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J Extracell Vesicles
2015 Nov 26;4:28734. doi: 10.3402/jev.v4.28734.
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Vesicles from different Trypanosoma cruzi strains trigger differential innate and chronic immune responses.
Nogueira PM
,
Ribeiro K
,
Silveira AC
,
Campos JH
,
Martins-Filho OA
,
Bela SR
,
Campos MA
,
Pessoa NL
,
Colli W
,
Alves MJ
,
Soares RP
,
Torrecilhas AC
.
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Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.
Fig. 1. Procedures employed for the production, fractionation and characterization of T. cruzi EVs from different strains.
Fig. 2. Flow cytometry strategy for intracellular cytokine production by spleen cells from infected mice. (a) Dot plots of CD4, CD8, CD19, F4/80/CD11b and MHCII/CD11c expression. The representative dot plots illustrating the frequency (%) of cytokines from spleen cells are shown for Colombiana and YuYu strains. (b) CD4+IL-10+ cells. (c) CD8+IL-10+. (d) CD19+TNF-α+. (e) MHC/CD11c+TNF-α+.
Fig. 3.
Trypanosoma cruzi (Y strain) trypomastigotes spontaneously shed vesicles from their entire membrane surface. Scanning electron microscopy (SEM) of parasite membrane shedding after incubation in culture medium (a–d, bars: 1–5 µm). Magnification: (a) 27,383×, (b) 25,242×, (c) 60,470× and (d) 92,084×.
Fig. 4. Nanoparticle tracking analysis of the EVs isolated from different strains of T. cruzi. Graphic demonstration of size distribution and concentration for Y (a), Colombiana (b), CL-14 (c) and YuYu (d) strains. The average size (nm) (e) and concentration (particles/mL) (f) of the vesicles for all strains are represented. Data are representative of 3 independent experiments.
Fig. 6. Nitric oxide (NO) production by murine macrophages stimulated by EVs from CL-14 and YuYu strains is dependent on TLR2. Murine macrophages (C57BL/6, TLR2 − /− and TLR4 − /−) were stimulated with different concentrations of T. cruzi EVs (1, 5 and 50 µg). Cells were pre-incubated with IFN-γ (100 U/mL) for 18 h prior to addition of EVs or LPS, LPG and live parasites MOI 10:1 (positive controls). Negative controls included medium and medium + INF-γ. LPG Lb, L. braziliensis LPG; LPS, lipopolysaccharide (LPS) from E. coli; T. cruzi (Y), live parasites of T. cruzi (Y strain). Bars express the mean value±SD of 2 separate studies (*p < 0.05).
Fig. 7. TNF-α production by murine macrophages stimulated by EVs from CL-14 and YuYu strains is dependent on TLR2. Murine macrophages (C57BL/6, TLR2 − /− and TLR4 − /−) were stimulated with different concentrations of T. cruzi EVs (1, 5 and 50 µg). Cells were pre-incubated with IFN-γ (100 U/mL) for 18 h prior to addition of the EVs, and controls (LPS, LPG and live parasites MOI 10:1) (positive controls). Negative controls included medium and medium + INF-γ. IFN-γ, gamma-interferon; LPG Lb, L. braziliensis LPG; LPS, lipopolysaccharide from E. coli; T. cruzi (Y), live parasites of T. cruzi (Y strain). Bars express the mean value±SD of 2 separate studies (*p < 0.05).
Fig. 8.
Trypanosoma cruzi EVs equally activate MAPKs (ERK 1/2, p38 and JNK) from J774.1 macrophages. Cells were stimulated with EVs (5 µg/mL) at different time points (5, 15, 30 and 45 min). Dually phosphorylated MAPKs were detected by western blot: (a) ERK 1/2, (b) p38 and (c) JNK. C-, negative control (medium); C+, positive control (LPS from E. coli) (100 ng/mL).
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