Trypanosoma cruzi SSP4 Amastigote Protein Induces Expression of Immunoregulatory and Immunosuppressive Molecules in Peripheral Blood Mononuclear Cells

Abstract

The acute phase of Chagas' disease in mice and human is marked by states of immunosuppression, in which Trypanosoma cruzi replicates extensively and releases immunomodulatory molecules that delay parasite-specific responses mediated by effector T cells. This mechanism of evasion allows the parasite to spread in the host. Parasite molecules that regulate the host immune response during Chagas' disease have not been fully identified, particularly proteins of the amastigote stage. In this work, we evaluated the role of the GPI anchored SSP4 protein of T. cruzi as an immunomodulatory molecule in peripheral blood mononuclear cells (PBMCs). rMBP::SSP4 protein was able to stimulate nitric oxide (NO) production. Likewise, rMBP::SSP4 induced the expression of genes and production of molecules involved in the inflammatory process, such as, cytokines, chemokines, and adhesion molecules (CAMs) as determined by RT-PCR and ELISA. These results suggest that the amastigote SSP4 molecule could play a key role in the immunoregulatory and/or immunosuppressive process observed in the acute phase of infection with T. cruzi.

Figure 1

rMBP::SSP4 induces NO synthesis in PBMC. PBMC were stimulated at 48 h with different stimuli. Cells cultured with medium alone were used as controls. Supernatants of cultured cells were harvested, and nitrite concentration was assayed. Data are expressed as means ± SD and are representative of three independent experiments. ***P < 0.0001 compared with non-stimulated cells and controls. NS: non-stimulated.

Figure 2

Expression of genes for cytokines and chemokines in PBMC stimulated with rMBP::SSP4. The cells were stimulated with recombinant protein for 12–96 h. The intensities of each band were quantified and plotted from the gels that are on top of each graph corresponding to the expression of genes for cytokines and chemokines. GAPDH was used as control housekeeping gene. ^{∗,∗∗,∗∗∗} P < 0.05, 0.001, and 0.0001, respectively, versus unstimulated cells.

Figure 3

Profile of cytokines and chemokines induced by rMBP::SSP4 in PBMC. PBMC was stimulated with the protein for 12–96 h, and cytokines and chemokines were measured in cells' culture supernatants by ELISA. Unstimulated cells (open bars), cells stimulated with rMBP::SSP4 (full bars), and cells stimulated with MBP (gray bars). Histograms show values in pg/mL (means ± SD) of three experiments run in duplicate. ^{∗, ∗∗, ∗∗∗} P < 0.05, 0.001, and 0.0001, respectively, versus unstimulated cells.

Figure 4

Effect of rMBP::SSP4 on gene expression of CAMs and TNFRs in PBMCs. RT-PCR analysis of CAMs and TNFRs mRNAs in PBMCs was performed as described (see Section 2). PBMCs were stimulated with the protein for 12–96 h, S/E (nonstimulated). The intensities of each band were quantified and plotted from the gels that are on top of each graph corresponding to the expression of genes. GAPDH was used as a control housekeeping gene. ^{∗∗, ∗∗∗} P < 0.001 and 0.0001, respectively, versus unstimulated cells.