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Abstract Background The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. Objectives We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. Methods We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. Results We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. Conclusion These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.

Abstract Background Chikungunya (CHIKV) virus is an important mosquito-borne virus causing outbreaks of acute febrile illness with arthropathy. The detection of specific antibodies against CHIKV is used for diagnosis after the acute viremic phase of the disease. However, a major challenge for serologic diagnosis of CHIKV and other alphaviruses is the cross-reactivity of antibodies to common antigens among these viruses. In the present study, we have developed an enzyme-linked immunosorbend assay using a recombinant envelope protein 2 of CHIKV produced in Escherichia coli system, as a capture antigen. Results High titers (1600 to 12,800) of anti-CHIKV antibodies were detected in human sera analyzed by the CHIKV assay, suggesting it may detect low levels of the antibodies presence. On the other side, cross-reactivity was not observed in mouse hyperimmune sera to Mayaro virus and other alphaviruses analyzed by the CHIKV immunosorbend assay, suggesting it is a CHIKV-specific test. Fifty-nine human serum samples of CHIKV infection suspected cases were tested for immunoglobulin G (IgG) and M (IgM) antibodies detection using the CHIKV immunosorbend assay. A total of 44% (26/59) of samples were positive for IgG to CHIKV, determining 89.66% sensitivity and 100% specificity when the assay is compared to a CHIKV-specific neutralization assay. In addition, 40.6% (24/59) of samples were positive for IgM, determining 92.48% sensitivity and 79.04% specificity by a Bayesian method in the absence of a gold standard. Moreover, CHIKV immunosorbend assay showed similar sensibilities to a commercial immunochromatography assay (Lumiquick, USA) for CHIKV IgG and IgM detection. Conclusion In short, we have developed a rapid, simple, specific and sensitive CHIKV immunosorbend assay for IgG and IgM detection and our results showed potential applicability on the diagnosis of infections by this virus.