Extracellular Vesicles Derived From Talaromyces marneffei Yeasts Mediate Inflammatory Response in Macrophage Cells by Bioactive Protein Components

Abstract

Extracellular vesicles (EVs) loaded with proteins, nucleic acids, membrane lipids, and other virulence factors could participate in pathogenic processes in some fungi such as Cryptococcus neoformans and Candida albicans. However, the specific characteristics of EVs derived from Talaromyces marneffei (TM) still have not been figured out yet. In the present study, it has been observed that TM-derived EVs were a heterogeneous group of nanosized membrane vesicles (30-300 nm) under nanoparticle tracking analysis and transmission electron microscopy. The DiI-labeled EVs could be taken up by RAW 264.7 macrophage cells. Incubation of EVs with macrophages would result in increased expression levels of reactive oxygen species, nitric oxide, and some inflammatory factors including interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor. Furthermore, the expression of co-stimulatory molecules (CD80, CD86, and MHC-II) was also increased in macrophages stimulated with EVs. The level of inflammatory factors secreted by macrophages showed a significant decrease when EVs were hydrolyzed by protease, while that of DNA and RNA hydrolase treatment remained unchanged. Subsequently, some virulence factors in EVs including heat shock protein, mannoprotein 1, and peroxidase were determined by liquid chromatography-tandem mass spectrometry. Taken together, our results indicated that the TM-derived EVs could mediate inflammatory response and its protein would play a key role in regulating the function of RAW 264.7 macrophage cells.

Keywords: Talaromyces marneffei; extracellular vesicles; inflammatory response; macrophage cells; proteomics.

FIGURE 1

The size distribution and morphology of extracellular vesicles (EVs) secreted by Talaromyces marneffei (TM). (A) Nanoparticle tracking analysis revealed the size distribution and particle concentration of isolated EVs. (B) The EVs were observed inside TM through transmission electron microscopy (TEM; black arrow). (C) It showed the moment of TM yeast cells releasing EVs (black arrow). (B,C) Scale bar = 200 nm. (D,E) TEM identified the purified EVs’ morphology with a cup-shaped structure. (D) Scale bar = 200 nm. (E) Scale bar = 50 nm.

FIGURE 2

Immunofluorescence images showed the process of RAW264.7 macrophage cells uptaking extracellular vesicles (EVs). The RAW264.7 macrophage cells were incubated with unstained EVs (A) and labeled vesicles (B) after 2 h of co-incubation. (C) RAW264.7 macrophage cells were incubated with actin polymerization inhibitors cytochalasin D for 2 h before the addition of EVs. EVs were labeled with DiI staining (red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). The fluorescent images are representative of three independent experiments (scale bar = 10 μm).

FIGURE 3

Determination of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory factor production after co-incubation of RAW264.7 macrophage cells with various concentrations of extracellular vesicles (EVs) derived from Talaromyces marneffei (TM). (A) ROS production was detected by flow cytometry after co-incubation of RAW264.7 macrophage cells with EVs for 12 h. From top to bottom, the groups were negative (green), lipopolysaccharide (LPS; orange), 5 μg/ml EVs (blue), and 10 μg/ml EVs (pink), respectively. (B) The change of NO was determined using the Griess reagent. The groups were negative, LPS, 5 μg/ml EVs, 10 μg/ml EVs, and 10 μg/ml EVs + SMT, respectively. (C–F) The RNA was isolated for qPCR after 24 h of stimulation of TM-derived EVs. The bar chart above shows the mRNA level of inflammatory factors. (G–J) The supernatants from the macrophages stimulated with EVs for 24 h were collected, and the cytokine levels were measured by ELISA. The bar chart below represents the ELISA result. *P < 0.05, **P < 0.001. LPS, positive. n = 3 independent experiments; Student’s t-test.

FIGURE 4

The expression of co-stimulatory molecules CD80, CD86, and MHC-II was examined with flow cytometry. The ordinate was the number of cells, and the abscissa was the fluorescence intensity. From top to bottom: negative, lipopolysaccharide (positive), 5 μg/ml EVs, and 10 μg/ml EVs. From left to right: CD80 (A), CD86 (B), and MHC-II (C). The experiments were repeated for three independent times.

FIGURE 5

Detection of nitric oxide (NO) in different extracellular vesicles (EV) treatment groups and functional classification and subcellular localization of proteins in EVs derived from Talaromyces marneffei (TM). (A) The NO levels in the supernatants were measured after EVs were added to RAW264.7 macrophage cells for 24 h. The three EV-treated groups are as follows: P, protease; D, DNA hydrolase; and R, RNA hydrolase. (B) The pie graphs approximately reflected the distribution of protein localization in TM-derived EVs, and different colors represented their different localization. (C) The vertical axis was the functional classification, and the horizontal axis was the percentage of every category of protein on the annotation. (D) The ordinate was the optical density (OD) value of MP1p ELISA among different groups. The result was positive if the OD value was greater than the cutoff value, and three independent experiments were performed. *P < 0.05.