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 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 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 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 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

C57BL/6 mice infected with the Colombian strain of <i>T. cruzi</i> develop chronic cardiomyopathy.

<p>Mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and parasitological and clinical parameters were evaluated. (<b>A</b>) Kinetics of parasitemia and cardiac parasitism. (<b>B</b>) Survival rate. (<b>C</b>) CK-MB activity levels in the serum of noninfected and <i>T. cruzi</i>-infected mice. (<b>D</b>) Positive correlation between cardiac tissue parasitism and CK-MB activity levels during the acute phase (r² = 0.982) but not during the chronic phase (r² = 0.039) of infection. (<b>E</b>) Representative ECG register segments of 1200 ms of sex- and age-matched noninfected (NI) controls and <i>T. cruzi</i>-infected C57BL/6 mice at 30, 45, 60, 90 and 120 dpi, showing arrhythmia and first and second degree atrioventricular block (AVB1, AVB2; arrows) in infected mice. (<b>F</b>) Relative heart weight (mg/g) of noninfected (NI, pool of three age-matched controls per analyzed point) and <i>T. cruzi</i>-infected C57BL/6 mice at 15, 30, 45, 60, 90 and 120 dpi. (<b>G</b>) Numbers of CD4⁺ and CD8⁺ cells in the cardiac tissue during acute and chronic <i>T. cruzi</i> infection were counted after immunohistochemistry staining. Each circle 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 NI controls and <i>T. cruzi</i>-infected mice.</p

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

Segregation of CD8⁺ T-cells into IFNγ⁺ and Pfn⁺ populations in <i>T. cruzi</i> infection.

<p>C57BL/6 mice were infected with 100 blood trypomastigotes of the Colombian strain of <i>T. cruzi</i> and the expression of IFNγ⁺ and Pfn⁺ by CD8⁺ cells in the peripheral blood and cardiac tissue was evaluated. (<b>A</b>) Representative histograms of flow cytometry analysis of peripheral blood CD8⁺ T-cells [R1 (SSCxFSC)/R2 (TCR)/R3 (CD8) gated] that were analyzed for IFNγ and Pfn expression in <i>T. cruzi</i>-infected mice (120 dpi). (<b>B</b>) Representative dot plots of flow cytometry analysis of peripheral blood CD8⁺ T-cells (R1/R2/R3 gated) expressing IFNγ and Pfn at 45 and 120 dpi. (<b>C</b>) Frequency of double-stained CD8⁺IFNγ⁺ and CD8⁺Pfn⁺ peripheral blood T-cells cells (R1/R2/R3 gated) at 45 and 120 dpi. (<b>D</b>) Representative histograms and dot plots of flow cytometry analysis of heart infiltrating CD8⁺ cells [R1 (SSCxFSC)/R2 (CD8) gated] expressing IFNγ and Pfn at 45 and 120 dpi. (<b>E</b>) Frequency of double-stained CD8⁺IFNγ⁺ and CD8⁺Pfn⁺ of heart infiltrating cells (R1/R2 gated) at 45 and 120 dpi. In (<b>C</b>) Bars represent the mean ± SD of five to eight mice per group. In (<b>E</b>) Bars represent the mean of two or three pools of 5 mice per group. These data represent two or 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 45 and 120 dpi.</p