Stress Induces Release of Extracellular Vesicles by Trypanosoma cruzi Trypomastigotes


Posted: 2021-10-04 19:00:00
J Immunol Res . 2021 Sep 23;2021:2939693. doi: 10.1155/2021/2939693. eCollection 2021. Affiliations Expand Affiliations 1 Departamento de Ciências Farmacêuticas, UNIFESP, Rua São Nicolau, 210, 09913-030, Diadema, São Paulo, Brazil. 2 Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria. 3 Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, Rua Pedro de Toledo, 669, 04039-032 São Paulo, Brazil. 4 Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, Rua Botucatu, 862, 04023-062 São Paulo, Brazil. 5 Instituto René Rachou/FIOCRUZ-MG, Av. Augusto de Lima, 1715, 30190-009 Belo Horizonte, Minas Gerais, Brazil. Item in Clipboard Camilla Ioshida Vasconcelos et al. J Immunol Res. 2021. Show details Display options Display options Format J Immunol Res . 2021 Sep 23;2021:2939693. doi: 10.1155/2021/2939693. eCollection 2021. Affiliations 1 Departamento de Ciências Farmacêuticas, UNIFESP, Rua São Nicolau, 210, 09913-030, Diadema, São Paulo, Brazil. 2 Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria. 3 Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, Rua Pedro de Toledo, 669, 04039-032 São Paulo, Brazil. 4 Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, Rua Botucatu, 862, 04023-062 São Paulo, Brazil. 5 Instituto René Rachou/FIOCRUZ-MG, Av. Augusto de Lima, 1715, 30190-009 Belo Horizonte, Minas Gerais, Brazil. Item in Clipboard CiteDisplay options Display options Format Abstract All extracellular forms of Trypanosoma cruzi, the causative agent of Chagas disease, release extracellular vesicles (EVs) containing major surface molecules of the parasite. EV release depends on several mechanisms (internal and external). However, most of the environmental conditions affecting this phenomenon are still unknown. In this work, we evaluated EV release under different stress conditions and their ability to be internalized by the parasites. In addition, we investigated whether the release conditions would affect their immunomodulatory properties in preactivated bone marrow-derived macrophages (BMDM). Sodium azide and methyl-cyclo-β-dextrin (CDB) reduced EV release, indicating that this phenomenon relies on membrane organization. EV release was increased at low temperatures (4°C) and acidic conditions (pH 5.0). Under this pH, trypomastigotes differentiated into amastigotes. EVs are rapidly liberated and reabsorbed by the trypomastigotes in a concentration-dependent manner. Nitrosative stress caused by sodium nitrite in acid medium or S-nitrosoglutathione also stimulated the secretion of EVs. EVs released under all stress conditions also maintained their proinflammatory activity and increased the expression of iNOS, Arg 1, IL-12, and IL-23 genes in IFN-γ and LPS preactivated BMDM. In conclusion, our results suggest a budding mechanism of release, dependent on the membrane structure and parasite integrity. Stress conditions did not affect functional properties of EVs during interaction with host cells. EV release variations under stress conditions may be a physiological response against environmental changes. Copyright © 2021 Camilla Ioshida Vasconcelos et al. Conflict of interest statement No potential conflicts of interest were reported by the authors. Figures Figure 1 Kinetics of EV release by… Figure 1 Kinetics of EV release by T. cruzi trypomastigote forms. 1 × 10 7… Figure 1 Kinetics of EV release by T. cruzi trypomastigote forms. 1 × 107 trypomastigotes were incubated in DMEM supplemented with 5% glucose at different temperatures. EV concentrations were determined at different time points by NTA (see Material and Methods). The panels show total EV concentration (a, b) and particle size (c) in the supernatants after 2 h of incubation at different temperatures (∗<0.05). SEM images of trypomastigotes incubated for 2 h at 4°C (d), 26°C (e), and 37°C ((f), size bars = 5 μm). Panel (g) indicates the correspondent relative fluorescence (RFU) of PrestoBlue viability reagent (mean and standard deviation, n = 3) showing no significant (NS) differences (p > 0.05). EVs labeled with PKH26 (1 × 108/mL and 109/mL) were incubated with 1 × 107 trypomastigotes/mL (37°C). The parasites were collected at different time points, and the median of fluorescence (h) or the percentage of labeled parasites (i) was evaluated by flow cytometry. Results are average values in triplicate measurements. Figure 2 EV release (particles/mL), size (nm),… Figure 2 EV release (particles/mL), size (nm), and membrane integrity under chemical stress. Trypanosoma cruzi… Figure 2 EV release (particles/mL), size (nm), and membrane integrity under chemical stress. Trypanosoma cruzi trypomastigotes were incubated in the absence or presence of methyl-beta-cyclodextrin (CBD) (25 μg/mL) or NaN3 (0.0001 to 1%) for 2 h at 37°C. EVs in the supernatant were quantified by NTA in triplicate (∗p < 0.05). The panels show EV concentrations (a, b) and size (nm) (c) in the presence of chemical agents. SEM of trypomastigotes preincubated with the indicated concentrations of NaN3 (d–g). Size bars are defined in each image. The relative fluorescence (RFU) of PrestoBlue viability reagent (mean ± standard deviation, n = 3) is shown in panel (h). Figure 3 pH effect on EV release.… Figure 3 pH effect on EV release. Trypomastigote (1 × 10 7 /mL) forms incubated… Figure 3 pH effect on EV release. Trypomastigote (1 × 107/mL) forms incubated (2 h, 37°C) in culture medium containing 5% glucose previously adjusted to the indicated pH values. EV concentrations (a) and sizes (b) were analyzed by NTA (∗p < 0.05). After incubation at pH 5 (c) or pH 7 (d), parasites were analyzed by SEM. In parallel, parasites were incubated at pH 5 (e) or pH 7 (f), stained with Giemsa, and 300 parasites were evaluated according to their form: trypomastigotes (Trypos), intermediate (Inter), or amastigote (Amas). Their percentages were quantified in triplicate. Figure 4 Effect of nitrosative stress on… Figure 4 Effect of nitrosative stress on EV release by T. cruzi trypomastigotes. 1 ×… Figure 4 Effect of nitrosative stress on EV release by T. cruzi trypomastigotes. 1 × 107 trypomastigotes were incubated for 2 h at 37°C in culture medium adjusted to pH 5.0 with different concentrations of NaNO2, including the control with the salt. After the incubation period, cell viability was evaluated by the PrestoBlue assay(a) and EV size (nm) (b) and EV concentration (particles/mL) (c) were measured by NTA (∗p < 0.05 and ∗∗p < 0.01). SEM of trypomastigotes releasing EVs incubated under different NaNO2 concentrations (d). Size bars = 5 μm. Alternatively, trypomastigotes were incubated at 37°C in DMEM supplemented with 5% glucose containing 100 μM SNOG. Parasite viability was evaluated at the indicated intervals by PrestoBlue assays (e), and the size (f) and concentration (g) of EVs in the supernatants were determined by NTA (n = 3). Panel (h) shows pictures of parasites stained by Giemsa after each incubation period. Figure 5 Immunomodulation of EVs obtained from… Figure 5 Immunomodulation of EVs obtained from T. cruzi submitted to different stress conditions. IFN-… Figure 5 Immunomodulation of EVs obtained from T. cruzi submitted to different stress conditions. IFN-γ-primed macrophages (BMDM) were incubated (24 h, 37°C) with T. cruzi EVs. LPS from E. coli was used as positive control. qRT-PCR was performed to detect the expression of immune response genes: Arg 1 (a), iNOS (b), IL-23 p19 (c), IL-12 p35 (d), IL-12 p40 (e), YM-2 (f), and IL-10 (g) (n = 3, ∗p < 0.05). References O World Health. Chagas Disease (Also Known as American Trypanosomiasis) 2021. https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(america...) Torrecilhas A. C., Soares R. P., Schenkman S., Fernandez-Prada C., Olivier M. Extracellular vesicles in trypanosomatids: host cell communication. Frontiers in Cellular and Infection Microbiology . 2020;10:p. 602502. doi: 10.3389/fcimb.2020.602502. - DOI - PMC - PubMed Campetella O., Buscaglia C. A., Mucci J., Leguizamon M. S. Parasite-host glycan interactions during Trypanosoma cruzi infection: trans -sialidase rides the show. Biochimica et Biophysica Acta. Molecular Basis of Diseases . 2020;1866(5):p. 165692. doi: 10.1016/j.bbadis.2020.165692. - DOI - PMC - PubMed TROCOLITORRECILHAS A., TONELLI R., PAVANELLI W., et al. Trypanosoma cruzi: parasite shed vesicles increase heart parasitism and generate an intense inflammatory response. Microbes and Infection . 2009;11(1):29–39. doi: 10.1016/j.micinf.2008.10.003. - DOI - PubMed Ribeiro K. S., Vasconcellos C. I., Soares R. P., et al. Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells. Journal of Extracellular Vesicles . 2018;7(1):p. 1463779. doi: 10.1080/20013078.2018.1463779. - DOI - PMC - PubMed Show all 51 references [x] Cite Copy Format: Send To [x]

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