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SARS-CoV2 ORF3a protein drives inflammation in COVID-19 through NLRP3 inflammasome activation
Pediatric Rheumatology ; 20(SUPPL 1), 2022.
Article in English | EMBASE | ID: covidwho-1677513
ABSTRACT

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.
Keywords

Full text: Available Collection: Databases of international organizations Database: EMBASE Language: English Journal: Pediatric Rheumatology Year: 2022 Document Type: Article

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Full text: Available Collection: Databases of international organizations Database: EMBASE Language: English Journal: Pediatric Rheumatology Year: 2022 Document Type: Article