Canine Macrophage DH82 Cell Line As a Model to Study Susceptibility to Trypanosoma cruzi Infection

Abstract

Trypanosoma cruzi is an obligatory intracellular protozoan parasite, and it is the etiological agent of Chagas' disease that is endemic in the Americas. In addition to humans, a wide spectrum of mammals can be infected by T. cruzi, including dogs. Dogs develop acute and chronic disease, similar to human infection. T. cruzi can infect almost all cell types and after cell invasion, the metacyclics trypomastigotes localize in the cytoplasm, where they transform into amastigotes, the replicative form of T. cruzi in mammals. After amastigote multiplication and differentiation, parasites lyse host cells and spread through the body by blood circulation. In this work, we evaluated the in vitro ability of T. cruzi to infect a canine macrophage cell line DH82 compared with RAW264.7, a murine tissue culture macrophage. Our results have shown that the T. cruzi is able to infect, replicate and differentiate in DH82 cell line. We observed that following treatment with LPS and IFN-γ DH82 cells were more resistant to infection and that resistance was not related reactive oxygen species production in our system. In this study, we also found that DH82 cells became more susceptible to T. cruzi infection when cocultured with apoptotic cells. The analysis of cytokine production has showed elevated levels of the TGF-β, IL-10, and TNF-α produced by T. cruzi-infected canine macrophages. Additionally, we demonstrated a reduced expression of the MHC class II and CD80 by infected DH82 cell line.

Keywords: Trypanosoma cruzi; chagas disease; dogs; immunomodulation; infection model; macrophages; susceptibility.

Figure 1

Infectivity of Trypanosoma cruzi in canine macrophage DH82 cell line. DH82 cells were cultured (2.5 × 10⁵/mL) with trypomastigotes forms of T. cruzi Dm28c clone. After overnight incubation, the cell culture was washed, and phagocyte was cultured for another 3 days with Dulbecco’s Modified Eagle Medium (DMEM) at 37°C. After this period, cells were stained and amastigotes inside the macrophages were counted under the light microscope (A) and set the percentage of infected cells (B). Infected macrophage displaying amastigotes after 3 days of infection with T. cruzi staining with Diff-Quick (C) and infection with CL strain green fluorescent protein (GFP) (D) observed by confocal microscope. To quantify trypomastigotes forms in the supernatants, the cells were infected with trypomastigotes forms of T. cruzi Dm28c clone. After overnight incubation the cell culture was washed and phagocyte were cultured for another 9 days with DMEM at 37°C. The trypomastigotes forms were quantified in the supernatant of the cultures of infected DH82 macrophages after 7 days (E) and 9 days (F). All cultures were performed in triplicate and bars show the mean + SD.

Figure 2

Curve of growth of Trypanosoma cruzi in DH82 and RAW264.7 cell line. DH82 and RAW 264.7 macrophages were cultured (2.5 × 10⁵/mL) with trypomastigotes forms of T. cruzi Dm28c clone. After overnight incubation, the cell culture was washed, and phagocyte was cultured for another 3 days with Dulbecco’s Modified Eagle Medium (DMEM) at 37°C. After 3 days, we start the quantification of the trypomastigotes forms in the supernatants of the cultures. All cultures were performed in triplicate and bars show the mean + SD. Statistical analysis was performed by t-test from representative results of three similar experiments (*p ≤ 0.05).

Figure 3

Activated DH82 macrophages decreased trypomastigotes release. DH82 macrophages were cultured (2.5 × 10⁵/mL). The macrophages were infected with Trypanosoma cruzi Dm28c clone. Some cultures were stimulated with LPS (400 ng/mL) and INF-γ (1.5 ng/mL) for 24 h. After 9 days of infection was quantified the number of the trypomastigotes forms in the supernatants of the cultures. All cultures were performed in triplicate and bars show the mean + SD. Statistical analysis was performed by t-test from representative results of three similar experiments (*p ≤ 0.05).

Figure 4

The production of reactive species of oxygen (ROS) does not affect the infectivity of Trypanosoma cruzi in DH82 cells. DH82 macrophages were cultured (5.0 × 10⁴/mL) and incubated with H2DCFDA, followed by washing and some cultures were stimulated with LPS (400 ng/mL) or LPS and INF-γ (1.5 ng/mL) in indicated time. All cultures were performed in triplicate are shown as the mean arbitrary fluorescence units (AU) + SD. Statistical analysis was performed by t-test from representative results of three similar experiments (*p ≤ 0.05).

Figure 5

Modulation of cytokines production in DH82 cells by Trypanosoma cruzi infection. DH82 macrophages (2.5 × 10⁵/mL) were infected with trypomastigotes forms of T. cruzi Dm28c clone. After infection the cells were washed and some cultures were stimulated with LPS (400 ng/mL) and INF-γ (1.5 ng/mL). After 24 h, the supernatant was collected and TNF-α (A), TGF-β (B), and IL-10 (C) were measured by ELISA. All cultures were performed in triplicate and bars show the mean + SD. Statistical analysis was performed by t-test from representative results of three similar experiments (*p ≤ 0.05, **p ≤ 0.01).

Figure 6

Measurement of MHC class II and CD80 expression in infected DH82 by Trypanosoma cruzi. DH82 macrophages (5.0 × 10⁵/mL) were infected with trypomastigotes forms of T. cruzi Dm28c clone. After infection the cells were washed and some cultures were stimulated with LPS (400 ng/mL) and INF-γ (1.5 ng/mL). After 24 h, the cells were prepared for analysis by flow cytometry. The M1 marker shows the percentage of cells identified by MHC calss II (A,B) or CD80 (C,D). The data are representative of three independent experiments.

Figure 7

Coculture of apoptotic cells with DH82 cells exacerbates infection by Trypanosoma cruzi. DH82 macrophages (2.5 × 10⁵/mL) were exposed to apoptotic T cells (five apoptotic cells for each DH82) 1 h before infection (Apo + T. cruzi) or infected and exposed to apoptotic T cells (T. cruzi + Apo). After 9 days, the trypomastigotes forms in the supernatants were counted. All cultures were performed in triplicate and bars show the mean + SD. Statistical analysis was performed by t-test from representative results of three similar experiments (*p ≤ 0.05).