice the concept that the release of primary mediators responsible for hypernociception is preceded by a cascade of cytokines.inflammation ͉ prostaglandins ͉ TNF ͉ hyperalgesia ͉ pain C ytokines are produced by various cells types in response to a variety of stimuli and constitute a link between cellular injury or recognition of nonself and the development of local and systemic signs and symptoms of inflammation (1-5). Acute inflammatory pain is characterized by hypernociception due to the sensitization of primary sensory nociceptive neurons, also referred to as hyperalgesia or allodynia (6). After tissue injury, specific primary mediators are released that act on membrane neuronal metabotropic receptors to trigger the activation of second messenger pathways. Eicosanoids and sympathetic amines are the most important primary mediators ultimately responsible for mechanical hypernociception in rats (7-9). These mediators activate second messenger pathways (cAMP, protein kinase A, and PKC) responsible for lowering the nociceptor threshold and increasing neuronal membrane excitability. In this state, nociceptor activation and impulse transmission by the primary nociceptive neurons are facilitated (7,(10)(11)(12)(13)(14)(15)(16)(17).In the last decade, it has been shown that inflammatory stimuli do not directly stimulate the release of primary hypernocic...

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