Vaccine-linked chemotherapy improves cardiac structure and function in a mouse model of chronic Chagas disease

Figures

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  Figure 1. Study Timeline and experimental treatment groups. Mice were infected by intraperitoneal injection at day 0. Infection was confirmed at 15dpi, then echocardiography and electrocardiograms (ECG) were performed at 28dpi, 53dpi and 70dpi. Mice randomly assigned to treatment groups as indicated were treated beginning 70dpi and follow up echocardiography and ECGs were performed at approximately 122dpi, 154dpi, and 208dpi. All mice were euthanized at approximately 209dpi. Image created with Biorender.

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  Figure 2. Echocardiography evaluation prior to treatment initiation. M-mode images were used to measure parameters at the end of both systole and diastole, including left ventricular posterior wall thickness (A, B), left ventricular end diameter (C, D), left ventricular volume (E, F), ejection fraction (G) and fractional shortening (H) at approximately 28dpi, 53dpi and 70dpi. At each timepoint, infected untreated mice were compared to naïve age matched controls*, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001.

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  Figure 3. Left ventricular posterior wall thickness (LVPW) after treatment. M-mode images from echocardiographic examination were used to measure LVPW in infected untreated and treated mice at 70dpi, 122dpi, 154dpi and 208dpi during both systole (A) and diastole (B). Panels (C) and (D) illustrate only a subset of treatment groups to better visualize treatment differences between key groups. The grey shaded area represents the mean ± SD of the LVPWs and LVPWd measurement from naïve age matched controls.

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  Figure 4. Left Ventricular End diameter (LVED) after treatment. M-mode images from echocardiographic examination were used to measure LVED in naïve, infected untreated and treated mice at 70dpi, 122dpi, 154dpi and 208dpi during both systole (A) and diastole (B). Panels (C) and (D) illustrate only a subset of treatment groups to better visualize treatment differences between key groups. The grey shaded area represents the mean ± SD of the LVEDs and LVEDd measurement from naïve age matched controls.

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  Figure 5. Left ventricular volume after treatment. M-mode images from echocardiographic examination were used to measure left ventricular volume (ESV and EDV) in naïve, infected untreated and treated mice at 70dpi, 122dpi, 154dpi and 208dpi during both systole (A) and diastole (B). Panels (C) and (D) illustrate only a subset of treatment groups to better visualize treatment differences between key groups. The grey shaded area represents the mean ± SD of the ESV and EDV measurement from naïve age matched controls.

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  Figure 6. Ejection fraction and fractional shortening after treatment. M-mode images from echocardiographic examination were used to measure left ventricular ejection fraction (EF, panel A) and fractional shortening (FS, panel B) in naïve, infected untreated and treated mice at 70dpi, 122dpi, 154dpi and 208dpi. Panels (C) and (D) illustrate only a subset of treatment groups to better visualize treatment differences between key groups. The grey shaded area represents the mean ± SD of the EF and FS measurements from naïve age matched controls.

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  Figure 7. Antigen specific TH1/TH2/TH17 cytokine release from splenocytes. Splenocytes were restimulated in vitro with recombinant Tc24-C4 protein or cRPMI alone (unstimulated) as described. Culture supernatants were harvested after 96 hours and stored at -80C until quantification of cytokines by Luminex assay. Data was analyzed for normality, then treatment groups were compared to controls using parametric or non-parametric ANOVA depending on normality. When comparing groups to the infected untreated control *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001. When comparing groups to E6020 only treatment $, p≤0.05. $$, p≤0.01; $$$, p≤0.001; $$$$, p≤0.0001.

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  Figure 8. Antigen specific cytokine producing T cells. Splenocytes were restimulated in vitro with recombinant Tc24-C4 protein or cRPMI alone (unstimulated), then stained with fluorescent antibodies for analysis by flow cytometry as described. Results are expressed as a ratio of antigen specific cells divided by unstimulated cells. (A) CD4+IFNγ+ cells, (B) CD8+IFNγ+ cells, (C) CD4+IL-4+ cells, (D) CD8+IL-4+ cells. When comparing groups to the infected untreated control *, p≤0.05; **, p≤0.01; ***, p≤0.001.

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  Figure 9. Cardiac Cellular infiltrate and fibrosis. Cardiac cellular infiltrate (A) was quantified from representative images of H&E stained tissue sections as described. Fibrosis (B) was quantified from representative images of Masson’s Trichrome stained tissue sections as described. When comparing groups to the infected untreated control *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001.

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  Figure 10. Representative images of H&E stained cardiac tissue sections from naïve (A), infected untreated (B) and treated (C–L) mice. Images were captured at 100X magnification. Scale bar represents 100µm.

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  Figure 11. Representative images of Masson’s Trichrome stained cardiac tissue sections from naïve (A), infected untreated (B) and treated (C–L) mice. Images were captured at 100X magnification. Scale bar represents 100µm.