Trypanosoma cruzi Mexican Strains Differentially Modulate Surface Markers and Cytokine Production in Bone Marrow-Derived Dendritic Cells from C57BL/6 and BALB/c Mice

Abstract

Dendritic cells (DCs) are a type of antigen-presenting cells that play an important role in the immune response against Trypanosoma cruzi, the causative agent of Chagas disease. In vitro and in vivo studies have shown that the modulation of these cells by this parasite can directly affect the innate and acquired immune response of the host in order to facilitate its biological cycle and the spreading of the species. Many studies show the mechanisms by which T. cruzi modulates DCs, but the interaction of these cells with the Mexican strains of T. cruzi such as Ninoa and INC5 has not yet been properly investigated. Here, we evaluated whether Ninoa and INC5 strains evaded the immunity of their hosts by modulating the biology and function of murine DCs. The CL-Brener strain was used as the reference strain. Herein, it was demonstrated that Ninoa was more infective toward bone marrow-derived dendritic cells (BMDCs) than INC5 and CL-Brener strains in both BMDCs of BALB/c and C57BL/6 mice. Mexican strains of T. cruzi induced different cytokine patterns. In BMDCs obtained from BALB/c mice, Ninoa strain led to the reduction in IL-6 and increased IL-10 production, while in C57BL/6 mice Ninoa strain considerably increased the productions of TNF-α and IL-10. Also, Ninoa and INC5 differentially modulated BMDC expressions of MHC-II, TLR2, and TLR4 in both BALB/c and C57BL/6 mice compared to Brazilian strain CL-Brener. These results indicate that T. cruzi Mexican strains differentially infect and modulate MHC-II, toll-like receptors, and cytokine production in DCs obtained from C57BL/6 and BALB/c mice, suggesting that these strains have developed particular modulatory strategies to disrupt DCs and, consequently, the host immune responses.

Figure 1

Trypanosoma cruzi infectivity in murine BMDCs from BALB/c and C57BL/6 mice. BMDCs with or without LPS stimulation (5 μg/mL) were incubated for 18 h with different T. cruzi strains (MOI 2 : 1) and stained with DAPI. (a) and (d) show the percentage of infected cells; in (b) and (e) the number of parasites per 100 cells and in (c) and (f) the ratio of parasites per infected cell are shown. (g) Representative images of BMDCs infected with T. cruzi after stimulation with LPS (5 μg/mL) and stained with DAPI. Statistical analysis, when applicable, was performed with the Kruskal-Wallis test with Dunn's posttest, where ^∗,∗∗p < 0.05.

Figure 2

Cytokine and chemokine production by BALB/c-derived BMDCs infected with CL-Brener, Ninoa, and INC5 strains of T cruzi. BMDCs with or without LPS stimulation (5 μg/mL) were incubated for 18 h with different T. cruzi strains (MOI 2 : 1), and the production of cytokines and chemokine was evaluated by CBA: (a) TNF-α, (b) IL-6, (c) IL-12p70, (d) CCL-2, and (e) IL-10. Statistical analysis, when applicable, was performed with the Kruskal-Wallis test with Dunn's posttest, where ^∗,∗∗p < 0.05.

Figure 3

Cytokine and chemokine production by C57BL/6-derived BMDC infected with CL-Brener, Ninoa, and INC5 strains of T cruzi. BMDCs with or without LPS stimulation (5 μg/mL) were incubated for 18 h with different T. cruzi strains (MOI 2 : 1), and the production of cytokines and chemokine was evaluated by CBA: (a) TNF-α, (b) IL-6, (c) IL-12p70, (d) CCL-2, and (e) IL-10. Statistical analysis, when applicable, was performed with the Kruskal-Wallis test with Dunn's posttest, where ^∗,∗∗p < 0.05.

Figure 4

Percentage of variation in the levels of pro-and anti-inflammatory cytokines and CCL-2 chemokine in the supernatant of BMDCs of BALB/c and C57BL/6 mice. The variation of cytokines was calculated by determination of percentage of increment or reduction in cytokines produced by T. cruzi-infected BMDCs previously stimulated with LPS and those maintained only with LPS (5 μg/mL). (f, h) Representation of the radar graph of the cytokine pattern in the dendritic cell culture supernatant. The lines highlight the change in cytokine levels in dendritic cells infected with different strains (MOI 2 : 1) in relation to the medium only with dendritic cells. (g, i) The pattern of cytokines in the dendritic cell culture supernatant previously stimulated with LPS. Data were obtained by calculating the ratio between T. cruzi-infected cells and their respective control. Statistical analysis, when applicable, was performed with the Kruskal-Wallis test with Dunn's posttest, where ^∗,∗∗p < 0.05.

Figure 5

Percentage of BMDCs expressing MHC-II, costimulatory molecules, and toll-like receptors after in vitro T. cruzi infection. The expression of molecules was evaluated by flow cytometry and represented as a variation of the percentage of BMDCs expressing (a) MHC-II, (b) CD80, (c) CD86, (d) TLR2, and (e) TLR4. Statistical analysis, when applicable, was performed with the Kruskal-Wallis test with Dunn's posttest, where ^∗p < 0.05.

Figure 6

Expression of MHC-II, costimulatory molecules, and toll-like receptors in BMDCs after in vitro T. cruzi infection. The expression of molecules was evaluated by flow cytometry and represented as a variation of the mean intensity of fluorescence (a) MHC-II, (b) CD80, (c) CD86, (d) TLR2, and (e) TLR4. (f, g) Representation of the cytokine secretion pattern in BMDC culture supernatant. The lines highlight the change in cytokine levels in LPS-stimulated BMDCs and infected with different strains of T. cruzi (MOI 2 : 1) in relation to uninfected LPS-stimulated BMDCs. Statistical analysis, when applicable, was performed with the Kruskal-Wallis test with Dunn's posttest, where ^∗p < 0.05.