As previously described, chemotherapy based on antiparasitic drugs is not able to stop or reverse the damage caused by the inflammatory response in CCC, although they can decrease or eradicate parasite load (Morillo et al., 2015). In this context, immunomodulatory drugs used alone or in combination with antiparasitic drugs can be a promising approach for the treatment of CCC. Both strategies seek to reduce inflammation, prevent organ deterioration and improve the quality of life and survival of CCC patients.

Several compounds of different pharmacological classes have already been shown to act as immunomodulatory agents in experimental models of mild and severe ( Table 1 ). These compounds were able to modulate inflammatory process in CCC, acting in different pathways ( Figure 2 ). Interestingly, none of compounds reactivated the parasitemia, proven that a moderate immunosuppression can be beneficial for CCC treatment.

Acetylsalicylic acid (aspirin), when tested in a BALB/c mouse model of chronic Chagas disease, decreased cardiac inflammatory infiltrate and cardiac fiber disarrangement (González-Herrera et al., 2017). These effects were associated with known actions of aspirin, which as reduction of thromboxane levels and adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, key molecules for the recruitment of monocytes and lymphocytes, which are involved in CCC pathogenesis (Molina-Berríos et al., 2013). Interestingly, simvastatin, a cholesterol-lowering statin, also decreased myocardial inflammation alone or in combination with benznidazole, as well as the area of fibrosis in the heart. Moreover, it inhibited endothelial cells (EC) activation, as shown by the reduced expression of endothelial cell adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin (González-Herrera et al., 2017). The effects of simvastatin in EC activation were partially reversed in 5-lipoxygenase knockout mice, suggesting a central role of 15-epi-lipoxin A4 production in the beneficial mechanisms of simvastatin in CCC (González-Herrera et al., 2017).

Another drug with potential use for treatment of CCC is pentoxifylline, a phosphodiesterase inhibitor with anti-inflammatory and cardioprotective effects (Sliwa et al., 2002; Shaw et al., 2009). Treatment of chagasic C57BL/6, which represents a model of mild CCC, with pentoxifylline reversed electrical abnormalities, decreased the number of inflammatory cells and reduced distances of connexin-43⁺ gap junctions in the heart. Moreover, it promoted the reduction of fibronectin area and CK-MB activity, markers of heart injury in T. cruzi-infected mice (Pereira et al., 2015). Interestingly, pentoxifylline also ameliorated the heart injury and electrical alterations in T. cruzi-infected C3H/He mice, which present a higher inflammation and heart parasitism than infected C57BL/6 mice, being considered a model of severe CCC (Pereira et al., 2014b). Furthermore, Vilar-Pereira et al. (2016) demonstrated that pentoxifylline associated with benznidazole was able to reduce the parasite load, myocarditis and fibrosis, in addition to restoring normal heart rate (QTc) corrected QT intervals in a C57BL/6 mice model for CCC.

In a different way, G-CSF, a pleiotropic cytokine, promoted a reduction of o inflammation and fibrosis in chronic chagas heart with improvement of heart function (Macambira et al., 2009; Vasconcelos et al., 2013). Correlating to the histological findings, a decrease in pro-inflammatory molecules such as, ICAM-1, galectin-3, IFN-γ, syndecan-4 and TNF was found In addition, G-CSF induced an increase of the anti-inflammatory cytokine IL-10 levels and the recruitment of Treg cells, which constitute an anti-inflammatory T-cell population (Belkaid et al., 2006). Interestingly, G-CSF also promoted a reduction in the parasite load in hearts of infected mice. Through in vitro experiments, a trypanocidal effect of G-CSF against T. cruzi was confirmed, characterizing the dual effect (antiparasitic and immunomodulatory) of this cytokine (Vasconcelos et al., 2013).

N-N-dimethylsphingosine (DMS), a pan sphingosine inhibitor, has also shown a dual nature of action in CCC, by reducing the parasitism, as well as heart inflammation and fibrosis in T. cruzi-infected C57BL/6 mice (Vasconcelos et al., 2017). The dual nature of DMS was supported by in vitro experiments showing that DMS inhibited lymphocyte proliferation, reduced nitric oxide and cytokine production in cultures of activated macrophage, having, in contrast, a direct effect on trypomastigotes and amastigotes forms of T. cruzi. Interestingly, DMS was shown to activate the inflammasome pathway, which contributes to its antiparasitic effect (Vasconcelos et al., 2017).

Lastly, BA5, an amide semi-synthetic derivative from the natural pentacyclic triterpenoid betulinic acid, was also proven to be a promising treatment for CCC. Initially, BA5 was characterized by its potent anti-T. cruzi activity, with values of IC₅₀ against trypomastigotes (IC₅₀ = 1.8 µM) and amastigotes (IC₅₀ = 10.6 µM) lower than benznidazole (IC_{50 trypomastigotes} = 10.6 µM; IC_{50 amastigotes} = 13.5 µM) (Meira et al., 2016). In a second investigation, BA5 promoted the reduction of important inflammatory mediators, such as nitric oxide and TNF, as well as inhibition of nuclear factor-кβ (NF-кβ) in cultures of activated macrophages and also show a protective effect against a lethal dose of LPS in a mouse model of endotoxic shock and decrease edema in a delayed type of hypersensitivity model (Meira et al., 2017). Finally, in a mouse model of CCC, BA5 attenuated attenuated heart inflammation and fibrosis in the hearts of infected mice. These effects were related to a reduction of pro-inflammatory molecules, such as IFN-γ, IL-1β, and TNF, and increased IL-10 production. Moreover, polarization to anti-inflammatory/M2 macrophage phenotype was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers, such as Arg1 and CHI3, in mice treated with BA5 (Meira et al., 2019).

In summary, immunomodulatory compounds may be an interest therapeutic tool to the management of CCC, especially those with dual effects (antiparasitic and immunomodulatory actions) or in combination with antiparasitic drugs, such as benznidazole (Cevey et al., 2017; González-Herrera et al., 2017).

As mentioned previously, APCs are essential elements for the immune system due to the connection established between innate and adaptive immunity and the unique ability to modulate the adaptive response, which can induce immunity or tolerance. These cells have been the target of several studies of immunotherapy aimed to promote immunomodulation (García-González et al., 2016; Moreau et al., 2017).

DCs have the ability to activate or induce T cell tolerance (Stagliano and Oppenheim, 2013), as determined by their state of maturation (Jansen et al., 2017). When mature DCs mediate immune responses in inflammatory conditions, these cells have a regulatory profile with the ability to induce immune tolerance, oppositely to DCs expressing an immature phenotype (Domogalla et al., 2017; Moreau et al., 2017).

Tolerogenic dendritic cells (tDCs) have been tested as a therapeutic tool to reduce or prevent autoimmune diseases (Hermansson et al., 2011; Lee et al., 2014; Choo et al., 2017; Huang et al., 2017; Aragão-França et al., 2018). Considering the pathogenetic mechanisms of Chagas disease and the important role of DCs in the regulation of immune responses, Santos et al. (2020) tested the therapeutic potential of tDCs in an experimental model of CCC. Administration of tDCs reduced cardiac inflammation and fibrosis, hallmarks of CCC. Furthermore, tDCs increased the frequency of Treg cells, elevated IL-10 production and inhibited the expression of markers associated with fibrosis, such as galectin-3, demonstrating a potential use of these cells in the immunotherapy for CCC.

Regenerative therapies have also been investigated for the treatment of chronic Chagas heart disease since stem cell-based therapies have emerged as an alternative to CCC treatment due to their regenerative and immunomodulatory properties ( Table 2 ). Initially, bone marrow mononuclear cells (BMMC), a cell fraction containing both mesenchymal and hematopoietic stem cells, were used due to their easy obtention, low cost and long experience in their use in other diseases, facilitating translation for clinical use. In 2004, Soares et al., observed a decrease in inflammatory cell number and percentage of fibroblasts in chronically infected BALB/c and C57BL/6 mice intravenously treated with BMMC, a result that lasted up to 6 months after transplantation. Later, Goldenberg et al. (2008) demonstrated a beneficial effect of BMMC therapy in the heart function of chronically infected C-129 mice, which represents a model of right ventricular (RV) cavity dilation. The authors observed a significant reduction in the RV dilation 3 months after treatment with BMMC. Additionally, BMC treatment prevented RV dilation when applied one month after the infection.

To investigate the mechanisms by which BMMC exerted its beneficial effects in CCC a cDNA microarray analysis was performed in hearts of chronically T. cruzi-infected mice, showing a large number of alterations in gene expression when compared to naïve mice (Soares et al., 2010; Soares et al., 2011). BMMC-transplanted infected mice, however, had a marked decrease in gene expression changes in the heart two months after cell therapy, showing a potent modulatory effect of BMC infusion (Soares et al., 2011). Of special interest, it was observed the down-regulation of several genes related with inflammation and fibrosis, including galectin 3, SDF-1, and TIMP-1, indicating a potent immunomodulatory action of transplanted BMMC.

The results found in the experimental models led to the development of a clinical trial to evaluate the safety and efficacy of BMC transplantation in patients with heart failure of Chagas etiology (Vilas-Boas et al., 2011). In this study, 28 patients were treated by the coronary route and evaluated 180 days later. The authors observed an improvement in the left ventricular ejection fraction, in the NYHA functional class, in the Minnesota quality of life score and in the six-minute walk test after autologous BMMC infusion, and no changes related to the procedure were found (Vilas-Boas et al., 2011). Subsequently, a randomized multicenter clinical trial was conducted with 183 patients divided into BMMC-treated and placebo groups (Santos et al., 2012). Patients were treated or not with BMMC by intracoronary route and evaluated 6 and 12 months after therapy. However, there were no statistically significant differences between groups when comparing several parameters, such as left ventricular ejection fraction (LVEF), NYHA functional class, Minnesota quality of life score, and six-minute walk test (Santos et al., 2012).

The reasons why the therapy with BMMC, despite producing good results in mice, was not observed in humans are unknown. One possible explanation may be o the differences between the mouse experimental models and the disease in humans, such as the lack of dilatation of right ventricular chamber in mice while humans present with left ventricular dysfunction. In addition, apical aneurysm and fatal arrhythmias occur in humans but not in mice, although T. cruzi-infected mice may show ECG changes, arrhythmias and conduction defects. Another factor to be considered is the number of cells used in the therapeutic scheme. To use in humans a dose equivalent to that used in studies with mice, it would be necessary to transplant 3.5 billion cells, while in the study performed on humans the treatment was done with 100 to 250 million cells (Carvalho et al., 2017).

In view of the above, other types of stem cells, such as mesenchymal stem/stromal cells (MSC), began to be studied as a better alternative to cell therapy in Chagas disease. MSC can be obtained from different sources, such as bone marrow, adipose tissue, and cardiac tissue, they are easy to obtain and expand, and possess immunomodulatory activity and low immunogenicity (Chamberlain et al., 2007). Such characteristics have made these cells a promising alternative for use in the treatment of CCC, due to its immunological pathogenetic mechanism.

Using bone marrow-derived MSC in the acute phase of Chagas disease, Jasmin et al. (2012) observed that, despite the low number of cells homing to the heart, MSC-treated animals had a decreased right ventricular internal diameter (RVID), suggesting a paracrine action of these cells. MSC may exert paracrine effects due to the secretion of different factors, such as growth factors, cytokines, microRNA (miRNA), and small molecules (collectively called secretome), which act in resident, as well as in immune cells, modulating their function and reducing fibrosis and inflammation, among other effects (Chang et al., 2021). When tested in the chronic phase, MSC showed similar results were observed in relation to homing and reduction of RVID (Jasmin et al., 2014). Other studies reinforced the hypothesis of paracrine effect of MSC, regardless of the source, as is the case of adipose-derived human mesenchymal stem cells (AD-MSC), transplanted by intraperitoneal route. In this case, these cells were found located in the abdominal or subcutaneous fat and, even so, the treated animals had a reduction in cardiac inflammation, parasitism and fibrosis, and prevented RV dilation (Mello et al., 2015). Additionally, Larocca et al. (2013), using the same route for MSC administration, observed a significant reduction in heart inflammation and fibrosis, but there was no improvement in arrhythmias in mice with CCC.

In another study, Silva et al. (2014) tested MSCs obtained from the mouse cardiac tissue. After treatment by intramyocardial injection in mice with CCC, a reduction in inflammation and TNF in cardiac tissue was observed two months after therapy. However, there was no significant reduction in the area of fibrosis, which correlated an increase in TGF-β expression. Although MSCs used were obtained from cardiac tissue, these cells were not able to differentiate into cardiomyocytes, since the few cells observed in cardiac tissue did not express specific cardiomyocyte markers such as troponin T and connexin 43.

Since the paracrine effects are an important mechanism of action of MSC, the genetic modification of these cells has been used as a tool to increase the production of specific factors that would enhance their therapeutic effect. Such a strategy allows bioactive molecules to be delivered systemically or directly to the injury site.

Genetic modification was carried out to induce the production of G-CSF, a growth factor known to mobilize bone marrow-derived stem cells to the peripheral blood (Anderlini and Champlin, 2008), and previously known to promote beneficial effects in the mouse model of CCC (Macambira et al., 2009; Vasconcelos et al., 2013). Silva et al. (2018) observed that G-CSF-genetically modified MSC were able to reduce inflammation and fibrosis and also reduce the production of inflammatory mediators (such as TNF and IFN-γ) and increase IL-10 levels in a more pronounced way the control MSC, when transplanted into mice with CCC. The authors attributed the observed effects to an increase in the ability to recruit suppressor cells, such as Treg cells and myeloid derived suppressor cells (MDSC).

Another growth factor tested by this approach was insulin like growth factor-1 (IGF-1), which increases the viability and differentiation of stem cells and progenitor cells (Rebouças et al., 2016). IGF-1 also improved the engraftment of MSC, which inhibits cardiomyocyte cell death in a model of myocardial infarction (Enoki et al., 2009). Using genetically modified MSC to overexpress IGF-1 (MSC-IGF-1) in a model of CCC, a reduction of inflammation and fibrosis in the heart and systemic TNF levels were found in mice treated both with IGF-1-modified or control MSC. However, MSC-IGF-1 therapy promoted a potent reduction of myofiber loss in skeletal muscle in T. cruzi-infected mice, compared to control groups (MSC or vehicle).

Although MSC have shown to promote beneficial effects in the mouse model of chronic Chagas heart disease, clinical trials are needed to demonstrate whether this therapy has beneficial effects in humans.

As previously described, fibrosis and cardiac inflammation are the main features of CCC and excessive fibrosis leads to ventricular dilation and heart failure (Rassi et al., 2010; Bern, 2015; Morillo et al., 2015). Thus, seeking interventions focused on improving cardiac fibrosis could reflect an improvement in cardiac function and even a reduction in the risk of mortality (Yuan et al., 2017). Some genes related to this disease process have already been identified, but there are still gaps in relation to regulatory mechanisms (Frey and Olson, 2003).

Within the gene processes related to remodeling pathways, there is the action of microRNAs (miR), which are short 18-25 nucleotide RNA sequences that specifically regulate gene expression and apparently act as regulators of gene expression, exerting, among its functions, the inhibition of translation or promotion of mRNA (messenger RNA) degradation (Van Rooij et al., 2006; Bartel, 2009; Shukla et al., 2011).

In fact, microRNAs have been described as regulators in several pathophysiological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis. Recently, miRs have been linked to several processes regulating heart disease, such as mMiR-29, miR-30 and miR-133, described as inhibitors of collagen expression (Van Rooij et al., 2008; Duisters et al., 2009), and miR-21, shown to be an important regulator in fibroblast proliferation and fibrosis (Thum et al., 2008; Yuan et al., 2017).

In order to modulate fibrosis and inflammation present in CCC, Nonaka et al. (2021) explored the potential of miR-21 as a therapeutic target. Initially, upregulation of circulating and cardiac miR-21 was identified in patients with CCC, as well as in T. cruzi-infected mouse hearts. In vitro and well as in silico analyses showed the pro-fibrotic action of miR-21. In the same study, using a mouse experimental model of CCC, the authors showed that miR-21 blockage using nucleic acid (LNA)-anti-miR-21 caused a reduction of inflammation and fibrosis in the heart tissue. Further studies involving gene therapy are needed to explore the potential of miR-21 and other microRNAs as therapeutic candidates for CCC.