Extracellular vesicles from Paracoccidioides brasiliensis induced M1 polarization in vitro

Abstract

Extracellular vesicles (EVs) released by eukaryotes, archaea, and bacteria contain proteins, lipids, polysaccharides, and other molecules. The cargo analysis of EVs shows that they contain virulence factors suggesting a role in the pathogenesis of infection. The proteome, lipidome, RNA content, and carbohydrate composition of EVs from Paracoccidioides brasiliensis and Paracoccidioides lutzii were characterized. However, the effects of P. brasiliensis EVs on the host immune system have not yet been investigated. Herein, we verified that EVs from P. brasiliensis induce the production of proinflammatory mediators by murine macrophages in a dose-dependent manner. Addition of EV to macrophages also promoted transcription of the M1-polarization marker iNOs and diminish that of the M2 markers Arginase-1, Ym-1, and FIZZ-1. Furthermore, the augmented expression of M2-polarization markers, stimulated by IL-4 plus IL-10, was reverted toward an M1 phenotype in response to secondary stimulation with EVs from P. brasiliensis. The ability of EVs from P. brasiliensis to promote M1 polarization macrophages favoring an enhanced fungicidal activity, demonstrated by the decreased CFU recovery of internalized yeasts, with comparable phagocytic efficacy. Our results suggest that EVs from P. brasiliensis can modulate the innate immune response and affect the relationship between P. brasiliensis and host immune cells.

Figure 1. EVs from P. brasiliensis induce the production of proinflammatory mediators by peritoneal macrophages.

Peritoneal macrophages (1.5 × 10⁶/mL) of C57BL/6 mice were incubated at 37 °C for 48 h with the indicated amounts of extracellular vesicles from P. brasiliensis (x axis). The medium and LPS (1 μg/mL) plus IFN-γ (2 ng/mL) were used as negative and positive controls, respectively. The culture supernatants were assessed for nitrite (a), TNF-α (b), IL-6 (c), IL-12p40 (d), IL-12p70 (e), IL-1α (f), and IL-1β (g) concentrations. The results, expressed as mean ± SEM, were compared to the levels in unstimulated cells (with the medium only). Differences were considered significant when p < 0.05 (*).

Figure 2. EVs from P. brasiliensis induce the production of proinflammatory mediators by the macrophage cell line J774A.1.

J774A.1 cells (1.5 × 10⁶/mL) were incubated at 37 °C for 48 h with the indicated amounts of extracellular vesicles from P. brasiliensis (x axis). The medium and LPS (1 μg/mL) plus IFN-γ (2 ng/mL) were used as negative and positive controls, respectively. The culture supernatants were assessed for TNF-α (a), IL-6 (b), and IL-12p40 (c) levels. The results, expressed as mean ± SEM, were compared to the levels in unstimulated cells (with the medium only). Differences were considered significant when p < 0.05 (*).

Figure 3. EVs from P. brasiliensis promote macrophage classical activation.

Macrophages (2 × 10⁶/mL) of C57BL/6 mice were incubated at 37 °C for 6 h with EVs (10 μg/mL) and IFN-γ (2 ng/mL) plus IL-12p40 (50 ng/mL) as M1 inducers (classical activation) or IL-10 plus IL-4 (50 ng/mL both) as M2 inducers (alternative activation). The medium was used as a negative control. Following RNA extraction and conversion into cDNA, the relative expressions of iNOS (a), FIZZ1 (b), Arginase-1 (c), and Ym-1 (d) were determined by real-time PCR. The results, expressed as mean ± SEM, were compared to those obtained from the negative control. Differences were considered significant when p < 0.05 (*).

Figure 4. EVs from P. brasiliensis induce switching from M2 to M1 macrophages.

(a) Peritoneal macrophages (2 × 10⁶/mL) were incubated at 37 °C with EVs (10 μg/mL; red), IL-4 plus IL-10 (50 ng/mL both; blue), and the medium as a negative control (green). After 24 h, the cells were washed and subjected to re-stimulation for 48 h as follows: the macrophages that were stimulated with IL-4 plus IL-10 (blue) during the first 24 hours were then re-stimulated with EVs (red); those stimulated for 24 h with EVs (red) were then re-stimulated with IL-4 plus IL-10 (blue). The culture supernatants were assessed for nitrite concentration (b), and cells were analyzed by real-time PCR for the relative expression of iNOS (c), Arginase-1 (d), and Ym-1 (e). The results, expressed as mean ± SEM, were compared to those obtained from the negative control. Differences were considered significant when p < 0.05 (*). An additional comparison was made between the results obtained at 24 and 48 h, represented as blue or red bars (#). Differences were considered significant when p < 0.05.

Figure 5. EVs from P. brasiliensis induce M1 macrophages under conditions that favor the development of M2 phenotype.

(a) Peritoneal macrophages (2 × 10⁶/mL) were incubated at 37 °C with EVs (10 μg/mL; red), IL-4 plus IL-10 (50 ng/mL both; blue), IL-12 plus IFN-γ (50 ng/mL both; black) and the medium as a negative control (green). After 24 h, the cells previously stimulated with IL-4 plus IL-10 (blue) were re-stimulated for 48 h with IL-12 plus IFN-γ (50 ng/mL both; black) or EVs (40 μg/mL; red). The cells were analyzed by real-time PCR for the relative expression of iNOS (b) and Arginase (c). The results, expressed as mean ± SEM, were compared to those obtained from the negative control (medium, green). Differences were considered significant when p < 0.05 (*).

Figure 6. EVs from P. brasiliensis enhanced the fungicidal activity of macrophages.

Peritoneal macrophages (1.5 × 10⁶/mL), treated for 24 h with EVs (10 μg/mL), IFN-γ (50 ng/mL), or the medium were washed and infected with P. brasiliensis (yeast-to-macrophage ratio of 1:1) and labeled with propidium iodide (PI), for 2 h. The macrophages were analyzed by flow cytometry. The percentage of PI-stained cells is shown in (a) (histogram) and (b) (bars). Alternatively, macrophages were infected with P. brasiliensis (yeast-to-macrophage ratio of 1:10), for 4 h. The cell supernatants were harvested and assessed for CFU, as a measurement of the presence of extracellular P. brasiliensis (c). After washing gently, the medium was added to the cells, and they were cultured for an additional 48 h. The cells were washed and lysed for the detection of viable intracellular yeasts by CFU (d). The results, expressed as mean ± SEM, were compared to those obtained from the negative control. Differences were considered significant when p < 0.05 (*).

Figure 7. A proposed model that demonstrate the effects of EVs from P. brasiliensis on murine peritoneal macrophages.

EVs from P. brasiliensis yeast have the ability to induce proinflammatory mediator’s production by murine peritoneal macrophages. Moreover, M1 polarization induced by EVs is associated with the increase in fungicidal activity. In addition, EVs from P. brasiliensis induce switching from M2 to M1 macrophages, a fact that suggests the EVs activity as a modulator of innate immune response.