Trypanosoma cruzi-induced immunosuppression: B cells undergo spontaneous apoptosis and lipopolysaccharide (LPS) arrests their proliferation during acute infection

Abstract

Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.

Fig. 1

Proliferative response of spleen mononuclear cells (SMC) from normal and Trypanosoma cruzi-infected mice following stimulation with LPS. SMC (2 × 10⁶/ml) obtained from normal (•) or 8 day-infected mice (▪) (a) or from 15- (▾) or 180 (chronic) -day-infected mice (✦) (b) were cultured with medium alone or stimulated with LPS (20 μ g/ml). (a,b) Thymidine incorporation was measured at different hours of culture and the results are presented as delta of the mean ct/min of triplicate cultures ± s.d. (see MATERIALS and METHODS). The data in (a,b) were obtained during different days and are representative of three independent experiments.

Fig. 2

Percentage of CD19⁺ cells on spleen mononuclear cells (SMC) from Trypanosoma cruzi-infected mice. SMC from normal (a) or 15-day-infected mice (b) were cultured with medium alone (left panels) or LPS (20 μ g/ml) (right panels) during 96 h. The cells were then analysed for CD19⁺ expression by flow cytometry using PE-labelled anti-mouse CD19 antibodies. Staining with control isotype is shown by open histograms. The relative cell number is plotted against fluorescence intensity.

Fig. 3

DNA fragmentation on cells obtained from Trypanosoma cruzi-infected mice. Spleen mononuclear cells (SMC) from normal 8-day, 15-day or 180-day-infected mice were cultured with medium alone or LPS (20 μ g/ml) during 6 h. DNA released into the supernatant by SMC was electrophoresed to monitor DNA fragmentation. Molecular size markers (Φλ DNA-EcoRI-Hind III digest) are shown in lane M. Data are representative of three independent experiments.

Fig. 4

Forward scatter^low population is enriched in spleen mononuclear cells (SMC) from Trypanosoma cruzi-infected mice. SMC from normal (a) or 15-day-infected mice (b) were cultured in medium alone (left panels) or 20 μ g/ml LPS (right panels) during 96 h. Cells were stained with PE-labelled anti-mouse CD19. CD19⁺ cells were gated and analysed for FSC versus SSC profiles. Density plots show three distinct populations (FSC^low or R1, FSC^int or R2 and FSC^high or R3) based on light scattering parameters. The percentages shown indicate the number of cells within each region.

Fig. 5

Incorporation of fluorescein-labelled dUTP into DNA strand breaks of purified B cells from Trypanosoma cruzi-infected and normal mice. B cells from normal (a) or infected mice at day 15 after infection (b) were cultured during 6 h (2 × 10⁶ cells/well) in medium alone (left panels) or with LPS (right panels). The cells were then incubated with FITC-labelled dUTP and terminal deoxitransferase according to Promega Kit directions. The percentages of apoptotic B cells are indicated. Staining without the TdT enzyme during TUNEL reaction is shown by open histograms The data are representative of two independent experiments.

Fig. 6

Flow cytometric analysis of Fas expression on B220⁺ spleen cells from Trypanosoma cruzi-infected and normal mice. Spleen mononuclear cells (SMC) from normal (a) or infected mice at day 15 after infection (b) were stained with PE-labelled anti-mouse B220⁺ and FITC-labelled anti-mouse Fas. B220⁺ cells were gated and the percentages of Fas⁺ cells are indicated. Staining with control isotype is shown by open histograms. The data are representative of three independent experiments.

Fig. 7

Cell cycle analysis of B220⁺ cells from Trypanosoma cruzi-infected and normal mice. Spleen mononuclear cells (SMC) from normal (a) or infected mice (b) were explanted at day 15 after infection and cultured during 6 h with medium alone (left panels) or with 20 μ g/ml LPS (right panels). Cells were double-stained with FITC-labelled anti-mouse B220 and propidium iodide. B220⁺ cells were gated and the percentages of B cells in G₀/G₁ (M1), S/G₂/M (M2) or sub G₀/G₁ (M3) cell cycle stages are indicated.