Mecanismos de secreção de eosinófilos: compartimentalização e tráfego vesicular de sintaxina-17, CD63 e interferon-gama

Abstract

Eosinophils are able to release numerous mediators that are pre-synthesized and stored within their cytoplasmic specific (secretory) granules. Secretion can occur by: (i) piecemeal degranulation (PMD), characterized by vesicle-mediated transport of products from granules to cell surface, involving mainly large carriers termed Eosinophil Sombrero Vesicles (EoSVs); (ii) classical exocytosis, characterized by fusion of individual granules with the plasma membrane; (iii) compound exocytosis, characterized by large channels formed by granule–granule fusions before granule content release and (iv) cytolysis, characterized by release of intact granules after cell lysis. Considering that the functional activity of eosinophils is based on their secretory capacity, the study of the molecules involved in eosinophil secretion, as well as their intracellular trafficking is important to understand eosinophil responses during allergic and inflammatory diseases. CD63, a tetraspanin family member, has been associated to eosinophil granules and secretion. Syntaxin 17 (STX17), a SNARE family molecule, is involved with vesicle fusion during constitutive secretion and potentially in cargo transport within specialized cells. High levels of interferongamma (IFN-γ), an important inflammatory cytokine, are constitutively expressed in human eosinophils. However, the intracellular trafficking of these molecules and their association with eosinophil secretory mechanisms remain to be established. In this work, we investigated the ultrastructural localization and the intracellular trafficking of CD63, STX17 and IFN-γ in human eosinophils stimulated or not with inflammatory mediators (CC-chemokine ligand 11- CCL11 - e tumor necrosis fator alpha -TNF-α-). Moreover, we investigated if human eosinophils can secrete extracellular vesicles (EVs), that might be involved in inflammatory responses. Pre-embedding immunonanogold and conventional transmission electron microscopy (TEM), flow cytometer and western blotting techniques were used to investigate these questions. First, our results showed that STX17 is localized on granules and EoSVs, important structures involved in eosinophil secretory mechanisms. The data also demonstrated that CD63 is strongly associated with eosinophil secretory processes (piecemeal degranulation and compound exocytosis) and EoSVs are translocating CD63 to/from granules. Results demonstrated that IFN-γ is localized on secretory granules and can be translocated to cell periphery by EoSVs after eosinophil stimulation. In addition, we demonstrated that human eosinophils can produce microvesicles and the number of these extracellular vesicles increased after stimulation with CCL11 or TNF-a. Altogether, our results shed light to eosinophil secretory mechanisms and their associated molecules, leading to new insights to understand how eosinophil respond to inflammatory conditions through vesicular trafficking of their granule contents secretion.