To prepare tissue culture cells for infection, DH82 and RAW264.7 cells (ATCC) were grown in 75 cm² tissue culture flasks (T75) (Nunc, Roskilde, Denmark) with Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Gibco), 100 μg/mL of streptomycin and 100 Units/mL of penicillin, 1% MEM non-essential amino acids (Sigma-Aldrich), and 2 mM of l-glutamine were cultured at 37°C with 5% CO₂. Subcultures of both cell lines were carried out once a week, when they reached a confluence of 95–100%. Cells were released with 0.25% of trypsin (Difco) and 1 mM of EDTA (Sigma-Aldrich), harvested, and washed twice in HBSS by centrifugation at room temperature for 10 min at 250g. The supernatant was discarded, cell pellet was resuspended in DMEM containing 10% FCS, cultured into new T75 flasks, and culture medium was changed after 3 days in culture. Prior to use in infectivity assays, cells’ viability was assessed by trypan blue exclusion in a hemocytometer.

Infection of DH82 and RAW264.7 cell lines were carried out in 24-well plates at a concentration of 2.0 × 10⁵ macrophages/well. Cells were infected overnight with chemically induced metacyclic trypomastigotes forms of T. cruzi clone Dm28c, obtained as previously described (24), at a 1:1, 3:1, and 5:1 parasite-to-cell ratios in 1 mL of DMEM 10% FCS and incubated at 37°C in 5% CO₂. In the following day (day 1), monolayers were extensively washed to remove extracellular parasites and cultured with complete culture medium containing 1% Nutridoma (Sigma-Aldrich) instead of FCS. Some cultures were stimulated with 400 ng/mL of lipopolysaccharides from Salmonella enterica serotype typhimurium (LPS, Sigma-Aldrich) and 1.5 ng/mL of recombinant canine or murine IFN-γ (Serotech) 24 h after infection. DH82 and RAW264.7 were also seeded in 24-well plate containing glass coverslips for parasite burden evaluation. Three days after infection, coverslips were washed with HBSS, fixed with methanol, and stained with Diff-Quick (Thermo Fisher, Waltham, MA, USA). Amastigotes were counted at 100× oil immersion on a (Olympus) microscope. The number of amastigotes was estimated in 100 infected cells per coverslip, and the frequency of infection was compared among six coverslips per time point. The number of viable trypomastigotes released in the supernatants was evaluated each 2 days of culture for 9 days, using a Neubauer chamber. We also conducted experiments using T. cruzi of CL strain transfected with green fluorescent protein (GFP) (25) obtained from Vero cells. The DH82 cells were infected overnight (ON). After that, the monolayers were washed, and DH82 cells were stained with DAPI 1.5 μg/mL (VECTASHIELD^®) at room temperature for 30 min before examination by confocal microscopy Zeiss Axioplan.

Intracellular levels of ROS were quantified by oxydation of non-fluorescent 2′,7′-dichlorofluorescein probe, delivered as diacetate form (DCFH-DA), to the fluorescent product 2′,7′-dichlorofluorescein (26). DH82 cells were seeded in 96-well plate (5.0 × 10⁴ cells/well), infected with metacyclic trypomastigotes at a ratio of 5:1 (parasite/cell), stimulated or not with LPS (400 ng/mL, Sigma-Aldrich) and recombinant IFN-γ (1.5 ng/mL, Serotech) and after 1, 2, 3, and 4 h later cells were washed and loaded with 10 μM DCFH-DA (Invitrogen) for 20 min at 37°C. Uninfected cells were used as negative control. In different checkpoints, the cells were washed, and fluorescence was measured (excitation = 485 nm excitation; λ emission = 535 nm) in an FLx800 Fluorescence Microplate Reader (BioTek).

Quantification of NO DH82 and RAW264.7 (2.5 × 10⁵/mL) was cultivated in the presence of LPS (200 or 400 ng/mL) and INF-γ (1.5 ng/mL). After 12 and 24 h of incubation, NO production was evaluated by the presence of the nitrite accumulated in the supernatant of cultures using Griess colorimetric method.

In order to evaluate the effect of apoptotic lymphocytes in T. cruzi growth in vitro, we have used two approaches. In both approaches, DH82 macrophages were seeded into 24-well plate at a concentration of 2.0 × 10⁵ cells/well, incubated for 1 h at 37°C in a humidified atmosphere with 5% CO₂ and the adherent monolayer was washed to remove non-adherent cells. Apoptotic cells were prepared as previously described (27). Briefly, single cell suspension of the Jurkat human lymphoblastoid cells was prepared and irradiated with 30 Gy (Apo-2) as previous described (27). We first performed experiments with T. cruzi-infected DH82 monolayers at a 5:1 parasite/cell ratio. Extracellular parasites were removed by washing the wells with HBSS and then apoptotic lymphocytes were added to the cultures at a 5:1 apoptotic lymphocyte:macrophage ratio, and kept at 37°C with 5% CO₂ for 9 days. In other experiments, prior to T. cruzi infection, the DH82 monolayer were incubated with apoptotic lymphocytes at a 5:1 apoptotic lymphocyte:macrophage ratio for 2 h at 37°C with 5% CO₂. After that period non-internalized apoptotic cells were removed and adherent macrophages were infected with T. cruzi at 5:1 parasite:macrophage ratio for ON at 37°C. After removing free parasites, the medium was replaced and infected DH82 cells were further incubated at 37°C with 5% CO₂. In both experiments, the extracellular motile trypomastigotes were determined in cell supernatants after 9 days in culture by counting parasites in Neubauer chambers (28).

Cell supernatants were collected at 24 h postinfection for cytokine determination. TNF-α, TGF-β, and IL-10 concentration was estimated by the method of sandwich immunoassay (ELISA) according to methodology recommended by the manufacturer (R&D). The optical density was evaluated by reading in a microplate spectrophotometer (Versamax Microplates Reader Molecular Devices, USA), with filter of 405 nm.

To assess the expression of MHC class II and CD80 on DH82 macrophage cell line by flow cytometric analysis, cells were infected or not with T. cruzi Dm28c and were activated or not in the presence of recombinant IFN-γ and LPS. Cells were detached at 24 h postinfection, washed, adjusted to a concentration of 5.0 × 10⁵ cells/tube and blocked with anti-CD16/CD32 (Calbiochem) at concentration of the 1 μg/10⁶ cells to prevent non-specific antibody binding to Fc-receptors. Cells were stained with MHC class II-FITC, and CD80-PE antibodies (Serotech). All washing steps were performed with phosphate-buffered saline containing 3% bovine serum albumin and 0.02% of sodium azide. Data were acquired (10,000 events), evaluated on FACSCalibur™ cytometer, and analyzed using Cell-Quest^® software (BD Biosciences, Heidelberg, Germany). Analyzed gates of scattered dot plots were adjusted not to exceed 2% of positive staining cells related to the particular negative controls (29).

Statistical analysis was performed in the program GraphPad InStat version 3.01 (San Diego, USA). Data were analyzed by the method of Student’s t-test. Differences at p-value 0.05 or lower were reported as significant for a given comparison.

Previously published studies report T. cruzi infection of various mouse macrophage cell lines to study the infectivity of T. cruzi strains in vitro (30), but infection of dog macrophages has not been showed. In order to evaluate the susceptibility of DH82, a canine macrophage cell line, to T. cruzi Dm 28c infection, cells were plated and infected at different parasite:cell ratios. We found that canine macrophages were infected and after 5 days in culture parasite intracellular multiplication was observed (Figures 1A–C). Analyzing the rate of infected cells, we found that 33% of DH82 cells were infected at a parasite-to-cell ratio of 5:1. (Figure 1D). After 7 days in culture a large number of trypomastigotes were counted in the supernatants (Figures 1E,F). To further confirm DH82 macrophage cell line infection, we have also used T. cruzi of CL strain transfected with GFP and observed the adhesion, internalization (data not shown) and differentiation into intracellular amastigotes 5 days after infection (Figure 1C). Our results showed, for the first time, that the DH82, a canine macrophage cell line is susceptible to infection with T. cruzi, being able to provide an enabling environment for replication and differentiation of the parasite and the release of viable infective forms.

Since we have found that dog DH82 tissue culture macrophages could be infected with T. cruzi Dm28c, we decided to compare the rate of infection, the replication of amastigotes, and the number of trypomastigotes released in cultures of dog DH82 and mouse RAW264.7 macrophages. For this purpose, DH82 cells and RAW264.7 cells were infected at a parasite/cell ratio of 10:1 and cultivated for 9 days. At the first 3 days of infection, motile trypomastigotes forms were observed in supernatants of DH82 macrophages cell line, but not in RAW264.7 cells. After 8 to 10 days postinfection, it was observed a higher number of trypomastigotes in DH82 culture supernatants than in RAW264.7. It is important to point out that canine DH82 cells infected with T. cruzi were able to support the infection for the entire infection period. In contrast, T. cruzi replication inside murine macrophage RAW264.7 cells died 7 days after infection whereas the DH82 cells showed loss of viable morphology after 9 days of infection. These findings indicate that DH82 cells are more susceptible to infection by T. cruzi than RAW264.7 cell line (Figure 2).

The outcome of infection is determined by the activation status of macrophages. In the present study, we have used classical macrophage activators such as LPS and INF-γ. Therefore, DH82 cells were plated, pretreated or not with LPS or LPS + IFN-γ, and were infected with T. cruzi. Following incubation for 9 days, the trypomastigotes forms released in the supernatants were counted. We demonstrated that LPS stimulated macrophages showed a reduced parasite burden in 50%. Furthermore, we demonstrated that pretreatment of mouse macrophages with LPS and IFN-γ resulted in low levels of macrophage infection by T. cruzi, suggesting that previous macrophage stimulation was able to effectively control parasite replication (Figure 3).

Since ROS production is a crucial event associated with in vitro microbicidal activity in macrophages (17), we measured the production of ROS to evaluate if it could interfere in intracellular parasite replication of DH82 cells infected by T. cruzi. The amount of ROS produced was measured in cultures that were infected or not, in the presence or absence of LPS and IFN-γ. Increased ROS production was similar in activated and non-activated infected cells as well as in uninfected and activated cells (Figure 4). Taking into consideration that stimulation of DH82 canine macrophages with LPS and IFN-γ restricted intracellular replication and ROS production was elevated, associated with the fact that ROS release was also observed in T. cruzi-infected and unactivated DH82 cells at similar level suggests that, in our system, ROS is not involved in limiting parasite replication inside DH82 cells infected by T. cruzi.

Cytokines play a fundamental role in mediating interaction between macrophages, T lymphocytes, and other cells from the immune system in order to establish an immune response to control disease progression or in maintaining a persistent infection. Depending on the type of cytokines produced, intracellular parasites can be eliminated or can favor its permanence in the infected host (31). Here, we analyzed the TNF-α, TGF-β, and IL-10 cytokines production in the supernatants of T. cruzi-infected DH82 cells after overnight stimulation or not with LPS and IFN-γ. Our results showed that secretion of TNF-α production was detected in T. cruzi-infected DH82 macrophages at similar levels observed in stimulated macrophages (Figure 5A). However, with stimulation by LPS and IFN-γ, infected cells exhibited TNF-α production and reached higher levels as seen in Figure 5A. With regard to IL-10, maximal production was also detected in stimulated and infected DH82 macrophages (Figure 5C). LPS and IFN-γ treatment of DH82 resulted in TGF-β secretion but T. cruzi-infected and stimulated macrophages triggered a substantially higher level of release TGF-β in DH82 cell than was observed with in T. cruzi-infected macrophages only (Figure 5B). The increased TGF-β production observed in infected DH82 cells indicate that TGF-β might be involved in the success of DH82 cell infection by T. cruzi.

The ability to express MHC class II and CD80 on surface confer to macrophage the status of antigen-presenting cell (APC). To be efficient APC during infection is very important to maintain unchanged the expression levels of these molecules (32). In order to study possible alterations resulting from T. cruzi infection that could lead to any modification in these molecules expression, DH82 canine macrophage cell line was infected or not and cultured in the presence or not of some activating factors, such as LPS and IFN-γ. After 24 h in culture, cells were harvested and labeled with monoclonal antibodies directed against MHC-II and CD80 and the expression was evaluated by flow cytometry analysis. The results showed that upon stimulation, the levels of surface MHC-II molecules increased but when DH82 macrophages were infected with T. cruzi the cells expressed lower levels of MHC II (Figures 6A,B). Upregulated surface expression of CD80 was also observed in DH82 stimulated and uninfected cells. On the other hand, low level of CD80 expression could be detected in T. cruzi-infected cells (Figures 6C,D). These data suggest that parasite infection inhibit the ability of DH82 canine macrophage cells to up regulate the expression of both molecules crucial to act as effective APCs.

Taken together, our results indicate that the DH82 canine macrophage cell line is susceptible to infection by T. cruzi, able to sustain the infection for long time when compare with RAW264.7, a murine macrophage cell line, support replication and differentiation to the parasite, culminating with the release of the infective forms that can be used as in vitro study to explore parasite-host interaction. Furthermore, the infection of activated cells induced production of cytokines known as deactivating factors for macrophage like TGF-β and IL-10, that when associated with decreased expression of MHC II and CD80 represent a powerful mechanism to modulate parasite growth in this cell line.

Phagocytosis of apoptotic cells by T. cruzi-infected macrophages has the potential to down modulate macrophages leading to an anti-inflammatory response that results in increased parasite replication (22). In this context, we tested the modulatory effect of apoptotic cells on T. cruzi-infected DH82 canine macrophages. DH82 cell monolayers were cocultivated with apoptotic Jurkat T cells before or after infection with T. cruzi. As seen in Figure 4, the quantification of viable parasites released in the supernatants was made 9 days after infection. Cultures that received apoptotic cells before or after T. cruzi infection showed a significant increase in the number of released parasites (Figure 7), indicating similar modulation as previously described for mouse peritoneal macrophages.