Caracterização da etose de eosinófilos em doenças eosinofílicas e em resposta à infecção por SARS-CoV-2

Abstract

Eosinophils are multifunctional leukocytes of the innate immune system traditionally associated with defense against parasitic infections and the pathogenesis of allergic diseases. However, the functional complexity of these cells extends to numerous inflammatory conditions, in which these cells infiltrate tissues and release products stored in their granules through various secretory mechanisms. One such mechanism is ETosis, a form of cytolytic cell death resulting from eosinophil activation, which leads to the release of DNA traps and secretory granules into the extracellular environment. Eosinophil ETosis (EETosis) has been increasingly recognized in eosinophil-associated diseases, but its early identification and in vivo dynamics are still poorly understood. Furthermore, there is limited knowledge about the occurrence of this process in viral infections like COVID-19, a disease caused by the SARS-CoV-2 coronavirus. The variation in eosinophil counts during this illness has attracted significant attention. This thesis aimed to evaluate the recruitment and activation of eosinophils, with a focus on ETosis, during human eosinophilic diseases and in SARSCoV-2 infection. To achieve this, we used Transmission Electron Microscopy (TEM) to identify the morphological changes undergone by eosinophils in biopsies from patients with eosinophilic diseases (rhinosinusitis, ulcerative colitis, hypereosinophilic syndrome, dermatitis, and Schistosoma mansoni infection). Our results confirmed that ETosis is a dynamic process of cell death that frequently occurs in vivo. By analyzing extensive areas of inflamed tissue (66,000μm²), we identified for the first time the ultrastructural signature of EETosis in situ, characterizing the entire sequence of early and late morphological changes of this process. Furthermore, quantitative TEM and electron tomography analyses to investigate the number, density, integrity, and threedimensional structure of eosinophil sombrero vesicles (EoSVs) demonstrated that EETosis leads to the release of these vesicles into the extracellular environment. EoSVs, until now associated only with the intracellular transport of products derived from eosinophil granules, are deposited in the extracellular matrix, where they remain intact after cell death, potentially acting as a cell-specific population of extracellular vesicles. The ability of human eosinophils to release DNA traps in response to SARSCoV-2 was also investigated in an in vitro model using the inactivated virus. We demonstrated that eosinophils respond rapidly (1h) to the viral stimulus by releasing DNA traps. Finally, to evaluate the clinical relevance of eosinophils, we conducted a retrospective study in a cohort of hospitalized COVID-19 patients, examining the dynamics of blood eosinophil counts and their association with disease outcome. The analysis revealed that an increase in the eosinophil count during hospitalization is associated with higher chances of patient survival. Together, our findings identify ETosis as a central mechanism in the pathogenesis of eosinophilic inflammation and, possibly, in the response to viral infections. We identified the characteristics of EETosis in vivo and reinforce the secretory nature of ETosis as a process of extracellular release of intact EoSVs, with implications for the propagation of eosinophil responses after cell death. Our data also indicate EETosis as a mechanism associated with the pathogenesis of COVID-19. The correlation between the increase in eosinophils and patient recovery reinforces their importance as a prognostic biomarker