Introduction

Chagas disease, or American trypanosomiasis, is a zoonotic disease caused by infection with the parasite, Trypanosoma cruzi. It is endemic in Mexico, Central, and South America, where transmission of T. cruzi is maintained by insect vectors (triatomines) and domestic and wild mammals that serve as reservoirs [1]. Other routes of transmission of T. cruzi include blood transfusion or transplantation of organs from infected donors, maternal-fetal transmission, and acquisition of infection via the oral route [2]. Due to increased immigration, individuals with Chagas disease have been identified in the USA, Canada, Europe, Australia, and Japan [3]. It is estimated that 300,000 persons living in the USA are chronically infected with T. cruzi [4]. In recent years, vectorial transmission of T. cruzi and autochthonous cases of Chagas disease has also been reported in the southern states of the USA [5].

The clinical course of Chagas disease is divided into the acute and chronic phases. The acute infection is usually mildly symptomatic and often misdiagnosed as febrile illness of childhood. Approximately 1% of the acutely infected persons manifest lymphadenopathy, hepatosplenomegaly, myocarditis, pericardial effusion, and heart failure or meningoencephalitis. Parasitemia is evident and lasts for 2 to 4 months, and then infected individuals evolve into a chronic phase. While many remain in an indeterminate phase without any clinical symptoms, but having a positive serology, approximately 30% of the infected individuals progress into clinically relevant Chagas disease. Chronic cardiomyopathy is the most important clinical manifestation of Chagas disease because of its frequency, severity, and effects on morbidity and mortality. It is a complex disease that includes a wide spectrum of manifestations, ranging from minor myocardium involvement to left ventricular systolic dysfunction, dilated cardiomyopathy, arrhythmias, thromboembolic events, and terminal cardiac failure [6••]. Gastrointestinal (GI) manifestations, such as mega-syndromes involving tubular structures of the GI tract, though not commonly recorded, are frequent in certain geographic areas [7].

The virtual absence of parasites in the heart of chronically infected individuals has stimulated a discussion in the literature regarding the etiology of chronic Chagas disease. Different strains of the parasite have been associated with distinct clinical outcomes of infection in experimental models [8] and human disease [9]. Host factors, such as genetic background of the host, role of B and T cell immunity in control of T. cruzi and pathogenesis of chronic disease, autoimmunity, vascular compromise, and involvement of autonomic and central nervous system have also been associated with distinct clinical outcomes of Chagas disease and are discussed elsewhere. Herein, we briefly discuss the role of macrophages, mitochondrial dysfunction, and oxidative stress in T. cruzi infection and chronic Chagas disease.

Innate Immunity against T. cruzi Infection

The significance of innate immune responses to T. cruzi infection has primarily been studied by using experimental models and has provided important information regarding mechanisms of parasite control and disease processes.

Cytokine and Chemokine Response

As universal cells of innate immunity, the epithelium, macrophages, dendritic cells, and NK cells deserve attention in Chagas disease. Parasites enter their host through a skin lesion. T. cruzi infection of epithelial cells have been shown to enhance the expression of proinflammatory genes associated with toll-like receptor (TLR) pathway and TNF-α and TGF-β signaling pathways. The frequently upregulated chemokines in infected epithelial cells, e.g., CXCL1, CXCL2, CXCL3, CCL8, CCL20, and IL8, participate in the recruitment of professional phagocytic cells [10].

The interaction of T. cruzi with macrophages and other innate immune cells induces a substantial increase in the expression and secretion of proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) at a level similar to that seen in IFN-γ/LPS-induced proinflammatory macrophages ([11] and references therein). Toll-like receptors recognize the pathogen-associated molecular patterns and transmit a signal via cytoplasmic Toll/IL-1R domains for the recruitment of cytosolic adaptor molecules, including myeloid differentiation primary-response protein 88 (MyD88), and subsequently induce nuclear factor-κB (NF-κB) activation, leading to the production of inflammatory cytokines and linking the innate to the adaptive immune responses [12, 13]. T. cruzi-derived glycosylphosphatidylinositol (GPIs) and GPI-anchored mucin-like glycoproteins have been demonstrated to stimulate the synthesis of IL-12, TNF-α, and nitric oxide (NO) by innate immune cells [14•]. The mucin-linked GPI anchors induced TLR2-dependent leukocyte recruitment via CCL2 [15]. The parasite expresses cruzipain, a kinin-releasing cysteine protease, which induces dendritic cell maturation via the activation of bradykinin (BK) B2 receptors (B2R) [16, 17]. TLR2 activation by T. cruzi also signals dendritic cell-driven mechanisms that stimulate Th1 responses via the cruzipain/kinin/B2R pathway [18]. TLR4 and TLR9 likely recognize parasite-derived GIPLs and DNA, respectively, and cooperate in the activation of host innate immune response against T. cruzi infection [19]. TLR2−/−, TLR9−/−, and MyD88−/− mice exhibited increased susceptibility to T. cruzi infection, and macrophages of these mice were defective in eliciting proinflammatory response [20•, 21•], thus, suggesting that TLR2 and TLR9 are the major TLRs involved in T. cruzi recognition by innate immune cells. In addition to MyD88, TRIF-dependent induction of type 1 IFNs (especially IFN-β) has also been documented to contribute to resistance to T. cruzi infection in dendritic cells and macrophages [22]. The Myd88−/−Trif−/− mice exhibited TLR-independent, NFATc1-dependent Th1 responses and dendritic cell maturation after T. cruzi infection [23], thus suggesting that NFATc1 complements TLR-dependent innate immune responses in T. cruzi infection.

T. cruzi-infected macrophages produce IL-12, a key mediator of IFN-γ production through activation of NK cells and induction of Th1 cell development. IFN-γ is required to activate the macrophage expression and activation of inducible nitric oxide synthase (iNOS) and NADPH oxidase (NOX2) and production of nitric oxide (NO) and reactive oxygen species (ROS), respectively [24••]. TNF-α may also provide a second signal stimulating NO/ROS production in IFN-γ-activated macrophages, as well as in infected cardiac myocytes [25, 26], and, thus, enhance the trypanocidal function. In the absence of IFN-γ, mouse and human macrophages produced insufficient amounts of ROS and NO and failed to clear the parasite [11]. Others have shown the splenic enrichment of Ly6C+ dendritic cells—like inflammatory cells that produced TNF-α and NO to kill parasite but also produced IL-10 that negatively affected the development of anti-parasite T cell response in infected mice [27]. Ponce et al. [28] suggested that a transient increase in CD39/CD73 enzyme pair (hydrolyzes extracellular adenosine) attenuated the inflammatory macrophage response to T. cruzi, and inhibition of CD73 was beneficial in establishing the proinflammatory macrophages’ predominance and reduce parasite load in the myocardium of acutely infected mice.

Nucleotide-binding oligomerization domain-like receptors (NLRs) are characterized by the presence of a central NACHT domain and a C-terminal leucine-rich repeats (LRRs) domain of variable length (20–29 amino acids). The N-terminal effector binding region consists of a protein-to-protein interaction domain, i.e., pyrin domain (PYD), a caspase recruitment domain (CARD), and a baculovirus inhibitor of an apoptosis protein repeat (BIR) domain, and based on this, NLRs are classified as NLRP, NLRC, and NAIP, respectively. The multimeric protein macromolecules formed by NLRs are named inflammasomes [29]. The most studied NLRP1 and NLRP3 inflammasomes recruit ASC (apoptosis-associated, speck-like protein containing a CARD domain) and caspase-1 proteins. The ASC-dependent activation of caspase-1 is essential for the cleavage of pro-IL-1β and pro-IL-18 into their functional form and initiation of the inflammatory cytokine response [30]. Recent studies showed that a deficiency of caspase-1/ASC and NLRC1 (also called NOD1) inflammasomes attenuated the activation of IL-1β/ROS and NF-κB-dependent cytokine gene expression for T. cruzi control in human and mouse macrophages [31,32,33]. However, NLRP3-mediated IL-1β/NF-κB activation was dispensable because NLRP3−/− macrophages produced high amount of ROS that provided efficient control of T. cruzi replication and survival in macrophages [33].

The cytokines synthesized during T. cruzi infection are capable of inducing or regulating the production of chemokines in macrophages and cardiac myocytes both in vitro and in vivo. The enhanced expression and release of chemokines and their receptors affected T cell proliferation, Th1/Th2 differentiation, and resistance to infection in mice [25, 34]. The chemokine receptors, CCR5 and CXC3, are immunological preferential markers of Th1 response, and CCR3 and CCR4 are preferentially associated with Th2 response [35]. CCR5 recognizes CCL3, CXCL10, and CCL5 chemokines. Chagasic patients with a point mutation in CCR5 promoter resulting in low levels of CCR5 expression in leukocytes exhibited attenuated heart disease [36]. Treatment of mice with CCR5 antagonist (Met-RANTES) decreased the tissue infiltration of CD4+ or CD8+ T cells and chronic myocarditis [37]; however, CCR5-deficient mice were susceptible to acute infection [38]. These results indicated an important role for CCR5 in the control of acute infection as well as in chronic immunopathology (reviewed in [39]).

In addition to innate immune cells, endothelial cells, cardiac myocytes, and vascular smooth muscle cells (VSMCs) can also sense and respond to pathogens (or PAMPs) [40,41,42,43]. T. cruzi induced the expression and release of IL-1β, TNF-α, and IL-6 in endothelial cells and cardiac myocytes [41, 44]. Trans-sialidase, a released surface protein of T. cruzi, induced IL-6 production in isolated endothelial cells [45]. VSMCs exhibited increased proliferation and upregulation of ERK-cyclin D1-endothelin-1 pathway in response to T. cruzi infection [46]. Others have shown that TGF-β plays a role in parasite invasion. Treatment of cardiac myocytes with SB-431542, which inhibits the TGF-β type 1 receptor, impaired the parasite invasion and replication and prevented heart damage in acute Chagas disease [47]. Thus, depending on the cell type, cytokine and signaling responses by the non-immune cells serve as a component of innate defense, a bystander effect to T. cruzi infection, or a mechanism exploited by parasite for invasion of a variety of cells.

Lipid Mediators

The precursor arachidonic acid (AA) is metabolized by a series of enzymes into a variety of biologically and clinical relevant eicosanoids and their metabolites. AA is metabolized by 5-lipoxygenase (5-LO) enzyme for the synthesis of leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE4). 5-LO is primarily expressed in macrophages, granulocytes, and mast and dendritic cells. Leukotrienes are produced during experimental T. cruzi infection, by tissue-resident and recruited leukocytes, and LTB4 synthesis activates intracellular killing of T. cruzi in macrophages [47]. 5-LO deficiency significantly increases acute parasitemia; however, 5-LO knockout mice were still able to control tissue parasites and exhibited decreased mortality and cardiac damage [48, 49]. The absence of LXA4 in 5-LO null mice modulated the expression of suppressor of cytokine signaling (SOCS2) in spleen and heart of infected mice. The SOCS2 deficiency enhanced the number of T regulatory cells and decreased the levels of proinflammatory cytokines; however, SOCS2-deficient macrophages were hyper-responsive to IFN-γ, produced increased levels of NO, and dealt with infection efficiently [50].

The cyclooxygenases COX-1/COX-2 convert AA to prostaglandin H2 (PGH2) that is further metabolized by thromboxane A2 (TXA2) synthase into TXA2. It has been demonstrated that the parasite also has a synthase capable of producing TXA2. The Tanowitz and Ashton laboratories found that T. cruzi likely utilizes host-derived PGH2 to produce TXA2 and that T. cruzi-derived TXA2 is the major source of TXA2 detected in serum of infected mice [48, 51]. TXA2-regulated vasospasm, thrombosis, vascular permeability, and endothelial cell dysfunction are observed in acute infection [44]. Interestingly, TXA2 receptor knockout mice displayed increased mortality, tissue parasitism, and myocardial inflammation upon infection [51] leading to suggestions that autocrine/paracrine TXA2 receptor activation provides a quorum sensor that regulates intracellular amastigote proliferation, providing opportunities to survive from infection. TXA2 and its receptor contribute to innate immunity by virtue of the fact that it mobilizes inflammatory cells and results in the release of proinflammatory cytokines. Furthermore, activation of TXA2 receptors on naïve T cells enhances chemokinesis, prevents adhesion of antigen presenting cells such as dendritic cells, and inhibits T cell proliferation to negatively modulate acquired immunity [49]. As such, TXA2 release by the parasite likely prevents the full development of host immunity, choosing short-term over long-term responses, and may contribute to the transition to the chronic state and the persistence of the infection.

Reactive Oxygen Species

In addition to cytokines/chemokines, activated macrophages exert cytotoxic effects against microbes by production of reactive oxygen (ROS) and nitrogen species. NADPH oxidase (NOX2), a multimeric complex, utilizes NADPH as substrate and reduces O2 to produce superoxide (O2˙−) that is then further dismutated into stable and diffusible H2O2 pro-oxidant. The plasma membrane-associated components gp91phox and p22phox together form flavocytochrome-b558 that is responsible for enzymatic stability and activity of the NADPH oxidase. Phosphorylation of cytosolic components (p47phox, p67phox, and p40phox), and small Rho GTPases, in response to exogenous or endogenous stimuli initiates their translocation to the cell membrane, and NOX2 activation [50]. T. cruzi-generated stimuli that initiate translocation of cytosolic factors and NOX2 assembly in infected macrophages are not identified. However, cytochemical detection of NOX2 components at the plasma membrane of peritoneal mouse macrophages exposed to T. cruzi is noted [51, 52]. Others have used an in vitro assay system or animal models and shown that NOX2-dependent O2•− formation is required for parasite control in macrophages and splenocytes [53, 54]. In addition to direct killing, NOX2/ROS also signaled the development of antigen-specific CD8+ T cell response that was required for control of tissue parasites in infected mice [55].

Inducible nitric oxide synthase (iNOS or NOS2) is induced by immunological stimuli in a Ca+2-dependent manner, and it utilizes L-arginine and O2 for the synthesis of L-citrulline and nitric oxide (NO) in a complex oxidoreductase reaction [56]. NF-κB and ISGF3 transcription factors act sequentially and cooperatively at the Nos2 promoter to signal iNOS expression and NO production in macrophages. The reaction of NO with O2˙− produces peroxynitrite that is a strong cytotoxic oxidant shown to promote killing of T. cruzi in macrophages [57, 58]. However, the extent of NOX2-dependent ROS response and iNOS-dependent NO response in human and mouse macrophages infected with T. cruzi is significantly lower than that observed in LPS/IFN-γ-induced proinflammatory macrophages [11], suggesting a potential mechanism for survival and dissemination of parasite by macrophages.

Notably, trypanosomes have evolved an elaborate antioxidant system to prevent ROS/NO-mediated killing. Trypanosome antioxidant defense utilizes trypanothione (T(SH)2) that shuttles the reducing equivalents to peroxidases through tryparedoxin intermediate [59]. Of the five tryparedoxin peroxidases (TXNPxs) identified in T. cruzi, cytosolic and mitochondrial TXNPxs were shown to increase during differentiation from the non-infective to the infective forms of the parasite and were found to be present at higher levels in the virulent isolates compared with the attenuated strains [59, 60•]. Parasite isolates overexpressing cytosolic and mitochondrial TXNPxs were able to infect and multiply more efficiently in macrophages, thus suggesting that TXNPxs provide at least one mechanism to provide survival benefits to parasite in macrophages and other cells [58, 61]. Importantly, trypanothione synthase is unique to parasites and rated as the most promising target to achieve selective inhibition of parasite [62].

At low levels, ROS are critical signaling intermediates involved in NF-κB-dependent expression of proinflammatory cytokines (e.g., TNF-α, IL-1β) by macrophages and dendritic cells (DCs). Low levels of ROS are produced during T. cruzi infection, and if scavenged, resulted in inhibition of inflammatory cytokines’ production in macrophages. The in vitro observations were confirmed by studies in mice. Garg and group initially utilized chemical antagonists of NOX2 and ROS scavenging antioxidants to demonstrate that blocking NOX2/ROS arrested the activation and proliferation of splenic phagocytes and production of inflammatory cytokines (e.g., IL-1, IL-6, IFN-γ, TNF-α) in infected mice [53]. These findings were also confirmed in p47phox−/− mice that also exhibited increased susceptibility to T. cruzi and succumbed to infection [55]. Whether T. cruzi-induced NOX2/ROS in macrophages signal the nuclear transport or assembly of transcription factors (e.g., NF-κB and AP-1) for promoting cytokine gene expression is not fully delineated. However, NF-κB activation has been described in a number of other cell types, including epithelial cells, endothelial cells, myocytes, and fibroblasts infected with T. cruzi (or T. cruzi-derived proteins, e.g. trans-sialidase) [63,64,65,66, 67••]. NF-κB activation increased the resistance to infection in many of these cell types. A majority of these studies, however, did not attempt to determine the source of ROS and its role in signaling NF-κB-dependent cytokine gene expression in non-phagocytic cells invaded by T. cruzi.

Interestingly, in cardiac myocytes, T. cruzi signals ROS production through mitochondria. It was found that T. cruzi invasion of cardiac myocytes disturbed the mitochondrial membrane potential and enhanced the release of electrons to O2 resulting in O2˙− production at the complex I and complex III of the respiratory chain [68]. The T. cruzi-induced mitochondrial ROS (mtROS), like NOX2, induced ROS in macrophages and signaled nuclear translocation of Rel A (p65) and activation of NF-κB-dependent cytokine gene expression in infected cardiac myocytes [41, 68]. The mtROS also provided secondary signal for cytokine gene expression; Garg and coworkers showed that ROS-induced DNA damage (e.g., 8-hydroxyguanine (8-oxoG) lesions) enhanced the expression and activation of a DNA repair enzyme polyadenosine ribose polymerase 1 (PARP-1) in infected cardiac myocytes. PARP-1 functions by poly ADP-ribosylation of nuclear proteins [69]. However, in the context of T. cruzi infection, PARP-1 had pathophysiological effects. This was evidenced by the observation that inhibition of PARP-1 by using RNAi or a chemical inhibitor (PJ34) was beneficial in blocking mtROS formation, DNA damage, and cytokine gene expression [41]. How PARP-1/PAR contribute to mitochondrial disturbance is not known, though PARP-1’s role in regulating cytokine gene expression in infected cardiac myocytes was described. PARP-1 does not directly interact with p65, and it does not signal RelA (p65) translocation to nuclei in infected cardiac myocytes. Instead, PARP-1 contributes to PAR modification of RelA (p65)-interacting nuclear proteins and assembly of an NF-κB transcription complex. Sirtuin 1, a highly conserved member of the family of NAD+-dependent Sir2 histone deacetylases, competes with PARP-1 for NAD+ substrate and integrates mitochondrial metabolism and inflammation. In a recent study, Wan et al. demonstrated that treatment with SIRT1 agonist (SRT1720) had no effect on parasite burden, but it suppressed the NF-κB transcriptional activity and reduced the oxidative and inflammatory stress in infected cells and mice [67••]. These studies point to the possibility that the ROS-PARP-1/PAR-RelA contribute to inflammatory pathology in T. cruzi infection, and it can be controlled by enhancing the SIRT1 activity.

Macrophages and Chronic Inflammation

The New York Heart Association (NYHA) functional classification of heart failure places patients in one of the four categories according to the severity of their symptoms. Chagasic patients with NYHA class III-IV display a proinflammatory transcriptomic and proteomic profile in peripheral blood mononuclear cells [70,71,72,73] and increased levels of TNFα+ monocytes [74,75,76] in the circulation. In comparison, IL10+ monocytes with an anti-inflammatory transcriptome were detected in peripheral blood of infected humans classified in NYHA class I-II with none-to-minimal left ventricular dysfunction [74,75,76]. The factors that drive macrophage phenotype in chronic infection are not described; however, these observations suggest that proinflammatory (vs. pro-healing) response of macrophages is a contributing factor in clinical evolution of Chagas disease.

Cells release diverse types of membrane vesicles into extracellular environment. These extracellular vesicles (EVs) selectively sort the biological information and carry out an important mode of intercellular communication in health and disease [77]. Recently, Garg and colleagues demonstrated that plasma EVs of NYHA class III-IV (vs. NYHA class I-II) patients elicited proinflammatory macrophage responses with upregulation of CD14+/CD16+ surface markers and inflammatory gene expression profile and cytokine release (IL-2 + IFN-γ > GCSF) [78••]. Similarly, sera components or plasma EVs of chronically infected (vs. control) mice elicited a strong ROS/NO response and proinflammatory cytokine profile in murine macrophages. In comparison, mice given a T. cruzi vaccine followed by a T. cruzi challenge elicited an M2-like macrophage phenotype [79]. Compositional analysis revealed that EVs of chronically infected mice and patients were composed of membrane vesicles of cardiac myocyte, macrophage, and leukocyte origin [78••]. In another study, Dhiman et al. demonstrated that cardiac proteins oxidized during T. cruzi infection serve as antigens, and treatment of infected rodents with phenyl-α-tert-butyl nitrone (antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats [80]. Though molecular markers on EVs are yet to be identified, these results strongly suggest that peripheral EVs consisting oxidized proteins of the cardiac and other cellular origin contribute to inflammatory state of macrophages in the setting of Chagas disease. Further studies may delineate the extracellular and intracellular immune receptors engaged by EVs in signaling inflammatory macrophages and further evaluate the potential benefits of PARP-1/SIRT1 balance in establishing resting homeostasis in peripheral and tissue macrophages in Chagas disease.

Mitochondrial Dysfunction and Oxidative Stress in Chronic Chagas Disease

Garg and colleagues were the first to report that T. cruzi invasion elicits Ca+2 overload, mitochondrial membrane potential transition [41, 68], and O2•− production in cardiac myocytes [81, 82]. In vivo studies showed that mitochondrial defects continue beyond the acute infection phase with consistently high levels of mtROS and oxidative adducts (e.g., protein carbonyls, lipid hydroperoxides) and a decline in oxidative phosphorylation capacity in the myocardium of chronically infected mice [81, 83]. A similar pro-oxidant milieu evidenced by a decline in the activities of the respiratory complex III and antioxidant enzymes (MnSOD and GPX) as well as in GSH contents and an increase in oxidative adducts has been reported in humans chronically infected with T. cruzi [84,85,86,87]. Moreover, treatment of T. cruzi-infected mice and rats with phenyl-α-tert-butyl nitrone, a spin-trapping antioxidant, tipped the balance in favor of preserving mitochondrial and left ventricular function associated with a significant decline in the myocardial oxidative adducts [86, 88]. Likewise, treatment with sildenafil, an inhibitor of phosphodiesterase 5, provided cardioprotection through preservation of cGMP/PKG activity and antioxidant/oxidant balance in chronically infected mice [89]. Others have shown a decline in oxidative stress in human chagasic patients given vitamin A [90]. Finally, Garg and colleagues demonstrated a significant control of myocardial oxidative adducts, preservation of mitochondrial and myofibrillar structure and arrangement, and improved mitochondrial and left ventricular function in MnSODtg mice equipped with an extra copy of MnSOD to scavenge cardiac mtROS [91••], thus conclusively establishing the pathological significance of mtROS and chronic oxidative stress in Chagas disease.

The re-expression of fetal cardiac genes (ANP, BNP, αsk-Actin, and β-MHC) is a hallmark of hypertrophic remodeling. Recent data suggest the involvement of ERK-1/2, small GTPase Ras, and NF-κB/ASK-1 in response to α-adrenergic agonist or angiotensin II stimulation in signaling cardiac remodeling [92]. Mice and cultured myoblasts infected with T. cruzi display increased expression of ERK, cyclin D1, and AP-1 whereas the expression of caveolins (Cavs), which negatively regulate ERK and cyclin D1, was decreased [93,94,95,96]. In that regard, Cav-1 and Cav-3 null mice as well as Cav-1/Cav-3 double-knockout mice display cardiac hypertrophy and interstitial fibrosis [97,98,99]. These observations, along with the finding that scavenging of ROS suppressed the hypertrophic gene expression and collagen content in chagasic mice, imply that ROS contributes to cardiac remodeling in chagasic disease. Further, the mtROS was found to be of pathological significance. The authors noted that treatment with benznidazole (anti-parasite drug) suppressed the classical mediators of inflammatory ROS (e.g., NOX2 and myeloperoxidase) but not the hypertrophic response in chagasic rodents, while the hypertrophic phenotype was depressed when mice or rats were treated with an antioxidant as well as in MnSODtg mice with enhanced mitochondrial antioxidant capacity [88, 91••].

Why antioxidant response is not triggered in the presence of continued oxidative stress in the chagasic myocardium was not understood until recently. NFE2L2 (also called Nrf2) is a transcription factor that regulates the expression of antioxidant proteins. Several drugs that stimulate the NFE2L2 pathway are being evaluated for treatment of diseases that are caused by oxidative stress. Garg and colleagues showed that NFE2L2 expression, nuclear translocation, and binding to cis-acting DNA regulatory antioxidant response elements (AREs) were significantly decreased and associated with a decline in antioxidants’ (e.g. γGCS, HO1, GCLM) expression in cardiac myocytes and myocardium of mice infected with T. cruzi. Importantly, inhibiting the T. cruzi-induced mtROS by overexpression of MnSOD in cardiac myocytes preserved the NFE2L2 transcriptional activity and antioxidant/oxidant balance, and MnSODtg mice also preserved the cardiac structure and function [91••]. This study provides evidence that mtROS inhibition of NFE2L2/ARE pathway constitutes a key mechanism in signaling the fibrotic gene expression and evolution of chronic chagasic cardiomyopathy.

Summary and Future Perspectives

The Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial was designed to evaluate the efficacy and safety of benznidazole in reducing the clinical outcomes among patients with established chagasic cardiomyopathy [100••]. Unfortunately, benznidazole treatment reduced the serum parasite detection but did not significantly reduce cardiac clinical deterioration through 5 years of follow-up. These disappointing results indicate that anti-parasite drugs are not effective in chronic Chagas disease, and new strategies are required in the treatment of this disease.

The current literature suggest that the inability of macrophages to elicit strong ROS/NO response, coupled with parasites’ ability to scavenge oxidants, contribute to long-term parasite persistence and mitochondrial oxidative stress in the heart. The cellular oxidative damage provides stimulus to macrophage activation and chronic inflammatory stress in chagasic disease. We propose that metabolic regulators, PARP-1/SIRT1, determine the disease outcome by modulating the mitochondrial and macrophage stress and antioxidant/oxidant imbalance and offer a potential new therapy against chronic Chagas disease.