SARS-CoV2 ORF3a protein drives inflammation in COVID-19 through NLRP3 inflammasome activation

Introduction: COVID-19 severe pneumonia has been associated to systemic inflammation and elevation of blood parameters and reminiscent of cytokine storm syndrome. Stimulation of PBMC from patients with severe COVID-19 have shown a high secretion of IL-1β, a pivotal cytokine driving inflammatory phenotypes, which maturation and secretion is regulated by NLRP3 inflammasome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients, however the mechanisms by which SARS-CoV2 virus triggers such an extensive inflammation remain unexplained. Objectives: The overall objective of this study was to investigate if SARS-CoV2 drives inflammation in COVID-19 patients through NLRP3 inflammasome activation and IL-1β secretion. Methods: Samples from SARS-CoV2 infected patients, were collected at day 0 and at 3 and 7 following treatment with anakinra. Fresh monocytes, purified through adherence, were cultured for 3, 6, 18 h in the presence or absence of LPS (100 ng/ml) and MCC950 (10μM). Release of IL-1β, IL-1Ra, IL-6, TNF-α, IL-18 was quantified by ELISA kit. Relative gene expression analysis of ORF3a gene was performed by RT-qPCR. THP-1 cells were transfected with a plasmid containing ORF3a sequence by nucleofection. NLRP3 inflammasome and ASC speck formation were detected by confocal microscopy and/or by FACS analysis. Results: In the present study we show that circulating monocytes from COVID-19 patients display ASC specks, index of NLRP3 activation, and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV2 protein, resulted in NLRP3- dependent ASC speck formation. The involvement of ORF3a in inflammasome activation was further supported by the detection by RT-PCR of ORF3a in monocytes from COVID-19 patients. Conclusion: In summary, these results provide a mechanistic explanation for the strong inflammatory manifestations associated to COVID-19 and further evidence that NLRP3 and IL-1β targeting could represent an effective strategy in this disease.

Asymptomatic peripheral neuropathy in patients with SARS-CoV-2 infection

Background and aims: Several neurological complications related to SARS-CoV-2 infection have been reported. The involvement of peripheral nervous system (PNS) consists in the development of immune-mediated neuropathies such as Guillain-Barrè Syndrome. In this study we aim at assessing the presence of asymptomatic abnormalities in peripheral nerves conduction during the acute phase of COVID-19 and, their correlation with blood circulating inflammatory markers. Methods: Thirty-nine patients with COVID-19 were assessed by electroneurographic study of lower limbs and blood tests within one week of hospital admission (T0) and after 30 ± 10 days (T1). Results: Electroneurographic changes were found at least on one nerve at T0 in 12 patients, consisting of axonal or demyelinating changes. Two biological markers were found to be significantly correlated with the presence of neuropathic changes: Reactive Protein C and lymphocyte count. Patients with pathological electrophysiology at T0 showed significant improvement of electrophysiological parameters at T1. The improvements in electroneurographic data were significantly correlated with the trend of laboratory parameters, in particular with fibrinogen, D-Dimer, ferritin, C Reactive protein and lymphocytes. None of the patients with neuropathic changes developed clinical evidence of a full-blown peripheral neuropathy over time. Conclusions: Our study shows that asymptomatic alterations of the PNS can be found during the acute phase of COVID-19. These alterations significantly improve after 20–40 days from the acute phase of infection and that the improvement correlates significantly with the trend of laboratory parameters. Further studies are needed to evaluate possible long-term neurological complications and the predictive value of subclinical damage of PNS in the acute phase of infection.