Cardiac alterations induced by Trypanosoma cruzi extracellular vesicles and immune complexes

Abstract

Background: Trypanosoma cruzi is a protozoan parasite responsible for American trypanosomiasis or Chagas disease (CD). This disease is characterized by the presence of cardiac or gastrointestinal symptoms in many patients during the chronic phase, with cardiac symptoms being the most common and severe, affecting approximately 30% of all patients. Although the origin of these pathologies remains unclear, several mechanisms have been proposed, involving factors related to T. cruzi and the host immune response. Extracellular vesicles (EVs) have been studied for their role in parasite-host cell communication, in modulating the host's immune response and more recently as diagnostic tools.

Methodology and main findings: In this study, we describe the role of EVs released by trypomastigotes and the immune complexes (ICs) they form with anti-T. cruzi IgGs (EVs-IgG) in the development of cardiac symptoms compatible with Chagas cardiomyopathy in mice. Autoantibodies detection, electrocardiographic, histopathological, and immunological analyses in mice's hearts were performed. The studies carried out revealed that, while the inoculation of EVs and ICs (seven intravenous injections of 2 µg of EVs and ICs over 21 days) did not elicit the appearance of autoantibodies, it led to ECG alterations (heart rate and PR interval), changes in heart cavity areas and wall thickness, and reduced expression of crucial proteins for heart function (connexin 43, tubulin, and dynein), as well as VCAM-1 and altered the cytokine expression profile in the heart. Finally, both EVs-inoculated and ICs-inoculated mice showed an increased presence of B-type natriuretic peptide (BNP) in serum, suggesting that EVs or ICs may participate in the onset of cardiac damage.

Conclusions: Our results confirm the ability of EVs shed by the infective forms of T. cruzi and the immune complexes they form with IgG to induce cardiac alterations in mice similar to those described in the literature, in T. cruzi-infected mice as well as in Chagas disease patients. This study highlights the role of EVs in the pathogenicity of Chagas disease and reinforces the importance of considering them as virulence factors in the development of Chagas disease.

Fig 1. Effects of EVs (green) or immune complexes (blue) on mice cardiac activity.

(A) Representative ECG registries of controls (PBS) and animals stimulated with EVs or ICs at different timepoints along the experiments. (B) Cardiac frequency in beats per minute of experimental mice/beats per minute of preinoculated mice; ICs-stimulated mice presented bradycardia. (C) PR interval of the ECG; Both EVs and ICs stimulation increased the PR interval in mice. The values represent the mean ± SEM, with significance set at p < 0.05(*).

Fig 2. Humoral immune response observed in mice after the inoculation with EVs (green) or ICs (blue).

Higher levels of specific antibodies against EVs were detected in mice receiving EVs compared to mice injected with the ICs. The values represent the mean ± SEM, with significance set at p < 0.05(*). Cut-off value = Mean of the PBS-injected mice absorbance + 3xSD.

Fig 3. Heart morphological alterations induced by EVs (green) and ICs (blue).

(A,B) Histological analysis of the cardiac longitudinal sections showed no relevant alterations or significant immune infiltration. (C,D) Measurement of H&E-stained mouse heart sections revealed an increase in the mean area of the right and left ventricles produced by the administration of EVs and ICs, respectively (C) Both EVs and ICs induce wall thinning in both ventricles as well as in the heart septum (D). The values represent the mean ± SEM, with significance set at p < 0.05 (*).

Fig 4. BNP serum levels obtained after inoculation of animals with EVs (green) and ICs (blue) when compared to the negative control (red).

The values represent the mean ± SEM, with significance set at p < 0.05 (*).

Fig 5. Analysis of connexin 43 expression in mice inoculated intravenously with EVs (green) or ICs (blue).

Confocal microscopy revealed decreased connexin 43 synthesis in mice injected with EVs and ICs (A). Fluorescence intensity was quantified using ImageJ, with values normalized to controls. Slides were stained with DAPI (nuclei in blue), and connexin43 was detected using monoclonal antibodies followed by labeling with a FITC-conjugated secondary antibody. (B). connexin 43 expression levels in mice injected with EVs or EVs-ICs were further assessed by RT-qPCR (C). The values represent the mean ± SEM, with significance set at p < 0.05 (*).

Fig 6. Decreased synthesis of VCAM-1, tubulin and dynein proteins in the heart of mice stimulated with EVs (green) or ICs (blue).

Confocal microscopy images of heart sections incubated with specific antibodies against VCAM-1 (A), tubulin (B) and dynein (C) showed a decrease in fluorescence signals in samples from the EVs and ICs groups. Fluorescence intensity was quantified by ImageJ, and values referred to the intensity present in controls (PBS) (D). The values represent the mean ± SEM, with significance set at p < 0.05 (*).

Fig 7. Relative expression of cytokines in the hearts of animals at the end of the experiment (21 days), following repeated inoculations with T. cruzi EVs (green) or ICs (blue).

Cytokine expression was normalized to the expression of gapdh as a housekeeping gene and compared to the expression in control PBS-injected mice (red). The values represent the mean ± SEM, with significance set at p < 0.05 (*).

Fig 8. Summary figures show the cellular alterations induced in the mouse heart after 21 days of inoculation with EVs and ICs, which could be related to the pathological manifestations of Chagas disease cardiomyopathy.

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