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.
ABSTRACT
SCIENTIFIC BACKGROUND: Environmental sampling of SARS-CoV-2 is a fundamental tool for evaluating the effectiveness of non-specific prophylaxis measures in counteracting virus spread. The purpose of our work was to evaluate the effectiveness of the different sampling methods in the hospital setting to assess their correlation with the structural, functional, and operational situation of the monitored departments and to define the dynamics of the spread of the virus in indoor environments. METHODS: The monitoring (air bubbling sampling, surface wipe test) was carried out at the San Martino Polyclinic Hospital (Genoa, Italy) in the period since April 2020 to June 2021. The presence of viral RNA in the collected samples was evaluated by qPCR. The infection capacity of the samples collected was also evaluated by an in vitro challenge test on cells sensitive to SARS-CoV-2 infection. RESULTS: The percentage of positivity with respect to the number of tests performed (sensitivity) were air bubbler 50%, wipe test 17%, and challenge test 11%. Only 20% of the samples tested positive in the wipe test and 43% of the samples tested positive in the bubbler sampling were also positive in the challenge test. All the positivity obtained was detected at a distance of less than 2 m and height of less than 1.5 from COVID-19 patients. CONCLUSIONS: Environmental contamination from SARS-CoV-2 detected at the San Martino Polyclinic Hospital is found lower than similar assessments performed in other hospitals both in Italy and abroad. Our study predicted that environmental monitoring of SARS-CoV-2 must be carried out in an integrated way by not using a single sampling method, as each individual test has a different biological significance and performance. However, the virus detected by wipe test only is often a degraded viral fragment and not an intact infecting virion.