Importance of the inflammasome in the development of forms severe cases of COVID-19 in patients and in an experimental model murine

Author(s)

Tamara Silva Rodrigues

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-12-19

Examining board members

Dario Simões Zamboni; Patricia Torres Bozza Viola

• Advisor: Dario Simões Zamboni

Abstract

Introduction: Severe cases of COVID-19 are characterized by a strong inflammatory process that can lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation through the cleavage and activation of key inflammatory molecules, including active caspase-1 (Casp1p20), IL-1β and IL-18. Activation of the NLRP3 inflammasome can occur via canonical or non-canonical pathways, when human caspase-4/CASP4 (mouse caspase-11/CASP11) is involved. Aim: The aim of this work was to investigate the activation of the inflammasome and its participation in the pathophysiology of COVID-19. Methods and Results: Initially, we infected primary human monocytes in vitro with SARS-CoV-2 and evaluated inflammasome activation. We observed that SARS-CoV-2 infection triggers caspase-1 activation, assessed through the luminescence assay, Caspase-Glo 1, and IL-1β production, quantified by ELISA. We also observed that SARS-CoV-2 triggers LDH release in monocytes and requires the presence of viable SARS-CoV-2. We then measured NLRP3 and ASC \"puncta\" formation and identified that only the viable SARS-CoV-2 triggers puncta formation. Subsequently, in vivo infections performed in transgenic K18-hACE2 humanized mice, CASP11 deficient or not, indicated that hACE2 Casp11-/- mice were protected from the development of the disease, with reduced body weight loss, less temperature variation, diminished lung parenchymal area, lower clinical disease score, and reduced mortality. Through the FAM-YVAD assay, which allows the labeling of active intracellular caspase-1, we evidenced a higher percentage of FAM-YVAD+ PBMCs in patients on day zero of admission compared to healthy controls. Furthermore, microscopic observation of these cells allowed the visualization of puncta of NLRP3 and ASC in PBMCs, indicating that there are active inflammasomes in cells from patients with COVID-19. Studying moderate and severe COVID-19 patients, we performed immunohistochemistry and immunofluorescence assays and found active NLRP3 inflammasome in PBMCs and in postmortem autopsies of fatal patients. Of note, inflammasome-derived products such as Casp1p20 and IL-18 present in patient sera correlated with markers of COVID- 19 severity, including IL-6 and LDH. Higher levels of IL-18 and Casp1p20 were associated with disease severity and worse clinical outcome. Evaluating samples from lethal COVID-19 cases, we also evidenced increased CASP4 gene expression by RT-PCR in nasopharyngeal swabs and lungs of patients, and this result correlated with the expression of inflammasome components and inflammatory mediators, including caspase-1 and IL-1β. Through western blotting assay, we also observed CASP4 activation in PBMCs from patients with moderate and severe COVID-19. Conclusion: We observed that SARS-CoV-2 induces the upregulation and activation of CASP4/CASP11 and this process contributes to inflammasome activation in response to SARS-CoV-2. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms can be a marker of disease severity and a potential therapeutic target for COVID-19. (AU)

• FAPESP's process: 19/21978-5 - Role of STING signaling pathway in recognizing and controling Leishmania spp infection
• Grantee: Tamara Silva Rodrigues
• Support Opportunities: Scholarships in Brazil - Doctorate