Stress Conditions Affect the Immunomodulatory Potential of Candida albicans Extracellular Vesicles and Their Impact on Cytokine Release by THP-1 Human Macrophages

Abstract

Human immune cells possess the ability to react complexly and effectively after contact with microbial virulence factors, including those transported in cell-derived structures of nanometer sizes termed extracellular vesicles (EVs). EVs are produced by organisms of all kingdoms, including fungi pathogenic to humans. In this work, the immunomodulatory properties of EVs produced under oxidative stress conditions or at host concentrations of CO2 by the fungal pathogen Candida albicans were investigated. The interaction of EVs with human pro-monocytes of the U-937 cell line was established, and the most notable effect was attributed to oxidative stress-related EVs. The immunomodulatory potential of tested EVs against human THP-1 macrophages was verified using cytotoxicity assay, ROS-production assay, and the measurement of cytokine production. All fungal EVs tested did not show a significant cytotoxic effect on THP-1 cells, although a slight pro-oxidative impact was indicated for EVs released by C. albicans cells grown under oxidative stress. Furthermore, for all tested types of EVs, the pro-inflammatory properties related to increased IL-8 and TNF-α production and decreased IL-10 secretion were demonstrated, with the most significant effect observed for EVs released under oxidative stress conditions.

Extracellular vesicle production: A bidirectional effect in the interplay between host and Candida fungi

“…For TNF-α, a proinflammatory cytokine, an increase in its level was shown for fEVs released under all tested conditions compared to untreated cells, although the greatest change was observed after stimulation with fEVs produced by fungi cultured under oxidative stress condition. As an indication of the rather proinflammatory properties of tested fEVs, generated under host-derived conditions, the production of IL-10 was also verified ( Kulig et al, 2023 ), and for all three types of fEVs the level of IL-10 was lower than in untreated cells. Furthermore, macrophage-like cells responded by producing a greater amount of IL-8 after contact with fEVs released under stress and control conditions, which was the most pronounced for fEVs produced under oxidative stress conditions ( Kulig et al, 2023 ), which could be explained by altered lipid composition of fEVs observed in other studies for fEVs produced by C. albicans strain ATCC 64,548 also in the presence of menadione ( Trentin et al, 2023 ).…”

“…For this purpose, conditions that mimic the natural host environment have been introduced for Candida growth, represented by oxidative stress, nutrient- or atmosphere-limited conditions, or host environment during infection, including the presence of antifungal drugs. In our previous work, the proinflammatory effect of fEVs released by yeast-like cells of C. albicans strain 10,231 under oxidative stress in the presence of menadione, host carbon dioxide concentration (5 %), or control conditions was demonstrated ( Kulig et al, 2023 ). The macrophage-like cells differentiated from THP-1 cells produced IL-1β, but no significant differences were observed for three types of fEVs tested.…”

“…For yeast-like cells of C. albicans strain SC5314, LDH release by immune cells was measured after exposure to fEVs and there were no significant changes compared to the control cells ( Martínez-López et al, 2022 ). For fEVs released by C. albicans strain 10,231 grown under stress conditions, due to oxidative stress or host concentration of carbon dioxide, their cytotoxicity was measured by the XTT assay, and no cytotoxic effect on THP-1 cells was demonstrated ( Kulig et al, 2023 ). Moreover, in the same work the internalization of fEVs ‒ both control and released under stress conditions ‒ by THP-1 cells was demonstrated with the use of vesicles labeled with tetramethylrhodamine-conjugated concanavalin A, being a lectin specific for mannose present in fungal glycoproteins ( Kulig et al, 2023 ).…”

“…For fEVs released by C. albicans strain 10,231 grown under stress conditions, due to oxidative stress or host concentration of carbon dioxide, their cytotoxicity was measured by the XTT assay, and no cytotoxic effect on THP-1 cells was demonstrated ( Kulig et al, 2023 ). Moreover, in the same work the internalization of fEVs ‒ both control and released under stress conditions ‒ by THP-1 cells was demonstrated with the use of vesicles labeled with tetramethylrhodamine-conjugated concanavalin A, being a lectin specific for mannose present in fungal glycoproteins ( Kulig et al, 2023 ). The use of this type of staining was possible due to previously confirmed presence of mannoproteins on the surface of fEVs produced by different Candida species ( Karkowska-Kuleta et al, 2020a ).…”

Host innate immune systems gather intel on invading microbes via pathogen-derived extracellular vesicles

“…For example, Candida albicans EVs induce pro-inflammatory cytokine release from murine macrophages and dendritic cells, such as IL-12, TGF-β, TNFα, and CCL2, in addition to free radicals like NO, 9 while IL-8 and TNFα production and IL-10 suppression occurs in human macrophages. 44 EVs generated by the closely related Candida auris similarly stimulate dendritic cells to produce IL-6. 45 Aspergillus flavus and Aspergillus fumigatus , the filamentous fungi that cause invasive and deadly aspergillosis in immunocompromised individuals, also secrete EVs that induce pro-inflammatory cytokine release of TNFα, IL-6, and IL-1β, as well as free radicals like NO, in a dose-dependent manner in macrophages.…”