Infected C57BL/6 mice show higher numbers of nT_reg cells than infected BALB/c mice.

<p>Non-infected (NI) BALB/c and C57BL/6 mice and mice infected with high inoculum were sacrificed at 0 (NI), 12 and 17 d.p.i. Thymocytes were counted and stained with antibodies against cell surface molecules and intracellular markers and analyzed with a flow cytometer. Data from BALB/c and C57BL/6 mice are represented by open circles and filled circles, respectively. (<b>A</b>) Anti-CD4 and anti-CD8 antibody staining of thymocytes from non-infected (NI) BALB/c mice at 12 and 17 d.p.i. (<b>B</b>) Anti-CD4 and anti-CD8 antibody staining of thymocytes from non-infected (NI) C57BL/6 mice at 12 and 17 d.p.i. (<b>C</b>) Total number of thymocytes per thymus in BALB/c (open circles) and C57BL/6 mice (filled circles). (<b>D</b>) Staining of the gated CD4⁺CD25⁺ population with anti-FoxP3 antibody in BALB/c mice. (E) Same as “D” for C57BL/6 mice. (F) Total number of FoxP3⁺ cells per thymus. Data represent the results of at least two independent experiments performed with samples pooled from 3 mice per experimental group.</p

IL-6 serum concentration significantly increased in infected BALB/c mice.

<p>BALB/c mice were infected with high inoculum (open bar) or low inoculum (open dashed bar). C57BL/6 mice were infected with high inoculum (filled bar) or low inoculum (filled dashed bar). IFN-γ and IL-6 concentrations were determined from sera extracted from non-infected (NI) mice and mice infected with (<b>A</b>) high inoculum or (<b>B</b>) low inoculum at 0 (NI), 7, 12 and 17 d.p.i. Data represent the results of at least two independent experiments performed with 3 mice per experimental group. Statistically significant differences between infected and non-infected mice (0 d.p.i.) and between BALB/c and C57BL/6 mice under each treatment are shown: *<i>p</i><0.05,**<i>p</i><0.01 and ***<i>p</i><0.001.</p

BALB/c mice infected with <i>T. cruzi</i> showed higher parasite loads and lower survival rates than infected C57BL/6 mice.

<p>BALB/c mice were infected with high inoculum (open circle) or low inoculum (open square). C57BL/6 mice were infected with high inoculum (filled circle) or low inoculum (filled square). (<b>A</b>) Survival was monitored from 0 to 100 d.p.i., (<b>B</b>) parasitemia was monitored from 0 to 35 d.p.i., and (<b>C</b>) the parasite load in heart tissue was determined at 7, 12, 17 and 22 d.p.i. in mice infected with high inoculum (top) or low inoculum (bottom) by extrapolation with parasite DNA standards. Data represent the results of at least two independent experiments performed with 3 mice per experimental group. Statistically significant differences between BALB/c and C57BL/6 mice are shown: *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001.</p

Infected BALB/c mice show a greater parasite load and more inflammation during the chronic phase than infected C57BL/6 mice.

<p>BALB/c and C57BL/6 mice were infected with the low inoculum and sacrificed at 100 d.p.i. (<b>A</b>) Specific <i>T. cruzi</i> PCR with DNA from the hearts of non-infected (NI) and infected BALB/c mice at 100 d.p.i.; parasite DNA was used as a positive control (C+) and H₂O as a negative control (C-). (<b>B</b>) As for “A” but for C57BL/6 mice. Quantitative RT-PCR from total heart tissue RNA of infected BALB/c mice (open dashed bars) and C57BL/6 mice (filled dashed bars) utilizing: (<b>C</b>) a <i>Cd4</i> probe, (<b>D</b>) <i>Ifng, Il1a, Tnf</i> and <i>Il2</i> probes, (<b>E</b>) <i>Il6</i> and <i>Il17a</i> probes and (<b>F</b>) <i>Foxp3</i>, <i>Fr4</i>, <i>Il10</i> and <i>Tgfb</i> probes. Data represent the results of at least two independent experiments performed with 3 mice per experimental group and were normalized with respect to NI mice (Fold change: 1). Statistically significant differences between infected and non-infected mice and between BALB/c and C57BL/6 mice under each treatment are shown: *<i>p</i><0.05,**<i>p</i><0.01 and ***<i>p</i><0.001.</p

Infected C57BL/6 mice showed greater numbers of cardiac infiltrating CD4⁺ T cells and more inflammation than infected BALB/c mice.

<p>BALB/c mice were infected with high inoculum (open bar) or low inoculum (open dashed bar). C57BL/6 mice were infected with high inoculum (filled bar) or low inoculum (filled dashed bar). (<b>A</b>) Immunofluorescence staining of heart tissue sections from BALB/c and C57BL/6 mice infected with high inoculum with anti-CD4 antibody at 14 d.p.i. (magnification: 630×). (<b>B</b>) Quantitative RT-PCR of total heart tissue RNA from non-infected (NI) mice and mice infected with either high inoculum (top) or low inoculum (bottom) at 7, 12, 17 and 22 d.p.i., utilizing the <i>Cd4</i> probe. (<b>C</b>) as for “B” utilizing the <i>Ifng, Il1a</i> and <i>Tnf</i> probes. Data were normalized with respect to NI mice (Fold change: 1) and represent at least two independent experiments performed with 3 mice per experimental group. Statistically significant differences between infected and non-infected mice (0 d.p.i.) and between BALB/c and C57BL/6 mice under each treatment are shown: *<i>p</i><0.05,**<i>p</i><0.01 and ***<i>p</i><0.001.</p

Th17 and T_reg cells were isolated from the hearts of BALB/c and C57BL/6 mice, respectively.

<p>BALB/c mice were infected with high inoculum (open bar) or low inoculum (open dashed bar). C57BL/6 mice were infected with high inoculum (filled bar) or low inoculum (filled dashed bar). (<b>A</b>) Percent of CD4⁺IL-17⁺ cells isolated from the hearts of BALB/c mice infected with high inoculum at 17 d.p.i. (<b>B</b>) Percent of CD4⁺CD25⁺FoxP3⁺ cells isolated from the hearts of C57BL/6 mice infected with low inoculum at 17 d.p.i. (<b>C</b>) Quantitative RT-PCR from total mouse heart tissue RNA from non-infected (NI) mice and mice infected with either high inoculum (top) or low inoculum (bottom) at 7, 12, 17 and 22 d.p.i., utilizing <i>Il6 and Il17a</i> probes. (<b>D</b>) as for (C) but utilizing <i>Tgfb, Il10</i> and <i>Il2</i> probes. (<b>E</b>) as for (C) but utilizing <i>Foxp3</i> and <i>Fr4</i> probes. For “A” and “B” the data represent the results from two experiments performed with 25 and 35 mice per group, respectively. For “C”, “D” and “E” the data were normalized with respect to NI mice (Fold change: 1) and represent the results from at least two independent experiments performed with 3 mice per experimental group. Statistically significant differences between infected and non-infected mice (0 d.p.i.) and between BALB/c and C57BL/6 mice under each treatment are shown: *<i>p</i><0.05,**<i>p</i><0.01 and ***<i>p</i><0.001.</p

<i>Trypanosoma cruzi</i> infection induces <i>Cox2</i>, <i>Tbxas1</i>, <i>Edn1</i> and <i>Nppa</i> in infected heart tissue.

<p>(A and B) C57BL/6 (<i>black circles</i>) and BALB/c (<i>white circles</i>) mice were infected with 2×10³ blood-trypomastigote forms of the Y strain. (A) Parasitemia expressed as the mean ± standard error of the mean (s.e.m.) of the number of parasites per 5 µl of blood. (B) Percent of mice survival. Results are representative of 2 independent experiments, each performed with 6 mice per group. (C) Tissue inflammation, parasitism and COX-2 expression in heart from uninfected (left panels) and <i>T. cruzi</i>-infected (21 days post-infection, right panels) mice. Representative results of histological analysis (Mason's trichrome staining) of cardiac tissue specimens from BALB/c and C57BL/6 mice (top and center panels, respectively) are shown. Bars = 100 µm. Bottom panels display representative results of COX-2 immunostaining (IS) in the hearts from C57BL/6 mice. Original magnification for microphotographs ×400. (D) <i>Cox2</i> (COX-2), <i>Tbxas1</i> (TXS), <i>Edn1</i> (ET-1) and <i>Nppa</i> (ANP) gene expression in the heart during the acute phase of infection in C57BL/6 and BALB/c mice. RNA from heart tissue at different days post-infection was used to perform RT-PCR with specific probes, and normalized to ribosomal 18S RNA as described in ‘<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002034#s2" target="_blank">Materials and Methods</a>’. Values are expressed as means ± s.e.m. from 3 independent infections, each performed with 3 mice per group. *<i>P</i><0.05. (E) Levels of circulating peptides (ET-1 and ANP) and eicosanoids (PGF_2α and TxB₂) in the sera of uninfected (<i>black bars</i>) and <i>T. cruzi</i>-infected (<i>grey bars</i>) C57BL/6 mice. Mouse sera were collected before and after 21 days of infection, and were assayed in triplicate by capture ELISA for ANP (top panel), ET-1 (central panel), PGF_2α and TxB₂ (bottom panel). Each bar represents the mean values for groups of 6 mice ± s.e.m. Similar results were obtained in two additional experiments. ^*<i>P</i><0.05; ^**<i>P</i><0.01.</p

Activation of the Ca²⁺/Calcineurin/NFAT intracellular signaling pathway in endothelin-1-stimulated and <i>Trypanosoma cruzi</i>-infected cardiomyocytes.

<p>(A) HL-1 cells, exposed or not to 0.3 nM ET-1, were loaded with the Ca²⁺ indicator Fura-2/M and changes in [Ca²⁺]_i upon <i>T. cruzi</i> infection were recorded. Uninfected cells were used as a control. Arrows indicate the time (min) when either culture medium (M) or <i>T. cruzi</i> trypomastigotes (T) was added. The results presented are representative of three independent experiments. (B) ET-1 stimulated and <i>T. cruzi</i>-infected HL-1 cardiomyocytes were disrupted and the protein expression of the four NFAT isoforms (c1 to c4) was analysed by immunoblotting. Alpha-tubulin protein levels were determined as a control of loading. (C) HL-1 cells were incubated for 2 h with ET-1 (0.3 nM) and subsequently infected with <i>T. cruzi</i> trypomastigotes for 3 h. For some experiments, FK506 (100 ng/ml) was added 1 h before stimulation. Fractionated extracts from both untreated and treated cells were analysed by immunoblotting with an antiserum to NFATc4. The phosphorylated cytosolic (P-NFATc4) or dephosphorylated nuclear (NFATc4) forms of the factor are indicated. Cyto, cytosolic extracts; Nucl, nuclear extracts. (D) Electrophoretic mobility shift assay (EMSA) analysis to determine NFATc4 binding to the NFAT sites of the <i>Cox2</i> gene (Cox-2 NFAT). HL-1 myocytes were stimulated with 0.3 nM ET-1 for 2 h and/or infected with <i>T. cruzi</i> trypomastigotes for 3 h. For some experiments, FK506 (100 ng/ml) was added 1 h before stimulation. Mock-treated cells were considered as controls. PMA (15 ng/ml) supplemented with 1 µM Ion was used as a standard stimulus. Nuclear extracts were analysed by EMSA using a Cox-2 NFAT radiolabeled probe. A 50-fold molar excess of unlabeled Cox-2 NFAT oligonucleotide (<i>T. cruzi</i>+ET-1+Cox-2 NFAT) was added to determine specific binding. NFATc4 antibody or normal rabbit IgG was added to the extracts before incubation with the probe. Arrows indicate specific supershifted complexes. This is representative of at least three independent experiments.</p

<i>Trypanosoma cruzi</i> infection of endothelin-1-pre-treated HL-1 cardiomyocytes induces cyclooxygenase-2 expression.

<p>(A) COX-2 protein expression in primary BALB/c cardiac myocytes infected with <i>T. cruzi</i>. Neonatal mouse heart cells were isolated and <i>ex vivo</i> infected with Y strain trypomastigotes (cell∶parasite ratio 1∶5) for 24 h. To obtain a positive control, the cells were incubated with 25 U/ml recombinant IFN-γ plus 1 µg/ml LPS. Uninfected cells (Mock) were used as controls. The levels of COX-2 and β-actin proteins were analysed by immunoblotting as described under ‘<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002034#s2" target="_blank">Materials and methods</a>’. (B) Effects of ET-1 pre-treatment and <i>T. cruzi</i> infection of HL-1 cardiomyocytes on <i>Cox2</i> mRNA expression. HL-1 atrial muscle cells were stimulated with 0.3 nM ET-1 for 2 h, and/or infected with <i>T. cruzi</i> trypomastigotes (cell∶parasite ratio 1∶5) for 3 h, and the levels of <i>Cox2</i> mRNA were assessed by reverse transcription and PCR; <i>Actb</i> (β-actin) was used as a loading marker. (C) Effects of ET-1 pre-treatment and <i>T. cruzi</i> infection of HL-1 cardiomyocytes on COX-2 protein expression. HL-1 atrial muscle cells were stimulated with 0.3 nM ET-1 for 2 h, and/or infected with <i>T. cruzi</i> trypomastigotes for 3 h, and the levels of COX-2 and α-tubulin proteins were analysed by immunoblotting. (D) Effects of ET-1 pre-treatment and <i>T. cruzi</i> infection of HL-1 cardiomyocytes on the inducibility of the <i>Cox2</i> promoter. Cells were transiently transfected with the P2-1900-Cox-2-LUC reporter construct, and then stimulated with 0.3 nM ET-1 for 2 h, and/or infected with trypomastigotes for 3 h. For some experiments, FK506 (100 ng/ml) was added to [<i>T. cruzi</i>+ET-1]-activated cardiomyocytes. PMA+Ion was used as a standard stimulus. Luciferase activity is expressed as fold induction relative to the transfection with empty expression vector. Data are the means ± s.e.m. of three independent experiments, each performed in triplicate. *<i>P</i><0.05. (E) Involvement of NFAT in <i>Cox2</i> induction by <i>T. cruzi</i> plus ET-1. HL-1 cells were transiently transfected with the P2-1900-Cox-2-LUC reporter construct, with the P2-274-Cox-2 promoter construct, or with the same construct containing distal and/or proximal NFAT sites (dNFAT and pNFAT, respectively), and/or actvated protein-1 (AP-1) site mutated (indicated by X). For some experiments, the cells were transiently co-transfected with the P2-274-Cox-2-LUC reporter plasmid along with a dominant-negative version of NFAT (dn-NFAT). Three hours later, the cells were stimulated with ET-1 (0.3 nM) for 2 h and infected with <i>T. cruzi</i> parasites for 3 h. Luciferase activity is expressed as percentage of induction (mean ± s.e.m.) relative to that achieved in P2-1900-Cox-2-LUC transfected cells. One out of three separate experiments performed is shown. *<i>P</i><0.05; ** <i>P</i><0.001 (respect to the P2-274 construct).</p

Time-course of <i>T. cruzi</i> infection in <i>Slamf1^−/−</i> mice and reduced <i>T. cruzi</i> infection in the hearts from <i>Slamf1−/−</i> animals.

<p>BALB/c or <i>Slamf1^−/−</i> mice were intraperitoneally infected with 2×10³ trypomastigotes of the <i>T. cruzi</i> Y strain and were sacrificed at different dpi. A) Survival. B) Serum CK levels. Analysis of <i>T. cruzi</i> presence in heart tissue from <i>T. cruzi</i> infected BALB/c or <i>Slamf1^−/−</i> animals: C) Quantification of <i>T. cruzi</i> DNA in the heart tissue of infected BALB/c- and <i>Slamf1^−/−</i> mice. <i>T. cruzi</i> DNA is expressed as the amount of parasite DNA obtained from a heart tissue sample (pg of parasite DNA/mg of heart tissue). Results are expressed as the mean values (±SD) for triplicates of pooled DNA from 5 different mice. A representative experiment of the 3 performed is shown. D) Histochemical analysis by Hematoxylin-Eosin stain. A representative field is shown. E) Quantification of the number of amastigote nests per 20 fields. F) Average number of amastigotes/nest per 20 fields. At least 20 fields were observed of each preparation (3 preparations/mouse and 3 mice per group). Results are expressed as the mean values (±SD) for 100 independent microscopic fields from 5 different mice (20 each). G) Blood parasitemia. (*) Statistically significant differences between <i>Slamf1^−/−</i> mice and BALB/c (p>0.05).</p