Extracellular Vesicles From Fungal Infection in Humans: A Key Player in Immunological Responses

Abstract

Fungal infections cause approximately 1.6 million deaths annually. Diagnosing and treating fungal infections is difficult due to limited access to diagnostic tests and rising antifungal resistance. Extracellular vesicles (EVs) facilitate interactions between fungal cells and hosts, significantly influencing the pathogen-host relationship. Owing to the complexity of fungal EVs and the lack of clinical studies on their roles in human infections, we analysed EVs from serum and urine samples of patients with infections caused by Candida albicans, Cryptococcus neoformans, and Paracoccidioides brasiliensis to determine their roles. Using mass spectrometry, we identified sterols, sphingolipids, and fatty acids as key metabolites in the EVs. We quantified cholesterol and ergosterol, confirming the presence of both host and fungal EVs in clinical samples. Our research investigated whether these EVs could modulate the host immune response. We observed a proinflammatory response in murine and human macrophages, characterized by increased cytokines, such as tumour necrosis factor-α, interferon-γ, and interleukin-6, and elevated expression of the inducible nitric oxide synthase gene, a marker of M1 macrophage response. Thus, circulating EVs in patients with fungal infections likely play a role in disease pathophysiology. These findings enhance our understanding of EVs in fungal infections, suggesting potential therapeutic targets for systemic mycoses.

Keywords: clinical samples; extracellular vesicles (EVs); fungal metabolites; immunomodulation; systemic mycoses.

FIGURE 1

Characterization of extracellular vesicles (EVs) from the clinical samples of healthy individuals, and patients with fungal infections caused by Candida albicans, Cryptococcus neoformans, and Paracoccidioides brasiliensis. NTA of the concentration (EVs/mL) and size (nm) of EVs from the serum and urine samples of healthy individuals (A and B), and patients with candidiasis (C and D), cryptococcosis (E and F), and paracoccidioidomycosis (G and H) from the University Hospital, Ribeirao Preto Medical School (HC‐FMRP). (I) Transmission electron microscopy was conducted on the clinical samples of healthy individuals and patients with fungal infections, and EVs with classic cup‐shaped morphology were detected. Scale bar: 200 nm. (J) Western blotting of the protein extract from EVs derived from infected and non‐infected patients with antibodies specific to the tetraspanin family (CD9 and CD63) and ALIX. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a loading control.

FIGURE 2

Statistical analysis of the metabolomics data of EVs from the serum and urine samples of healthy individuals and patients with fungal infections. (A) Data analysis using principal components analysis (PCA). (B) Annotation of the chemical compounds identified in EVs under experimental conditions using tandem MS/MS libraries, with representation of the molecular formula, retention time (Rt), theoretical and measured m/z, node, and reference library.

FIGURE 3

Annotations of secondary metabolites in the serum EVs of healthy individuals and patients infected by C. albicans, C. neoformans, and P. brasiliensis. Molecular networks generated by the GNPS platform (nodes with library correspondence in FBMN) and representation of the chemical structure of compounds belonging to the sterols (cholesterol and ergosterol) (A, D), sphingolipids (phytosphingosine and sphingosine) (E, H), and fatty acids (oleic acid) (I) classes. Statistical determination of the peak areas of sterols (B, C), sphingolipids (F, G), and fatty acids (J) in the serum EVs of healthy individuals and patients infected by C. albicans, C. neoformans, and P. brasiliensis. Healthy individuals were used as controls. Statistical difference: ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

Estimation of host‐to‐fungal EV ratios from serum of healthy individuals and patients with fungal infections. Quantification of the concentration (parts per million – ppm) of cholesterol (A) and ergosterol (B) content in EV preparations from the serum of healthy individuals and patients infected with C. albicans, C. neoformans, and P. brasiliensis. (C) Ratio of cholesterol and ergosterol concentration (ppm) in healthy individuals and patients with candidiasis, cryptococcosis, and paracoccidioidomycosis. Samples were analysed in triplicate. ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

Proinflammatory mediators produced by AMJ2‐C11 cells after stimulation with EVs from patients with candidiasis, cryptococcosis, and paracoccidioidomycosis. AMJ2‐C11 cells (1 × 10⁶ cells/mL) were stimulated with Pam3CSK4 (1 µg/mL), EVs from the serum and urine samples from healthy individuals (1 × 10⁶ EVs/mL), and patients with fungal infections (1 × 10⁶ and 1 × 10⁹ EVs/mL), or culture medium alone. After 24 and 48 h of incubation, levels of tumour necrosis factor (TNF)‐α, interferon (IFN)‐γ, interleukin (IL)‐6, and IL‐10 in the culture supernatant were determined. Statistically significant differences in the production of the analysed cytokines were evaluated after stimulation with serum and urine EVs from patients with candidiasis (A, B, E–J), cryptococcosis (C, D, K–T) and paracoccidioidomycosis (U and V). Values are expressed as the mean ± standard deviation (SD) compared to the levels in healthy individuals. Differences were considered significant at p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

FIGURE 6

Polarization profile of macrophages derived from THP‐1 human monocytes after stimulation with EVs from the serum and urine samples of patients with fungal infections. THP‐1 cells (1 × 10⁶ cells/mL) were stimulated with Pam3CSK4 (1 µg/mL), EVs from the serum and urine samples of healthy individuals (1 × 10⁶ EVs/mL) and patients with fungal infections (1 × 10⁶ and 1 × 10⁹ EVs/mL), or culture medium alone. After 24 and 48 h of incubation, levels of TNF‐α, IFN‐γ, IL‐6, and IL‐10 in the culture supernatant were determined. Statistically significant differences in the production of the analysed cytokines were evaluated after stimulation with serum and urine EVs from patients with candidiasis (A–D), cryptococcosis (E–P, S, and T), and paracoccidioidomycosis (Q, R, U, and V). Values are expressed as the mean ± SD compared to the levels in healthy individuals. Differences were considered significant at p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

FIGURE 7

EVs of patients infected by Candida albicans promote the classical activation of murine macrophages. AMJ2‐C11 cells (2 × 10⁶ cells/mL) were stimulated with EVs from the serum (1 × 10¹⁰ EVs/mL) and urine (1 × 10⁹ EVs/mL) samples of patients with fungal infections, EVs from the serum and urine samples of healthy individuals (1 × 10⁶ EVs/mL), Pam3CSK4 (1 ug/mL), IL‐4 (50 ng/mL), or medium alone for 24 h at 37°C. Relative expression levels of inducible nitric oxide synthase (iNOS) (A, B) and arginase 1 (Arg‐1) (C, D) were determined via quantitative real‐time polymerase chain reaction (qPCR). The results, expressed as the mean ± SD, were compared to those obtained on basal expression (statistical difference represented by the letter a) and healthy individuals (represented by the letter b).