Estudo do impacto das vesículas extracelulares liberadas por Paracoccidioides lutzii, após cultivo em condições de privação e suficiência de ferro, na fagocitose por macrófagos in vitro

Abstract

Introduction: Extracellular vesicles (EVs) are membrane-enclosed structures released by cells into the extracellular space and play fundamental roles in biological processes and in the regulation of the immune response. In the context of the fungus Paracoccidioides lutzii, the causative agent of paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Brazil, it was observed that the production and release of EVs are influenced by environmental conditions, such as iron restriction. Iron is essential for the metabolic functions of the fungus, and deprivation of this mineral appears to significantly impact both the production and content of EVs. These vesicles are crucial for the interaction of P. lutzii with the host, including immune cells such as macrophages, where they can modulate the immune response and the pathogenesis of the infection. Objectives: The aim of this study was to analyze the effect of EVs released by P. lutzii, after cultivation of the fungus under iron deprivation and sufficiency conditions, on the phagocytosis of the fungus by macrophages in vitro. Methodology: To investigate this hypothesis, P. lutzii were cultured weekly in Brain Heart Infusion agar and in solid Modified Minimal Medium (MVM), under iron deprivation (supplemented with bathophenanthroline disulfonic acid, BPS) and under iron sufficiency conditions (supplemented with Fe (NH4)2(SO4)2). Then, phagocytosis assays were conducted, where the ability of macrophages to inactivate the fungus (CMIF) and the intracellular proliferation index (IPR) were evaluated. Results: Stimulation with IFNγ increases the inactivation of P. lutzii in macrophages after 6 hours, evidencing its role in microbicidal activation. However, after 24 hours, VEsFe and VEsBPS induced an increase in intracellular fungal proliferation, suggesting a negative immunomodulatory effect. After changing the medium, only IFNγ maintained the significant reduction in fungal growth, while VEs did not alter the interaction with macrophages, indicating a limited effect in modulating the immune response. Conclusion: The results indicate that IFNγ has a fundamental role in the microbicidal activity of macrophages, significantly reducing the proliferation of P. lutzii. In contrast, extracellular vesicles (VEsFe and VEsBPS) present a negative immunomodulatory effect, favoringthe increase in fungal growth at 24 hours, which suggests that they may interfere with the ability of macrophages to control the infection. After changing the culture medium, only IFNγ continued to inhibit fungal growth. These findings highlight the therapeutic potential of IFNγ and the need for further studies on the effects of extracellular vesicles.