IFN-gamma expression is up-regulated by peripheral blood mononuclear cells (PBMC) from non-exposed dogs upon Leishmania chagasi promastigote stimulation in vitro

Abstract

While the response to Leishmania spp. is well characterized in mice and humans, much less is known concerning the canine immune response, particularly soon after exposure to the parasite. Early events are considered to be a determinant of infection outcome. To investigate the dog's early immune response to L. chagasi, an in vitro priming system (PIV) using dog naïve PBMC was established. Until now, dog PIV immune response to L. chagasi has not been assessed. We co-cultivated PBMC primarily stimulated with L. chagasi in vitro with autologous infected macrophages and found that IFN-gamma mRNA is up-regulated in these cells compared to control unstimulated cells. IL-4 and IL-10 mRNA expression by L. chagasi-stimulated PBMC was similar to control unstimulated PBMC when incubated with infected macrophages. Surprisingly, correlation studies showed that a lower IFN-gamma/IL-4 expression ratio correlated with a lower percentage of infection. We propose that the direct correlation between IFN-gamma/IL-4 ratio and parasite load is dependent on the higher correlation of both IFN-gamma and IL-4 expression with lower parasite infection. This PIV system was shown to be useful in evaluating the dog immune response to L. chagasi, and results indicate that a balance between IFN-gamma and IL-4 is associated with control of parasite infection in vitro.

Fig. 1

(A and B) L. chagasi-stimulated PBMC modulate parasite load in co-incubated autologous macrophages. The macrophages were obtained as described in Section 2. At day 5 of culture, macrophages were infected with L. chagasi stationary-phase promastigotes at a ratio of 10 parasites per macrophage. Twenty-four hours later, PBMC recovered from parallel cultures of non-adherent cells were added to L. chagasi-infected macrophages. Adhered macrophages were fixed and stained, and quantitative observations were performed to determine the percentage of infected cells and the number of parasite per cell to calculate the number of parasites per 100 macrophages. Data represent the individual mean values of the number of parasites per 100 macrophages submitted to the co-incubation with unstimulated (medium), L. chagasi-stimulated (L. chagasi), or ConA-stimulated PBMC at 2 and 4 days of infection. Each symbol represents a distinct dog donor: (■) Dog 1 (n = 4), (●) Dog 2 (n = 4), (▴) Dog 3 (n = 3), (▾) Dog 4 (n = 4), (♦) Dog 5 (n = 4), line = mean value; *p < 0.05, **p < 0.01 (one-way ANOVA).

Fig. 2

IFN-γ expression is up-regulated by PBMC from naïve dogs upon primary in vitro stimulation with L. chagasi. Quantitative mRNA of cytokine was determined by qRT-PCR of primary L. chagasi-stimulated PBMC and control unstimulated PBMC cultivated for 6 days and co-cultivated for an additional 4 days with infected macrophages as described in Section 2. Each symbol represents a distinct dog donor: (●) Dog 2, (▴) Dog 3, (▾) Dog 4, (♦) Dog 5, line = median value of IFN-γ (A), IL-4 (B) and IL-10 (C) mRNA concentrations normalized to the concentration of the housekeeping gene 18S RNA expression represented as log₂. To test statistical differences Friedman test followed by the Dunn's multiple comparisons post-test were used: L. chagasi-stimulated × medium = *p < 0.05.