Additional File 4: Figure S4.

Pre-treatment of fibroblast with rTNF does not affect infection by Trypanosoma cruzi. (a) Experimental scheme showing that L-929 fibroblasts were pre-treated with TNF (1 ng/mL) and subsequently infected with trypomastigote forms of the Colombian T. cruzi strain. (b-c) The percentage of infected cells and the number of parasites per cells were analyzed at 4 h (b) and 24 h (c) of infection, at a MOI of 1:1 or 10:1. Data are presented as mean ± SEM of duplicates. (TIFF 1430 kb

Additional File 7: Figure S7.

rTNF effects on Trypanosoma cruzi/astrocyte interaction are conserved in primary astrocyte cultures of C3H/He mice. (a) Monotypic primary astrocyte cell cultures of C3H/He mice were seeded, left untreated or treated with rTNF and infected with trypomastigote forms of the Colombian T. cruzi strain at a MOI of 1:1 or 10:1. (b) After 4 h of interaction, the frequencies of infected cells and the number in untreated (NT), rTNF-treated astrocytes are shown. (c) Data show the frequencies of infected astrocytes stratified by classes according to the number of amastigote-like forms harbored in the cytoplasm. Data are presented as means ± SEM of triplicates. *, p < 0.05, ***, p < 0.001 untreated (NT) vs. rTNF-treated astrocytes. #, p < 0.05, ##, p < 0.01, ###, p < 0.001 MOI 1:1 vs. MOI 10:1. (TIFF 1337 kb

Additional File 2: Figure S2.

Astrocytes from neonatal C57BL/6 mice are susceptible to in vitro infection by Trypanosoma cruzi. (a) Monotypic astrocyte cell cultures were allowed to interact during 4 or 24 h with Vero cells-born trypomastigote forms of the Colombian T. cruzi strain, at a MOI of 1:1 or 10:1. (b-e) Astrocyte infected with T. cruzi at a MOI of 1:1 and analyzed after 4 h (b, c, d) or 24 h (e) of interaction. (f) Parasites per 100 cells and frequency of T. cruzi-infected astrocytes after 4 and 24 h of T. cruzi/astrocyte interaction at a MOI of 1:1 or 10:1 Data are presented as means ± SEM of triplicates. *, p < 0.05 and **, p < 0.01 MOI 1:1 vs. MOI 10:1 in each analyzed interval; #, p < 0.05 and ##, p < 0.01 4 h vs. 24 h of infection in each analyzed MOI. (TIFF 1349 kb

Additional File 1: Figure S1.

Primary cell cultures of C57BL/6 cerebral cortex are enriched in astrocytes. CNS cells of seven to ten days of primary culture were seeded at a density of 105 cells per 13-mm-diameter coverslip and analyzed 24 h later. (a, b, c) Giemsa staining reveals cells resembling astrocytes with thin extensions, delicate branches, recognizable nucleoli and cytoplasm lightly stained. (d, e, f) Simultaneous immunohistochemical staining for GFAP and CD11b was used to determine the cell composition of primary astrocyte cultures. (e) Insert showing RAW 264.7 mouse macrophage lineage used as positive control for CD11b staining. (TIFF 1266 kb

Additional File 5: Figure S5.

Pre-exposing astrocyte cultures to rTNF enhances parasite load in Trypanosoma cruzi-infected astrocytes. C57BL/6 primary astrocyte cultures were seeded, exposed to rTNF and infected as described in Fig. 5a. At 24 and 48 h of infection, the frequencies of infected astrocytes were stratified by classes according to the number of amastigote-like forms harbored in the cytoplasm. Data are presented as means ± SEM of triplicates. *, p < 0.05, **, p < 0.01 untreated (NT) vs. rTNF-treated astrocytes. (TIFF 1045 kb

Additional File 6: Figure S6.

rTNF amplifies the pro-inflammatory profile of human astrocytes exposed to Trypanosoma cruzi infection. Human astrocytes were submitted to treatment with rTNF and left non-infected (NI) or infected by T. cruzi (MOI 1:1). At 4 h of infection, supernatants were collected and submitted to detection of cytokines by CBA. Data are presented as means ± SEM of triplicates. *, p < 0.05, untreated (NT) vs. rTNF-treated astrocytes. (TIFF 1375 kb

Pentoxifylline Reverses Chronic Experimental Chagasic Cardiomyopathy in Association with Repositioning of Abnormal CD8⁺ T-Cell Response

<div><p>Background</p><p>Chronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8⁺ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8⁺ T-cell abnormalities and cardiac alterations using a model of experimental Chagas’ heart disease.</p><p>Methodology/Principal Findings</p><p>C57BL/6 mice chronically infected by the Colombian <i>Trypanosoma cruzi</i> strain and presenting signs of CCC were treated with PTX. The downmodulation of T-cell receptors on CD8⁺ cells induced by <i>T</i>. <i>cruzi</i> infection was rescued by PTX therapy. Also, PTX reduced the frequency of CD8⁺ T-cells expressing activation and migration markers in the spleen and the activation of blood vessel endothelial cells and the intensity of inflammation in the heart tissue. Although preserved interferon-gamma production systemically and in the cardiac tissue, PTX therapy reduced the number of perforin⁺ cells invading this tissue. PTX did not alter parasite load, but hampered the progression of heart injury, improving connexin 43 expression and decreasing fibronectin overdeposition. Further, PTX reversed electrical abnormalities as bradycardia and prolonged PR, QTc and QRS intervals in chronically infected mice. Moreover, PTX therapy improved heart remodeling since reduced left ventricular (LV) hypertrophy and restored the decreased LV ejection fraction.</p><p>Conclusions/Significance</p><p>PTX therapy ameliorates critical aspects of CCC and repositioned CD8⁺ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas' heart disease and to improve prognosis.</p></div

IFNγ⁺ and Pfn⁺ cells differentially invade the cardiac tissue of <i>T. cruzi</i>-infected C57BL/6 mice.

<p>Mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and the presence of IFNγ⁺ and Pfn⁺ cells in the cardiac tissue was evaluated by IHS. (<b>A</b>) Numbers of IFNγ⁺ cells infiltrating the cardiac tissue at 15, 30, 45, 60, 90 and 120 dpi. (<b>B</b>) Numbers of Pfn⁺ cells infiltrating the cardiac tissue at 15, 30, 45, 60, 90 and 120 dpi. (<b>C</b>) Relationship between IFNγ⁺ cells infiltrating the cardiac tissue and heart parasitism or CK-MB activity in serum. (<b>D</b>) Relationship between Pfn⁺ cells infiltrating the cardiac tissue and heart parasitism or CK-MB activity in the serum. (<b>E</b>) Immunohistochemistry staining of IFNγ⁺ (green arrows) and Pfn⁺ (red arrows) cells infiltrating the cardiac tissue at 60 dpi. In (<b>A</b>) and (<b>B</b>), each circle represents an individual mouse. In (<b>C</b>) and (<b>D</b>), each circle represents the mean ± SD of the studied group (5–7 animals/time point). These data represent three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.01; and ^***, <i>p</i><0.001, comparing noninfected (NI) controls and <i>T. cruzi</i>-infected mice. ^#, <i>p</i><0.05, comparing <i>T. cruzi</i>-infected mice at 60 and 120 dpi.</p

Distinct migratory behavior and effector function of CD8⁺IFNγ⁺ and CD8⁺Pfn⁺ T-cells in <i>T. cruzi</i> infection.

<p>(<b>A</b>) Experimental scheme of CD8⁺ cell isolation from noninfected naïve <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors, <i>in vivo</i> reconstitution of the CD8⁺ cell compartment of naïve <i>cd8</i>^−/− mice, infection with 100 bt of the Colombian strain at 15 days after cell transfer (dact) and analysis at 30 days post-infection. Parasitemia, survival rate, cardiac parasitism and CK-MB activity levels in the serum and colonization of the cardiac tissue by IFNγ⁺ and Pfn⁺ cells were evaluated. (<b>B</b>) Parasitemia and (<b>C</b>) survival curve of <i>cd8</i>^−/− mice non-reconstituted (NR) or reconstituted with CD8⁺ cells from <i>ifnγ</i>^+/+<i>pfn</i>^−/− and <i>ifnγ</i>^−/−<i>pfn</i>^+/+ donors. In independent experiments, the animals were analyzed at 30 dpi when 100% of the mice in all the experimental groups were alive (arrow). (<b>D</b>) Number of amastigote nests in 100 microscopic fields of cardiac tissue sections. (<b>E</b>) Cardiomyocyte lesion evaluated by CK-MB activity in serum samples. The number of (<b>F</b>) IFNγ⁺ and (<b>G</b>) Pfn⁺ cells in 100 microscopic fields of cardiac tissue sections from mice non-reconstituted (NR) or reconstituted with CD8⁺ cells from <i>ifnγ</i>^+/+<i>pfn</i>^−/− and <i>ifnγ</i>^−/−<i>pfn</i>^+/+ donors, at 30 dpi. Each symbol represents an individual mouse. These data represent three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.01; and ^***, <i>p</i><0.001, comparing <i>cd8</i>^−/− infected mice non-reconstituted and reconstituted with CD8⁺ cells from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors.</p

CD8⁺ T-cells recognizing the VNHRFTLV ASP2 <i>T. cruzi</i> peptide are enriched in the cardiac tissue.

<p>C57BL/6 mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and the anti-parasite immune response in the spleen and cardiac tissue was assessed. (<b>A</b>) Representative spots formed after stimulation of spleen cells from noninfected (NI) and <i>T. cruzi</i>-infected mice at 45 and 120 dpi with H-2K^b-resctricted VNHRFTLV peptide. The number of CD8⁺IFNγ⁺ as determined by <i>ex vivo</i> ELISpot were analyzed and compared with parasitemia and CK-MB activity levels in the serum. (<b>B</b>) Representative histogram profiles of <i>in vivo</i> cytotoxicity assay showing the specific lysis of H-2K^b-resctricted VNHRFTLV peptide-labeled CFSE^high cells from NI controls and <i>T. cruzi</i>-infected C57BL/6 mice at 45 and 120 dpi. The frequencies of <i>in vivo</i> specific lysis of with H-2K^b -resctricted VNHRFTLV peptide-labeled target cells in <i>T. cruzi</i>-infected C57BL/6 mice at 15, 30, 45, 60, 90 and 120 dpi were analyzed and compared with parasitemia and CK-MB activity levels in the serum. The peak of parasitemia and the maximum CK-MB activity are highlighted (dotted line). Each circle and vertical lines represent the mean ± standard deviation (SD) of the studied group (5–7 animals/time point). These data represent three independent experiments. (<b>C</b>) Frequency of double-positive CD8⁺ H-2K^b /VNHRFTLV⁺ cells [R1 (SSCxFSC) gated] of spleen and heart of NI and <i>T. cruzi</i>-infected C57BL/6 mice at 40 dpi. (<b>D</b>) Frequencies of double-positive CD8⁺ H-2K^b /VNHRFTLV⁺ cells [R1 (SSCxFSC) gated] of spleen, peripheral blood and heart of NI and <i>T. cruzi</i>-infected C57BL/6 mice at 30, 45 and 120 dpi. Representative flow ctometry profiles and mean ± SD of two or three animals per group. Bars represent the mean of two or three pools of 5 mice per pool.</p