Response to Trypanosoma cruzi by Human Blood Cells Enriched with Dentritic Cells Is Controlled by Cyclooxygenase-2 Pathway

Abstract

Chagas disease (Cd) or American human trypanosomiasis is caused by Trypanosoma cruzi and affects ~7 million people, mostly in Latin America. The infective trypomastigote forms of the parasite can invade several human blood cell populations, including monocytes and dendritic cells (DC). Although these cells display a wide functional diversity, their interactions with T. cruzi via cyclooxygenase (COX) and cyclic adenosine monophosphate (cAMP) dependent pathways have not been analyzed. To exploiting this mechanism, DC-enriched peripheral human blood mononuclear cell populations (DC-PBMC) were used as our model. Our results showed that the treatment of these cell populations with celecoxib (CEL), a cyclooxygenase-2 selective inhibitor or SQ 22,536, an adenilate cyclase inhibitor, significantly caused marked inhibition of T. cruzi infection. In contrast, aspirin (ASA, a non-selective COX-1 and COX-2 inhibitor) treatment did not inhibit the infection of the cells by the parasite and was independent of nitric oxide (NO) production. The expression of co-stimulatory molecules CD80 and CD86 were similar on cells treated or not with both COX-inhibitors. The infection stimulated the release of TNF-α, IL-1β, IL-6, IL-8, and IL-10 production by infected cells. Treatment with ASA or CEL did not affect TNF-α, IL-6, IL-8, IL-10, and NO production by infected cells, but increased IL-1β production by them. Our results suggest a key role of COX-2 and cAMP pathways in T. cruzi invasion process of human blood cells and these pathways may represent targets of new therapeutic options for Cd.

Keywords: Trypanosoma cruzi; aspirin; celecoxib; cell invasion; human monocyte-derived dendritic cells.

Figure 1

T. cruzi-infected DC-PBMC. DC-PBMC presented different susceptibilities to T. cruzi infection. DC-PBMC were incubated with T. cruzi trypomastigotes at parasite-to-cell ratio (5:1). After 18 h, the cultures were washed to remove free parasites and DC-PBMC were fixed with methanol and stained with Giemsa stain. Percentages of infected DC-PBMC (A) and mean numbers of amastigotes per infected DC-PBMC (B) were recorded after microscopy examination of at least 200 cells. Results are the mean ± standard error for six independent experiments with six different blood donors.

Figure 2

Inhibition of ciclooxigenase-2 impairs T. cruzi entry into DC-PBMC Percentage of infected cells from the interaction process between DC-PBMC treated for 1 hour with ASA (A) or CEL (B) [0.312, 0.625, and 1.25 μM] and exposed to 5:1 trypomastigotes of T. cruzi (Y strain) during 18 h. After DC-PBMC were fixed with methanol and stained with Giemsa stain. Quantification was carried out under a light microscope where the number of intracellular parasites was counted in a total of least 200 cells. MTT assay to measure cell viability in DC-PBMC after treatment with ASA (C) or CEL (D). H₂O₂ (1,000 μM) was used as positive control. Values are the mean ± standard error of two experiments. DMSO, Dimethyl sulfoxide 0.05%. Results are the mean ± standard error for duplicate determinations, with six blood donors in each experiment. Means not sharing letter are significantly different (P < 0.05, one-way ANOVA with Tukey's post-test).

Figure 3

Trypanosoma cruzi infection levels of DC-PBMC. Number of infected DC-PBMC with nine amastigotes per cell decreased with the treatment for both COX-inhibitors used. Only 1.25 mM of ASA was able of provoked this reduction (A). High (1.25 mM) and low concentrations of CEL (0.625 mM) provoked a decrease in the number of cells containing nine amastigotes per cell (B). Results are the mean ± standard error for duplicate determinations, with three blood donors in each experiment. Means not sharing letter are significantly different (P < 0.05, one-way ANOVA with Tukey's post-test).

Figure 4

PGE₂ restored the invasiveness of T. cruzi in DC-PBMC previously treated with CEL. DC-PBMC were treated for 30 min with separately either with or without PGE₂ (1 or 10 μM) alone or in combination with ASA (A) or with CEL (B) and exposed to 5:1 trypomastigotes of T. cruzi (Y strain) during 18 h. After cells were fixed with methanol and stained with Giemsa stain. Results are the mean ± standard error for duplicate determinations, with three blood donors in each experiment. Means not sharing letter are significantly different (P < 0.05, one-way ANOVA with Tukey's post-test).

Figure 5

Adenylate-cyclase activity regulates the entry of T. cruzi into DC-PBMC. DC-PBMC were treated for 30 min separately either with or without SQ 22536 (20 μM) and exposed to 5:1 trypomastigotes of T. cruzi (Y strain) during 18 h. Inhibition of adenylate-cyclase decreases T. cruzi infection (A). Number of infected DC-PBMC with nine amastigotes per cell decreased with the treatment (B). Results are the mean ± standard error for duplicate determinations, with five (A) and six (B) blood donors in each experiment. Means not sharing letter are significantly different (P < 0.05, one-way ANOVA with Tukey's post-test).

Figure 6

The activation of adenylyl cyclase with forskolin reverted CEL effects on T. cruzi entry. DC-PBMC were treated for 30 min with forskolin (10 μM) alone or in combination with ASA (A) or with CEL (B) and exposed to 5:1 trypomastigotes of T. cruzi (Y strain) during 18 h. The activation of adenylyl cyclase with forskolin did not affect cell invasion by trypomastigotes from Y strain (P > 0.05) (A,B), but reverted CEL effects (P < 0.05) (B). Results are the mean ± standard error for duplicate determinations, with three blood donors in each experiment. Means not sharing letter are significantly different (P < 0.05, one-way ANOVA with Tukey's post-test).

Figure 7

Effects of ASA and CEL on innate inflammatory response of DC-PBMC cells infected with T. cruzi. The levels of IL-8, IL-1β, IL-6, IL-10, and TNF-α were measured following a 24-h treatment of DC-PBMC infected or not with T. cruzi. Infection induced cytokine production in DC-PBMC (B–J). The treatment with ASA or CEL did not affect TNF-α, IL-6, IL-8, IL-10 production by DC-PBMC (A,C–J), but increased IL β production by them (B,G). Representative results from at least two independent experiments are shown, with three blood donors in each experiment. Means not sharing letter are significantly different (P < 0.05, non-parametric, Kruskal-Wallis test, Dunn's post-test).

Figure 8

Nitric oxide (NO) production by DC-PBMC infected with T. cruzi. DC-PBMC were treated for 1 h with ASA (A) or CEL (B). After treatment, the cells were washed and incubated with 5:1 trypomastigotes for 24 h. Nitrite levels in the supernatant were measured by Griess reaction. Results are the mean ± standard error of duplicate determinations. Three independent experiments were performed, with six individuals in each experiment. Means not sharing letter are significantly different (P < 0.05, one-way ANOVA with Tukey's post-test).