<i>Trypanosoma cruzi</i> Needs a Signal Provided by Reactive Oxygen Species to Infect Macrophages

<div><p>Background</p><p>During <i>Trypanosoma cruzi</i> infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during <i>T</i>. <i>cruzi</i> infection and the results obtained are sometimes contradictory. <i>T</i>. <i>cruzi</i> has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress.</p><p>Methodology/Principal Findings</p><p>In order to investigate the contribution of ROS in <i>T</i>. <i>cruzi</i> survival and infection, we utilized mice deficient in the gp91^phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from <i>Escherichia coli)</i> or TcMTH (from <i>T</i>. <i>cruzi</i>), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and <i>in vivo</i>.</p><p>Conclusions</p><p>Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to <i>T</i>. <i>cruzi</i> growth inside the cells.</p></div

Inhibition of ROS production in macrophages impairs <i>T</i>. <i>cruzi</i> proliferation.

<p>Inflammatory macrophages from C57BL/6 WT and Phox KO mice were incubated with 300μM of apocynin (APO), 1mM of N-acetyl-cysteine (NAC), 25U of superoxide dismutase-polyethylene glycol (SOD) and catalase-polyethylene glycol (CAT) 2 hours before the infection with different parasites. Parasites were added for another 2 hours, the cells were washed to remove extracellular parasites, and ROS inhibitors were added back to the cultures. <b>(A,B)</b> Slides were stained and counted to determine the infection index 72 hours after infection. A minimum of 200 macrophages were counted per group in triplicates. The number of Y strain parasites (<b>C, D</b>) and TcWT, TcMTH <b>(E)</b> in supernatants was quantified four days after infection <b>(C)</b> or between the third and sixth day after infection <b>(D, E)</b>. Data shown are representative one of three independent experiments performed in triplicates. All data are presented as the means ± standard deviation. In <b>A</b>, bars labeled with different letters are statistically different, same letters indicate that the values are not different statistically. * indicates statistical differences between marked bars <b>(B,C)</b> or between apocynin-treated and non-treated cultures <b>(D, E)</b>. Means were considered different if p<0.05, by two-way ANOVA test and Bonferroni post-test.</p

Production of reactive oxygen species by macrophages stimulated with Y strain of <i>T</i>. <i>cruzi</i>.

<p>Thioglycolate-elicited macrophages were harvested from the peritoneal cavity of C57BL/6 WT and Phox KO mice 4 days after stimulation. Reactive oxygen species production by macrophages was detected by luminol. Macrophages previously incubated with apocynin (APO), catalase (CAT), superoxide dismutase (SOD) and N-acetyl-cysteine (NAC) for 2 hours were incubated with 0.5mM of luminol in culture medium and exposed to <i>T</i>. <i>cruzi</i> trypomastigotes. Chemiluminescence was continuously measured immediately after <i>T</i>. <i>cruzi</i> addition to the macrophage monolayer, and the area under the obtained curves was calculated. (A, C) Graphs showing area under curve, data represent mean of triplicates ± S.D. of total counts in 120 min. (B, D) Graphs showing chemiluminiscence rates, data represent means of triplicate counts in 120 min, standard deviations were omitted for clarity. The graphs are representative of five independent experiments performed in triplicate (cells were pooled from three mice for each replicate). * refers to significant differences from the infected non-treated macrophages. Bars marked by different letters are statistically different (p<0.05, one-way ANOVA test with Bonferroni post-test).</p

The enzyme MTH prevents 8-oxodGTP incorporation.

<p>Epimastigotes were treated with 200μM H₂O₂ for 30 minutes, fixed and 8-oxoG incorporation in DNA was evaluated with streptavidin-Alexa Fluor 488 by flow cytometer. Histogram overlays and MFI values plotted in the table are from one representative experiment three independent experiments with similar results.</p

Parasitemia and mortality from C57BL/6 WT and Phox KO mice infected with TcWT and TcMTH parasites.

<p>Mice were infected into the peritoneum with 1x10⁶ parasites. <b>(A)</b> Mean parasitemia (n = 4) and <b>(B)</b> Mortality (n = 5). Results are representative of three independent experiments, * indicates significant differences between points, p<0.05.</p

Growth of parasites with enhanced 8-oxod-GTP pyrophosphohydrolase activity in macrophages.

<p>Inflammatory macrophages obtained from peritoneal cavity of C57BL/6 WT mice were infected with wild type and modified parasites. The cells were washed to remove extracellular parasites and either fixed or re-incubated with medium for different times. <b>(A)</b> Number of parasites per macrophage. <b>(B)</b> Number of infected macrophages per total macrophages. <b>(C)</b> Infection index for each parasite population. <b>(D)</b> Number of parasites released into the macrophage culture supernatant between the third and seventh day after infection. Data shown are from one representative of three independent experiments performed in triplicate (cells were pooled from three mice for each replicate). All data are presented as the mean ± standard deviation of triplicates. * indicates significant differences between marked bars or points, p<0.05, two-way ANOVA test with Bonferroni post-test. A replicate of this experiment is presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004555#pntd.0004555.s001" target="_blank">S1 Fig</a>.</p

<i>T</i>. <i>cruzi</i> needs a signal provided by ROS to multiply efficiently.

<p><b>(A)</b> Inflammatory macrophages obtained from Phox KO and C57BL/6 WT mice were incubated with 1mM of N-acetyl-cysteine (NAC) and 100μM H₂O₂ 2 hours before the infection with different parasites. Cells were washed to remove extracellular parasites and either fixed or re-incubated with medium or medium plus NAC or H₂O₂ for different times. The slides were stained and counted to determine the infection index. <b>(B)</b> Inflammatory macrophages from C57BL/6 WT and Phox KO mice were infected with CL Brenner strain of <i>T</i>. <i>cruzi</i> previously treated with 50μM H₂O₂ for 30 minutes. Cells were washed to remove extracellular parasites and either fixed or re-incubated with medium. The slides were stained and counted to determine the infection index. Data shown are representative one of three independent experiments performed in triplicate. A minimum of 200 macrophages were counted per group in triplicate. All data are presents as the means ± standard deviation. * indicates significant differences between marked bars or points, p<0.05, two-way ANOVA test with Bonferroni post-test.</p

ROS in lower concentrations promote parasite increase in macrophages and mice.

<p><b>(A)</b> Inflammatory macrophages obtained from Phox KO and C57BL/6 WT mice were infected with <i>T</i>. <i>cruzi</i> previously treated with different concentrations of H₂O₂ for 30 minutes. After 2 hours of infection, cells were washed to remove extracellular parasites and re-incubated with medium for 72 hours. The slides were stained and counted to determine the infection index. A minimum of 200 macrophages were counted per group in triplicate. Data shown are representative one of three independent experiments performed in triplicate. All data are presents as the means ± standard deviation. * indicates significant differences between marked bars or points, p<0.05, two-way ANOVA test with Bonferroni post-test. <b>(B)</b> C57BL/6 WT mice were infected with 1x10⁵ bloodstream parasites of Y strain treated or not with H₂O₂ for 30 minutes before infection. Parasitemia levels were evaluated (n = 5). Results are representative of three independent experiments, * indicates significant differences between points, p<0.05.</p

TcMTH and EcMutT parasites effectively decreased DHR oxidation and show increase in antioxidant enzyme expression.

<p><i>T</i>. <i>cruzi</i> epimastigotes (TcWT, EcMutT and TcMTH) were treated with 200μM H₂O₂ for 30 minutes. <b>(A)</b> Parasites were pre-loaded with DHR (50 μM), exposed to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1, 0.1mM) and intracellular RH 123 was measured by flow cytometry. Histogram overlays and MFI values plotted in the table are representative one of three independent experiments with similar results. <b>(B)</b> Cytosolic tryparedoxin peroxidase in parasite extracts was detected by Western Blot using an anti-TcCPX specific antibody. Results are expressed as the density of bands in the Western Blot, pooled results from three experiments performed. A representative Western Blot is in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004555#pntd.0004555.s002" target="_blank">S2 Fig</a>. <b>(C)</b> Mitochondrial tryparedoxin peroxidase, <b>(D)</b> superoxide dismutase B and <b>(E)</b> cruzipain were detected by anti-TcMPX, anti-SODB, and anti-cruzipain specific antibodies, respectively, by flow cytometry. Histogram overlays and MFI values plotted in the table are representative one of three independent experiments with similar results.</p

NO production by Phox KO and C57BL/6 WT macrophages.

<p>Inflammatory macrophages obtained from peritoneal cavity of C57BL/6 WT and Phox KO mice were stimulated with IFN-γ/LPS and infected with <i>T</i>. <i>cruzi</i>. After 48 hours, NO production was quantified by the Griess reaction.</p