Treatment With Suboptimal Dose of Benznidazole Mitigates Immune Response Molecular Pathways in Mice With Chronic Chagas Cardiomyopathy

Abstract

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe form of Chagas disease, a neglected tropical illness caused by the protozoan Trypanosoma cruzi, and the main cause of morbimortality from cardiovascular problems in endemic areas. Although efforts have been made to understand the signaling pathways and molecular mechanisms underlying CCC, the immunological signaling pathways regulated by the etiological treatment with benznidazole (Bz) has not been reported. In experimental CCC, Bz combined with the hemorheological and immunoregulatory agent pentoxifylline (PTX) has beneficial effects on CCC. To explore the molecular mechanisms of Bz or Bz+PTX therapeutic strategies, C57BL/6 mice chronically infected with the T. cruzi Colombian strain (discrete typing unit TcI) and showing electrocardiographic abnormalities were submitted to suboptimal dose of Bz or Bz+PTX from 120 to 150 days postinfection. Electrocardiographic alterations, such as prolonged corrected QT interval and heart parasite load, were beneficially impacted by Bz and Bz+PTX. RT-qPCR TaqMan array was used to evaluate the expression of 92 genes related to the immune response in RNA extracted from heart tissues. In comparison with non-infected mice, 30 genes were upregulated, and 31 were downregulated in infected mice. Particularly, infection upregulated the cytokines IFN-γ, IL-12b, and IL-2 (126-, 44-, and 18-fold change, respectively) and the T-cell chemoattractants CCL3 and CCL5 (23- and 16-fold change, respectively). Bz therapy restored the expression of genes related to inflammatory response, cellular development, growth, and proliferation, and tissue development pathways, most probably linked to the cardiac remodeling processes inherent to CCC, thus mitigating the Th1-driven response found in vehicle-treated infected mice. The combined Bz+PTX therapy revealed pathways related to the modulation of cell death and survival, and organismal survival, supporting that this strategy may mitigate the progression of CCC. Altogether, our results contribute to the better understanding of the molecular mechanisms of the immune response in the heart tissue in chronic Chagas disease and reinforce that parasite persistence and dysregulated immune response underpin CCC severity. Therefore, Bz and Bz+PTX chemotherapies emerge as tools to interfere in these pathways aiming to improve CCC prognosis.

Modulation of miR-145-5p and miR-146b-5p levels is linked to reduced parasite load in H9C2 Trypanosoma cruzi infected cardiomyoblasts

“…The infection caused by T. cruzi elicits an immune response that is driven by pro-inflammatory cytokines such as IFN-γ and TNF, chemokines and enzymes and has been shown in several studies that etiological treatment contributes to parasite load reduction and rearrangement of the dysregulated immune response in patients 71 , 72 and experimental models 15 , 71 . Chagas disease cardiomyopathy is a knowingly immune dysregulated disorder 46 , and recent study by our group, using C57BL/6 mice chronically infected with the same Colombian T. cruzi strain used in the present study, showed global immune dysregulation with upregulation of key CD cytokines such as IFN-γ, CSF2, IL-12, IL-2 and chemokines such as CCR4, CCL3 and CCL5 73 corroborating that infection with T. cruzi is in fact causing increased production of inflammatory mediators that could be triggering the upregulation of miR-145-5p and, especially miR-146b-5p that is highly dependent of inflammatory stimulus 44 . Besides its trypanosomicidal activity, Bz has also been explored as having anti-inflammatory properties 14 .…”

“…In T. cruzi , actin-regulating molecules such as Rac1 CDc42 have been implicated in amastigote invasion, mediating actin recruitment and enhancing invasiveness 88 . It is known that miR-146b-5p is dependent on inflammatory stimulus of specific cytokines such as IFN-γ, TNF and IL-1β 44 , that are also dysregulated in CD 5 , as showed in recent study by our group in cardiac tissue using an experimental model of C57BL/6 mice chronically infected with the Colombian T. cruzi strain 73 . In murine T. cruzi infection, PTX was able to reverse CCC clinical signs, hampering the progression of heart injury, as improves connexin 43 expression and decreases fibronectin overdeposition, reducing CD8 + T-cells expressing activation and migration markers, and of activated blood vessel endothelial cells, and decreased the number of perforin-expressing cells invading the cardiac tissue 19 .…”

“…Additionally to its classically described mechanism of action, PTX can also inhibit a wide range of cytotoxic responses mainly by acting on cytokine production dynamics 78 , downregulating proinflammatory cytokines IFN-γ, GM-CSF and TNF 79 , attenuating cell surface expression of IL-2 receptor and production of IL-8 and CCL2 in pulmonary epithelial cells 80 . Recent study from our group on a 30-day treatment with Bz + PTX in the C57BL/6 T. cruzi chronic model, showed regulation of upregulated pro-inflammatory molecules as CSF2, IL-7 and IL-12, also substantially decreasing the production of IFN-γ, CSF2, IL-2 among other immunological molecules 73 suggesting that the immunomodulation caused by the combined Bz + PTX therapy might be affecting the upregulation of these two miRNAs. It is most likely that the combined Bz + PTX therapy was able to reverse miR-145-5p and miR-146-5p overexpression by combining the trypanosomicidal effect of Bz, successfully eliminating T. cruzi and, putatively, reducing the tissue damage caused by the parasites, as well as the immunomodulatory effect of PTX, preventing potential overproduction of cytokines that could culminate in the modulation of potential miRNA targets, that remains to be explored in the present experimental T. cruzi model.…”

“…In a model of primary cardiomyocyte culture infected with the RA T. cruzi strain, treatment with suboptimal doses has shown to decrease NOS2 expression, IL-1β and IL-6 production 11 . Additionally, recent study from our group on a 30-day treatment with Bz in the C57BL/6 T. cruzi chronic model, showed downregulation of altered pro-inflammatory molecules as CSF2, IL-7 and IL-12, also substantially decreasing the production of IFN-γ and IL-2 73 , which was not sufficient however, to reverse the upregulation of both miRNAs triggered by the infection, although it was effective in controlling parasite replication. A previous study of microRNA transcriptome profiling found miR-146b-5p to be upregulated and associated with parasitemia levels and electrical abnormalities in a model of acute Chagas’ heart disease induced by infection with the Colombian strain 33 , corroborating our data and supporting the importance of therapeutic intervention aiming to decrease the expression of this miRNA.…”

Extracellular Vesicles: Potential Role in Remote Signaling and Inflammation in Trypanosoma cruzi-Triggered Disease

“…Furthermore, EVs from infected cells stimulate the proinflammatory PARP-1-cGAS-NF-κB pathway and may play a role in fibrosis in the heart tissue, since T. cruzi infection promotes a profibrotic response in the myocardium and macrophages via the PARP-1/AP-1 pathway (Choudhuri and Garg, 2020b). Therefore, one can speculate that EVs shed by the parasites or from infected fibroblasts/cardiomyocytes may sustain a proinflammatory milieu and the renin-angiotensin system, favoring cardiac fibrosis and contributing to the progression of the cardiac form of CD (Laugier et al, 2020;Farani et al, 2021). Additionally, it is reasonable to speculate that EVs from the intracellular parasite forms may act from the inside of the host cells and regulate the infection and its consequences.…”