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

PTX modulated the expression of molecules involved in cell migration and reduced heart inflammation.

<p>(A) Frequency of CCR5⁺LFA-1⁺ cells among CD8⁺ T-cells in spleen. (B) Quantification of the ICAM-1⁺ area (%) detected by immunohistochemistry in the heart tissue. (C) Illustrative pictures of immunohistochemistry staining for detection of ICAM-1 in the heart tissue and blood vessels at 150 dpi. (D) Summary of quantitative data of immunohistochemistry staining for inflammatory cells in the heart tissue. The results represent three to five mice per experimental group. *** <i>p<</i>0.001, saline-injected <i>T</i>. <i>cruzi</i>-infected mice compared with noninfected (NI) controls. ^#<i>p<</i>0.05 and ^##<i>p<</i>0.01, saline-injected compared with PTX-treated <i>T</i>. <i>cruzi</i>-infected mice.</p

PTX treatment improved heart dysfunction in chronic experimental Chagas’ heart disease.

<p>(A) Heart weight/body weight (HW/BW) coefficient. (B) Left ventricular mass (LVM). (C) Representative echocardiography images of ventricular areas. (D) Percentage of left ventricular ejection fraction (LVEF). * <i>p<</i>0.05 and *** <i>p<</i>0.001, saline-injected <i>T</i>. <i>cruzi</i>-infected mice compared with noninfected (NI) controls. The results represent eight to thirteen mice per experimental group. ^#<i>p<</i>0.05 and ^##<i>p<</i>0.01, saline-injected compared with PTX-treated <i>T</i>. <i>cruzi</i>-infected mice.</p

PTX influenced the balance of inflammatory and cytotoxic profiles systemically and in the heart tissue.

<p>(A) ELISpot assay identifying the functional capacity of IFNγ-producing CD8⁺ T-cells recognizing <i>T</i>. <i>cruzi</i> crude extracts and the H-2K^b-restricted ASP2 (VNHRFTLV) immunodominant peptide. (B) Frequencies of IFNγ⁺, IFNγ⁺CD107a⁺ and CD107a⁺ CD8⁺T-cells in spleen. (C) Frequencies of IFNγ⁺, IFNγ⁺PFn⁺ and PFn⁺ CD8⁺T-cells in spleen. Dotted lines show mean frequencies in noninfected (NI) controls. (D) Immunohistochemistry for detection of IFNγ⁺ and Pfn⁺ cells in the heart tissue. The results represent three to five mice per experimental group in three independent experiments. * <i>p<</i>0.05, ** <i>p<</i>0.01 and *** <i>p<</i>0.001, saline-injected <i>T</i>. <i>cruzi</i>-infected mice compared with NI controls. ^#<i>p<</i>0.05 and ^##<i>p<</i>0.01, saline-injected compared with PTX-treated <i>T</i>. <i>cruzi</i>-infected mice.</p

PTX therapy rescued TCR expression and influenced naïve/memory/activation phenotypes of CD8⁺ T-cells.

<p>(A) Flow cytometry analysis of CD8⁺ T-cells in the spleen. (B) Frequency of TCRαβ^Low cells and mean fluorescence intensity (MFI) of TCR in CD8⁺ T-cells. (C) Frequencies of splenic TCR⁺CD8⁺ cells expressing CD45RA⁺CCR7⁺ (naïve), CD45RA^-CCR7⁺ (central memory) and CD45RA^-CCR7^- (effector memory). (D) Frequencies of CD44^-CD62L⁺ (naïve), CD44⁺CD62L⁺ (central memory) and CD44⁺CD62L^- (activated) CD8⁺ T-cells. The results represent three to five mice per experimental group in three independent experiments. ** <i>p<</i>0.01 and ***<i>p<</i>0.001, saline-injected <i>T</i>. <i>cruzi</i>-infected mice compared with noninfected (NI) controls. ^#<i>p<</i>0.05, saline-injected compared with PTX-treated <i>T</i>. <i>cruzi</i>-infected mice.</p

Heart parasitism and electrical abnormalities are reduced in rAdVax-vaccinated mice challenged with the Colombian strain.

<p>(<b>A</b>) Mice were primed-boosted (s.c.) with 2 × 10⁸ plaque-forming units (PFU) of rAdCtrl or a mixture of 10⁸ PFU of each recombinant adenovirus vaccine construction (rAdASP2+rAdTS; rAdVax) or the vehicle control saline at 6-week intervals. Four weeks after the last immunization, the mice were challenged (i.p.) with 100 blood trypomastigotes (bt) of the Colombian <i>T. cruzi</i> Type I strain and analyzed during the acute (50 dpi) and chronic (150 dpi) phases of infection. (<b>B</b>) Relative spleen weight (mg of spleen/g of body). (<b>C</b>) Quantitative immunohistochemical staining data for <i>T. cruzi</i> parasitism (nests/100 microscopic fields) in the heart tissue. (<b>D</b>) Representative electrocardiogram (ECG) register segments of sex- and age-matched noninfected (NI) controls and mice injected with saline, rAdCtrl or rAdVax, challenged with <i>T. cruzi</i> and analyzed at 150 dpi. (<b>E</b>) Summary of the group data from NI controls and saline-injected, rAdCtrl- or rAdVax-immunized and <i>T. cruzi</i>-infected mice showing the frequency of mice presenting arrhythmias (ART), second-degree atrioventricular block (AVB2) and afflicted with ECG alterations, at 150 dpi. The data are presented as the means ± SD of seven to ten mice per group. *<i>P</i> <0.05, experimental groups compared with NI controls. ^ΨΨ<i>P</i> < 0.01, rAdVax-immunized compared with saline-injected <i>T. cruzi</i>-infected mice. ^§§<i>P</i> < 0.01, rAdVax-immunized compared with rAdCtrl-injected <i>T. cruzi</i>-infected mice.</p

rAdVax immunotherapy recovered the injured heart tissue of chronically <i>T. cruzi</i>-infected mice.

<p>(<b>A</b>) Chronically Colombian <i>T. cruzi</i> strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 10⁸ plaque-forming units (PFU) of rAdCtrl or a mixture of 10⁸ PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. (<b>B</b>) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). (<b>C</b>) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for <i>T. cruzi</i> parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). (<b>D</b>) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically <i>T. cruzi</i>-infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. (<b>E</b>) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or <i>T. cruzi</i>-infected mice pre- and post-therapy with rAdVax. (<b>F</b>) Evaluation of CK-MB activity in the serum of NI controls and <i>T. cruzi</i>-infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** <i>P</i> <0.01 and ***<i>P</i> <0.001, experimental groups compared with NI controls. ^#<i>P</i> <0.05 and ^##<i>P</i> <0.01, rAdVax-immunized compared with pre-therapy <i>T. cruzi</i>-infected mice.</p

rAdVax immunotherapy modulated serum NOx levels and cardiac iNOS expression in chronically <i>T. cruzi</i>-infected mice.

<p>Chronically Colombian-infected mice were immunized with 2 × 10⁸ plaque-forming units (PFU) of rAdCtrl or a mixture of 10⁸ PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax) in a prime-boost homologous protocol. The hearts and sera of noninfected (NI) controls, infected mice pre-therapy (120 dpi) and rAdCtrl- or rAdVax-immunized mice were collected at different time-points, accordingly with the experimental design. (<b>A</b>) Concentration of NO_x in the serum NI controls and infected mice at 190 dpi (70 days post-therapy; dpt). (<b>B</b>) Concentration of NO_x in the serum of NI controls, chronically <i>T. cruzi</i>-infected mice pre-therapy (at 120 dpi) and after immunization with rAdCtrl or rAdVax: 1 dose (at 160 dpi; 40 dpt), 2 doses (at 190 dpi; 70 dpt) and at the endpoint (post-therapy; at 230 dpi; 110 dpt). (<b>C</b>) Relative quantification of iNOS mRNA expression quantified using qRT-PCR in the heart tissue of NI controls and chronically <i>T. cruzi</i>-infected mice pre-therapy (at 120 dpi) and post-therapy with rAdVax (at 230 dpi; 110 dpt). The results represent three to seven mice per experimental group. * <i>P</i> <0.05, ** <i>P</i> <0.01 and ***<i>P</i> <0.001, experimental groups compared with NI controls. ^#<i>P</i> <0.05, rAdVax-immunized compared with pre-therapy <i>T. cruzi</i>-infected mice. ^§<i>P</i> < 0.05 and ^§§<i>P</i> <0.01, rAdVax-immunized compared with rAdCtrl-injected <i>T. cruzi</i>-infected mice.</p

rAdVax immunotherapy increased IFNγ expression in the heart tissue and in the serum.

<p>Chronically Colombian-infected mice were immunized with 2 × 10⁸ plaque-forming units (PFU) of rAdCtrl or a mixture of 10⁸ PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax) in a prime-boost homologous protocol. The hearts and sera of noninfected (NI) controls, infected mice pre-therapy (120 dpi) and rAdCtrl- or rAdVax-immunized mice were collected at 190 dpi (70 days post-therapy; dpt) or 230 dpi (110 dpt). (<b>A</b>) immunohistochemical staining (IHS) quantification of Pfn⁺ and IFNγ⁺ cells in the cardiac tissue; relative ratios of IFNγ⁺ /Pfn⁺ cells are shown in the upper-box. (<b>B</b>) Expression of IFNγ mRNA in the heart tissue detected by qRT-PCR. The data are expressed as the mRNA fold-increase in relation to NI controls. (<b>C</b>) Concentration of IFNγ in the serum of NI controls and <i>T. cruzi</i>-infected pre-therapy and rAdVax-immunized mice. The results represent three to five mice per experimental group. * <i>P</i> <0.05, ** <i>P</i> <0.01 and ***<i>P</i> <0.001, experimental groups compared with NI controls. ^#<i>P</i> <0.05 and ^##<i>P</i> <0.01, rAdVax-immunized compared with pre-therapy <i>T. cruzi</i>-infected mice. ^§<i>P</i> <0.05 and ^§§§<i>P</i> <0.001, rAdVax-immunized compared with rAdCtrl-injected <i>T. cruzi</i>-infected mice.</p

rAdVax immunotherapy ameliorated electrical abnormalities of chronically <i>T. cruzi</i>-infected mice.

<p>(<b>A</b>) Chronically Colombian-infected mice (120 dpi) were primed-boosted with 2 × 10⁸ plaque-forming units (PFU) of rAdCtrl or a mixture of 10⁸ PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax) and analyzed for electrocardiogram (ECG) abnormalities at 70 and 110 days post-therapy (dpt). (<b>B</b>) Heart rate (beats per minutes, bpm) and P wave duration (per ms). The data are shown as the means ± SD per group of 7–8 mice. (<b>C</b>) Representative ECG register segments of sex- and age-matched noninfected (NI) controls and <i>T. cruzi</i>-infected mice injected with saline or vaccinated with rAdCtrl or rAdVax at 160 dpi (40 days post-prime) and 230 dpi (110 dpt). (<b>D</b>) Summary of the group data from NI controls and chronically <i>T. cruzi</i>-infected mice injected with saline or vaccinated with rAdCtrl or rAdVax, showing the frequency of mice presenting arrhythmias (ART), second-degree atrioventricular block (AVB2) and afflicted with ECG alterations. * <i>P</i> <0.05, **<i>P</i> <0.01 and ***<i>P</i> <0.001, experimental groups compared with NI controls. ^#<i>P</i> <0.05, ^##<i>P</i> <0.01 and ^###<i>P</i> <0.001, rAdVax-immunized compared with pre-therapy <i>T. cruzi</i>-infected mice. ^§<i>P</i> <0.05, ^§§<i>P</i> < 0.01 and ^§§§<i>P</i> <0.001, rAdVax-immunized compared with rAdCtrl-injected <i>T. cruzi</i>-infected mice.</p