In vitro inflamassome activation by SARS-CoV-2 variants in infected monocytes and macrophages.

• Grant number: 24/04121-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2024
• End date: December 31, 2024
• Field of knowledge: Biological Sciences - Microbiology
• Principal Investigator: Eurico de Arruda Neto
• Grantee: Leticia Maria Ribeiro Bassi
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 19/26119-0 - Emerging and re-emerging viruses: biology, pathogenesis and prospection, AP.TEM

Abstract

SARS-CoV-2 is the virus responsible for the COVID-19 disease, a severe acute respiratory syndrome. One key aspect in understanding SARS-CoV-2 infection is the activation of the NLRP3 inflammasome. Our study aims to assess this activation in response to infection by clinically relevant variants of SARS-CoV-2 (Wuhan, Gamma, Delta, and Omicron) in THP1 monocytic lineage cells. To do so, we will differentiate THP1 cells into macrophages in vitro using 200nM PMA for 48 hours and validate the process through the labeling of macrophage-specific antibodies such as CD68 using immunofluorescence. Infections will be conducted in both differentiated and undifferentiated THP-1 cells with the different virus strains, with a Multiplicity of Infection (MOI) of 1. The resulting infection will be analyzed by absolute quantification of the viral genome present in cell pellets using qPCR after 24 and 48 hours. Furthermore, we will assess in vitro infection by labeling viral proteins NP (nucleoprotein) and NSP-16 (non-structural protein 16) using monoclonal antibodies through confocal immunofluorescence. Subsequently, we will quantify NLRP3 expression and a panel of pro-inflammatory cytokines to understand the relationship between viral replication, NLRP3 inflammasome activation, and the expression of relevant inflammatory cytokines during infection with SARS-CoV-2 variants. This study is crucial for understanding how the immune system responds to SARS-CoV-2 infection across different variants. In summary, the results of this study have the potential to enhance our understanding of inflammation during COVID-19 and drive the search for more effective therapies.